Effects of γ-irradiation on elongation and indole-3-acetic acid level of maize (Zea mays) coleoptiles

The effects of γ-irradiation on elongation and the level of indole-3-acetic acid (IAA) of maize (Zea mays) coleoptiles were investigated. When 3-day-old seedlings of maize were exposed to γ-radiation lower than 1 kGy, a temporal retardation of coleoptile elongation was induced. This retardation was at least partly ascribed to a temporal decrease in the amount of free IAA in coleoptile tips on the basis of the following facts: (1) the reactivity to IAA of the elongating coleoptile cells was not altered by irradiation; (2) endogenous IAA level in the tip of irradiated coleoptiles was at first unchanged, but then declined before returning to nearly the same level as that of the non-irradiated control; and (3) the amount of IAA that diffused from coleoptile tip sections showed a similar pattern to that of endogenous IAA. The rate of conversion between free and conjugated IAA was not significantly affected by irradiation. These results suggest that a temporal inhibition of maize coleoptile elongation induced by γ-irradiation can be ascribed to the reduction of endogenous IAA level in the coleoptile tip, and this may originate from the modulation in the rate of IAA biosynthesis or catabolism.     

Is pathogenicity of Ustilago maydis (huitlacoche) strains on maize related to in vitro production of indole-3-acetic acid?

Corn smut caused by Ustilago maydis (DC) Corda on maize (Zea mays L.) is characterized by gall (tumour) formation on aerial parts of the plant. Young galls on immature corn ears are called huitlacoche and are highly appreciated as a food delicacy. Several reports have suggested that production of the auxin indole-3-acetic acid (IAA) by U. maydis might be involved in tumour formation. Because strains showing defects in IAA biosynthesis (Iaa^– phenotype) would be valuable in elucidating the role of IAA in pathogenicity, in a previous study we isolated and analysed several such mutants. In the present work, we have crossed a null Iaa^– auxotrophic mutant with compatible wild-type strains. The main objective of the present work was to evaluate the pathogenicity of crosses involving wild-type and Iaa^– strains, in order to examine its relation to IAA production. Significant differences were found in growth, IAA production and ability to survive in water suspension among wild-type and mutant progeny strains. In general, high levels of pathogenicity were associated to high levels of IAA production by the strains, which supports the hypothesis that U. maydis strains require the ability to produce IAA in order to induce tumours in the host.     

Indole-3-methanol—a metabolite of indole-3-acetic acid in pea seedlings

Summary Degradation of indole-3-acetic acid was investigated in etiolated pea shoots; the study was limited to indolic metabolites. The products formed were fractionated by column chromatography and identified by thin-layer chromatography and chemical methods. The pathway of indole-3-acetic acid degradation involving indole-3-aldehyde was found to be more significant than stated in literature, and indole-3-methanol was established as the major indolic metabolite.The following abbreviations will be used: IAA: indole-3-acetic acid; IM: indole-3-methanol; IAld: indole-3-aldehyde; ICA: indole-3-carboxylic acid.     

Biosynthesis of indole-3-acetic acid in tomato shoots: Measurement,mass-spectral identification and incorporation of −2H from −2H2O into indole-3-acetic acid,d- and l-tryptophan,indole-3-pyruvate and tryptamine

Indole-3-acetic acid (IAA) and its putative precursors, l- and d-tryptophan, indole-3-pyruvate, and tryptamine were isolated from tomato (Lycopersicon esculentum (L.) Mill.) shoots, identified by mass spectrometry, and measured using capillary gas chromatography with an electron capture detector and radioactive internal standards. Average amounts present were 7.9ng · (g FW)^–-1 IAA, 5.7ng · (g FW)^–-1 indole-3-pyruvate, 132 ng · (g FW)^–-1 tryptamine, 103 ng · (g FW)^–-1 d-tryptophan, and 2250 ng · (g FW)^–-1 l-tryptophan. Indole-3-acetaldoxime was not found; detection limits were less than 1ng · (g FW)^–-1. When tomato shoots were incubated for 6, 10 and 21 h in 30% ^–2H₂O, up to four positions in IAA, l- and d-tryptophan, tryptamine and indole-3-pyruvate became labelled with ^–2H. Compounds became labelled rapidly with 10% of IAA molecules containing ^–2H after 6 h. The percentage of labelled molecules of IAA and l-tryptophan increased up to 10 h but then decreased again, correlating with an increase in the total shoot tryptophan and presumably a result of protein hydrolysis in the excised, slowly senescing tissue. The amount of ^–2H in d-tryptophan also showed an increase followed by a decrease, but the proportion of labelled molecules was much less than in l-tryptophan and IAA. Tryptamine became labelled initially at a similar rate to IAA but continued to accumulate ^–2H up to 21 h. We conclude that tryptamine is synthesized from a different pool of tryptophan from that used in IAA synthesis, and is not a major endogenous precursor of IAA in tomato shoots. Indole-3-pyruvate was the most heavily labelled compound after 6 and 10 h incubation (21-h data not available). Furthermore, the proportion of ^–2H-labelled indole-3-pyruvate molecules was quantitatively consistent with the amount of label in IAA. On the other hand, a quantitative comparison of the IAA turnover rate and the rate of ^–2H incorporation into both l- and d-tryptophan indicates that IAA is not made from the total shoot pool of either l- or d-tryptophan. Instead IAA appears to be synthesized from a restricted pool which is turning over rapidly and which has access to both newly synthesized tryptophan and that from protein hydrolysis.Abbreviations GC-ecd gas chromatography with electroncapture detector - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - IAOX indole-3-acetaldoxime - IPyA indole-3-pyruvate - PFB pentafluorobenzyl - R_T retention time - TNH₂ tryptamine - Trp tryptophan - SIM selected ion monitoring We wish to thank Ms. Sue Alford for running the mass spectra and Dr Harry Young for advice with the mass spectrometry. The work was supported by grants from the University of Auckland Research Committee and the C. Alma Baker Trust fund. The mass spectrometer was supported jointly by the University Grants Commitee (NZ) and the DSIR Division of Horticulture and Processing.     

Indole-3-methanol-β-D-glucoside and indole-3-carboxylic acid-β-D-glucoside are products of indole-3-acetic acid degradation in wheat leaf segments

The formation of glucosides of indole-3-methanol (IM) and indole-3-carboxylic acid (ICA) after application of radio-labelled indole-3-acetic acid (IAA) and IM to leaf segments of wheat and other members of the Gramineae is described. The results suggest that the decarboxylative pathway of IAA degradation leading to ICA via IM may occur in the leaf segments.     

Effect of exogenous abscisic acid (ABA) on the morphology,phytohormones, and related gene expression of developing lateral roots in ‘Qingzhen 1’ apple plants

Plant Cell, Tissue and Organ Culture (PCTOC) - Lateral roots (LRs) are critical for plant stress tolerance and productivity. Understanding how hormones and genes interact in a fluctuating...     

Endogenous abscisic acid and 2-trans-abscisic acid in alternate bearing ‘Wilking’ mandarin trees

The relationship of abscisic acid (ABA) and 2-trans-abscisic acid (t-ABA) to alternate bearing has been examined in Wilking mandarin (Citrus reticulata Blanco) trees. Leaves, stems and buds of trees loaded with fruit (on trees) had 4.3, 6.0 and 2.2 fold higher ABA levels than the corresponding organs from off trees. Leaves had higher ABA levels than stems and buds in both on and off trees. t-ABA was non-detectable in Wilking leaf, stem and bud tissue. Amounts of t-ABA not exceeding 40% of the ABA content, were found in Shamouti and Valencia orange buds and in Wilking fruit peel.The elevated levels of ABA in on tree organs may reflect a stress imposed by the fruit overload.     

Effect of potassium humate on apple cv. ‘Golden Delicious’ cultured in vitro

The effects of humic substances on in vitro culture of Golden Delicious apple are reported. Potassium humate (KH) when used in proliferation showed a negative interaction with BA while it enhanced rooting when IBA was not present in the culture medium. In the presence of IBA, KH increased root number and reduced root growth. The highest concentration tested, 500 mg l^-1, caused a drastic reduction in root system development. 50 mg l^-1 KH hastened rooting and plants grew more rapidly when transferred to soil.     

Genetic and biochemical studies on perchloroethylene ‘in vitro’ and ‘in vivo’

Perchloroethylene (PCE) was tested in a diploid strain (D7) of the yeast Saccharomyces cerevisiae in suspension tests with and without a mammalian microsomal activation system (S9) and ‘in vivo’ by the intrasanguineous host-mediated assay. In addition, enzyme alteration studies were performed in mice non-pretreated or pretreated with phenobarbital + β-naphthoflavone. PCE did not induce any genetic effect either ‘in vitro’ or ‘in vivo’. In the suspension test, PCE was more toxic without metabolic activation and less toxic with mammalian microsomal activation. The enzymatic determinations showed an increase of the aminopyrine demethylase activity and of the level of cytochrome P-450.     

Effects of gibberellic acid and abscisic acid on α-amylase activity and ultrastructure of aleurone cells ofAvena sativa L.

The effects of GA₃ and/or ABA on the α-amylase activity and the ultrastructure of aleurone cells in halves of seeds without embryos (embryo-less half seeds) of oats (Avena sativa L.) were studied. α-Amylase activity was detected by the starch-agar gel method in the aleurone layers of embryo-less half seeds soaked in 1 μM GA₃ solution or 100 μM GA₃+10 μM ABA solution but not in those of seeds soaked in distilled water, 10 μM ABA solution, or 1 μM GA₃+10 μM ABA solution. Ultrastructural examinations of aleurone cells with α-amylase activity showed a decrease in the number of sphaerosomes, the appearance of flattened saccules pressed to the surface of aleurone grains, and the development and transformations of the rER from a slender form to the one with wide inner spaces. In the aleurone cells in which the enzyme activity was not detected, components of the rER showed only slender profiles. The number of sphaerosomes did not decrease, and no flattened saccules appeared in the aleurone cells treated with 10 μM ABA or 1 μM GA₃+10 μM ABA.     

Induction of karyopherin α1 expression by indole-3-acetic acid in auxin-treated or overproducing tobacco plants

Macromolecules may transfer between the cytoplasm and the nucleus only through specific gates—the nuclear pore complexes (NPCs). Translocation of nucleic acids and large proteins requires the presence of a nuclear localization signal (NLS) within the transported molecule. This NLS is recognized by a class of soluble transport receptors termed karyopherins α and β. We previously characterized the expression pattern of the tomato karyopherin α1 (LeKAPα1) promoter in transformed tobacco plants. Expression of LeKAPα1 was mainly observed in growing tissues where cell division and extension is rapid. The expression pattern of LeKAPα1 resembled that of auxin-responsive genes. This led us to suggest that auxin participates in the regulation of LeKAPα1 expression. Here we characterized the correlation between auxin level and the activity of the LeKAPα1 promoter. To this end, transgenic tobacco plants carrying the GUS reporter gene under the control of the LeKAPα1 promoter were treated with various levels of exogenous auxin. We also studied transgenic plants in which we increased the endogenous levels of auxin. For this, we expressed in plants both the LeKAPα1 promoter-GUS reporter and the Agrobacterium tumefaciens iaaM gene, which increases the endogenous levels of auxin. The results indicate that the auxin indole-3-acetic acid (IAA) can induce LeKAPα1 expression. We also identified that the sites and levels of LeKAPα1 expression correlated with the endogenous pathways of polar auxin transport.Key words: auxin, karyopherin α1, nuclear pore complex, TYLCV, plant virus     

The relationship between leaf water potential ψ leaf and the levels of abscisic acid and ethylene in excised wheat leaves

The amount of diffusible ethylene from excised wheat leaves (Triticum aestivum L. cv. Eclipse) increased when they were subjected to water stress. The quantity of ethylene produced was related to the severity of the stress, reaching a maximum at a leaf water potential _leaf of approximately-12 bars. Irrespective of the severity of the stress, the maximum rate of ethylene production usually occurred between 135–270 min after applying the stress and then the rate declined. Part of the decline may have been due to an oxygen deficiency in the leaf chambers. In excised water-stressed leaves there was a sigmoid relationship between increasing ethylene and abscisic acid (ABA) levels and decreasing leaf water potential values. The two curves were displaced from each other by approximately 1 bar, with ethylene evolution leading that of ABA accumulation. The maximum rate of increase in ethylene occurred between-8 and-9 bars and for ABA between-9 and-10 bars. A significant increase in the levels of these two plant growth regulators was found when the _leaf decreased outside the normal diurnal _leaf range by 1 bar for ethylene and 2 bars for ABA. Because of the sigmoid nature of the curves there was no distinct threshold _leaf value triggering-off an increase in ethylene or ABA, but with ABA the curve became very steep at a _leaf value of-9.3 bars and this could be looked upon as a kind of threshold value.It seems unlikely that the stress-induced ethylene evolution in excised wheat leaves stimulated the accumulation of ABA, because when the leaves were subjected to a substantial water stress (e.g. _leaf bars) ABA increased immediately and at a faster rate than ethylene.Abbreviations ABA abscisic acid - GLC gas-liquid chromatography - RWC relative water content - TLC thin-layer chromatography - _leaf leaf water potential     

Quantitative determination of indole-3-acetic acid in yeasts using high performance liquid chromatography—tandem mass spectrometry

High performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was applied to the comprehensive analysis of the ability of yeast strains to synthesize a plant hormone indole-3-acetic acid (IAA). A total of 124 strains (37 species) of yeasts isolated from various regions and substrates were studied. Testing of IAA production showed that 92% strains were capable of IAA synthesis. The results indicated that, in general, ascomycetous yeasts were more active auxin producers than basidiomycetous ones. Geographically, strains from tropical regions were the most active IAA producers. Analysis of the substrate variability of the strains showed higher auxin production (on average) by the yeasts isolated from plants compared to the soil isolates, indicating a specific regulatory role of the plant yeast population.     

Regulatable endogenous production of cytokinins up to ‘toxic’ levels in transgenic plants and plant tissues

The effects of expressing a chimeric gene consisting of a soybean heat shock gene promoter and a sequence that encodes an enzyme catalyzing the synthesis of a potent phytohormone, the cytokinin iPMP, have been analyzed in transgenic tobacco plants. The production of cytokinin endogenously produced several effects previously undocumented. The differentiation of shoots independent of exogenous cytokinin from heat-treated transgenic plant leaf explants demonstrates that long-term heat treatments do not interfere with complex developmental processes. This extends the potential usefulness of heat shock gene promoters to conditionally express genes during windows of development that span several weeks.     

Effects of 4-CPPU on in vitro multiplication and sustainable generation of Hibiscus rosa-sinensis L. ‘White Butterfly’

There are more than nine thousand cultivars of Hibiscus rosa-sinensis L., with a series of flowers with shapes, colors and new cultivars continues as generated through both traditional and modern breeding techniques. In this study, advanced biotech methods of in vitro culture have been used to identify a technique for the efficient mass multiplication of H. rosa-sinensis ‘White Butterfly’, using phenyl urea, N-(2-Chloro-4-pyridyl)-N′-phenylurea (4-CPPU). For the first time, the effects of 4-CPPU for stimulating axillary shoot proliferation and multiple shoot regenerations from nodal explants were evaluated, and the optimal nutrient media deduced. From the diverse concentrations as 0.1, 0.5, 2.5, 5.0 & 10.0 µM of 4-CPPU, the highest frequency of shoots was recorded at 2.5 µM supplied in Murashige and Skoog (MS, pH-5.8) medium. After eight-weeks of culture, on an average of 6.7 shoot were obtained on this media with shoot heights of 4.2 cm from each explant. With the involvement of 0.5 µM-IBA (indole-3-butyric acid) in MS medium the regenerated shoots were rooted and followed by successful acclimation to ex vitro conditions. The ploidy consistency among the micro-plants was analyzed using flow cytometry and compared with ex vitro grown plants. No differences in the ploidy levels were observed among the 4-CPPU induced plants, when compared with the donor plants.     

Effects of γ-rays on Poly-L-glutamic acid and Poly-D,L-glutamic acid in the solid state

Summary Poly-L-glutamic acid and poly-D,L-glutamic acid, as models of proteins, were irradiated with⁶⁰Co--radiation in air and under vacuo to examine whether or not the changes caused by the exposure to ionizing radiation depend on the conformations of polypeptides.It was found that theG- values (yield of main-chain scissions per 100 eV of energy absorbed) of both polypeptides are approximately equal for the irradiation in air, while under vacuo theG- value of poly-D,L-glutamic acid is larger than that of poly-L-glutamic acid. This observation for irradiation under vacuo was ascribed to the stabilizing effect of intramolecular hydrogen bond bridges in poly-L-glutamic acid. It was also found that the-helical structure of poly-L-glutamic acid is destroyed by the exposure to ionizing radiation.     

Effectiveness of indoleacetic acid,indolebutyric acid and naphthaleneacetic acid during adventitious root formation in vitro in Malus ‘Jork 9’

The production of an antifungal spirostanol saponin designated SC-1 has been detected in cell suspension cultures of the Mexican species Solanum chrysotrichum. Batch cultures of a cell suspension obtained from hypocotyl derived calluses of this species were grown for 25 days in shake flasks containing Murashige & Skoog (MS) medium. Throughout the growth cycle, fresh and dry weight, SC-1 yield, and uptake of sucrose, glucose and fructose were determined. The effects of inoculum size and sucrose concentration on the biomass accumulation and synthesis of the active metabolite, were studied. The maximum SC-1 production, above 14 mg.g⁻¹ (which was fifty times that of field grown plants), was reached after 20 days using a 2% inoculum and complete MS medium supplemented with 2 mgl⁻¹ 2,4-D, 2 mg l⁻¹kinetin, and sucrose between 30 and 45 gl⁻¹. . This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of ornithine on neutrophil (PMN) free amino acid and α-keto acid profiles and immune functions in vitro

Summary. The objective of this study was to determine the effects of ornithine on polymorphonuclear leucocyte (PMN) free amino- and -keto acid profiles, superoxide anion (O₂^–) generation, hydrogen peroxide (H₂O₂) formation and released myeloperoxidase acitivity (MPO). Exogenous ornithine significantly increased PMN asparagine, glutamine, asparatate, glutamate, arginine, citrulline, alanine, -ketoglutarate and pyruvate as intracellular ornithine increased. Concerning PMN immune function markers ornithine increased H₂O₂-generation and MPO acitivity while O₂^–-formation was decreased. We believe therefore that ornithine is important for affecting PMN susceptible free amino- and -keto acid pool although the mechanisms are not yet clear. This may be one of the determinants in PMN nutrition considerably influencing and modulating PMN host defense capability.