Physiological effects of betalains upon higher plants

Red and yellow betalains isolated from red beetroots by means of gel filtration were strong inhibitors of indole-3-acetic acid oxidase; 50% inhibition was obtained at 5 × 10^?7 M and 3 × 10^?7 M respectively. Concentrations of 10^?4 M betanin had no effect upon ATP production in mitochondria. The red pigment relieved the inhibitory effects upon wheat root elongation caused by indole-3-acetic acid but not the inhibition caused by 2,4-dichlorophenoxyacetic acid.     

Differences in endo/exogenous auxin profile in cuttings of different physiological ages

The process of physiological ageing in woody plants is a very important factor influencing adventitious rooting. However, there is a lack of knowledge of biochemical backgrounds triggering ageing and consequently, rhizogenesis. Experiments with Prunus subhirtella ‘Autumnalis’ leafy cuttings of three different physiological ages (adult (over 40-year-old stock plants), semi-adult (5-year-old cutting plants) and juvenile (5-year-old in vitro plants)) were conducted in 2009. Half of the cuttings were banded ca. 3 cm above the bottom of the cutting with aluminum wire prior to insertion into the substrate to block the polar auxin transport. IBA, which was exogenously applied to the cuttings, could only be detected in the base of the cuttings on the first day after severance. Juvenile cuttings tended to have the highest values, but the effect was age specific. Later, the detection was not possible, regardless of the age. The IAA profile in cutting bases was similar for all physiological ages, reaching the peak on the first day after severance. Juvenile cuttings, in which the stems had been banded before insertion, contained more IAA in their bases on day 1 compared to the stems, which were not banded. These cuttings presumably transported absorbed auxin mainly via phloem, and not via mass flow like semi-adult and adult cuttings, where IAA concentrations were similar or even greater in non-banded cuttings compared to banded ones. These cuttings also tended to exhibit the best rooting results. The IAA-Asp accumulation was especially strong in adult cuttings, which contained significantly more aspartate on the first and third days after severance when compared with semi-adult and juvenile cuttings.     

Effects of sodium diethyldithiocarbamate, solvent, temperature and plant extracts on the stability of indoles

A study has been made of the effects of solvent, temperature, and the antioxidant, sodium diethyldithiocarbamate, on the breakdown of indole-3-pyruvic acid to indole-3-acetic acid (IAA). In addition, the degradation of tryptophan, tryptamine, indole-3-pyruvic acid, indole-3-acetaldehyde and indole-3-ethanol to IAA during the purification and analysis of extracts from Pinus sylvestris L. needles, in the presence and absence of sodium diethyldithiocarbamate, has been investigated. The data obtained indicate that if the antioxidant is supplied throughout the analytical sequence there is a marked reduction in the spontaneous formation of IAA from other indolic compounds and, by inference, the stability of indoles in general is enhanced.     

Effects of putrescine and inhibitors of putrescine biosynthesis on organogenesis inEuphorbia esula L.

Summary Exogenous putrescine (≤5 mM) had little effect on root or shoot formation in aseptically isolated hypocotyl segments of leafy spurge (Euphorbia esula L.) grown on full-strength B5 medium. Unexpectedly, putrescine inhibited root and shoot formation in hypocotyl segments grown on B5 medium diluted 10-fold. In the full-strength medium, root and shoot formation were inhibited by 0.5 mM concentrations ofdl-α-difluoromethylornithine (DFMO) anddl-α-difluoromethylarginine (DFMA). DFMO and DFMA are inhibitors of the ornithine decarboxylase and arginine decarboxylase pathways, respectively, of putrescine biosynthesis in plants. Exogenous putrescine (0.5 to 5 mM) did not reverse either the DFMO-or DFMA-induced inhibition of shoot formation. However, the DFMA-induced inhibition of root formation was partially reversed by exogenous putrescine. The auxin, indole-3-acetic acid (IAA), reduced the inhibitory effects of DFMO+DFMA (applied together) on both roots and shoots. In the first few days of culture, the endogenous levels of putrescine and spermidine, but not of spermine, increased in the presence of IAA. The levels of putrescine and spermidine in the tissues did not correlate well with either root or shoot production in the later stages of organ formation; especially in tissues treated with IAA. These results show that there were no obvious correlations between polyamine levels and organogenesis in leafy spurge hypocotyl segments, although residual putrescine or spermidine or both in the tissues at the time of excision may be indirectly involved in the early stages of root formation.     

Chlorophyll-protein levels and degree of thylakoid stacking in radish chloroplasts from high-light, low-light and bentazon-treated plants

Lemna gibba plants were incubated aseptically on medium containing labelled 10^-7 M indole-3-acetic acid (IAA-1-¹⁴C). Most of the radioactivity disappeared from the culture medium during a 24 h light period. A high percentage of the loss was due to photolysis and only a low percentage of the radioactivity was recovered in the plants. Uptake of ¹⁴C by the plants was strongly stimulated by light. The radioactivity taken up by the plants was the sum of photosynthetically taken up ¹⁴CO₂ and ¹⁴C taken up in IAA. Analyses with the indolo-α-pyrone fluorescence method revealed that the free IAA content was almost the same in plants grown in control and in IAA media for 5 h, whereas the amount of IAA which could be liberated by alkaline hydrolysis was doubled by the presence of IAA in the medium.     

Comparative effect of IBA, BSAA and 5,6-Cl2-IAA-Me on the rooting of hypocotyl in mung bean

Mung bean hypocotyl cuttings were treated with indole-3-butyric acid (IBA), 3-(benzo[b]selenienyl)acetic acid (BSAA) and 5,6-dichloroindole-3-acetic acid methyl ester (5,6-Cl2-IAA-Me) at different concentrations, respectively. Each chemical produced the maximum number of adventitious roots at a different concentration. Compared with IBA treatment, 5,6-Cl2-IAA-Me and BSAA treatments significantly increased root numbers on hypocotyl cuttings at lower concentration, particularly of 5,6-Cl2-IAA-Me treatment. Combinations of paclobutrazol (PB) with either 5,6-Cl2-IAA-Me or BSAA significantly stimulated the production of more adventitious roots than either chemical alone or combined. Capillary electrophoresis analysis have shown that the levels of IAA, IBA and BSAA in IBA plus PB or BSAA plus PB treatments were higher than those of IBA or BSAA alone. It was suggested that the cause of the synergistic effect of IBA (or BSAA) plus PB treatment might be due to increased endogenous auxin level. The activities of peroxidase and IAA oxidase in the rooting zone coincided with root development, indicating that the activities of these two enzymes were positively correlated to rooting. Peroxidase and IAA oxidase activity in all treatments started 24 h and 12 h after cutting, respectively. It is suggested that the major role of IAA oxidase differed from that of peroxidase in adventitious root formation.     

Effect of 2,5-norbornadiene on abscission and ethylene production in citrus leaf explants

Citrus ( Citrus sinensis L. Osbeck) leaf explants completely abscise within 48 h when exposed to saturating amounts of ethylene at 25°C. When 2,5-norbornadiene was added, 2000 μl 1⁻¹ reduced abscission of explants also exposed to 2 μl 1⁻¹ of ethylene to the level of the control, and 8000 μl 1⁻¹ reduced abscission in explants exposed to 10 μl 1⁻¹ of ethylene to the level of the control, but abscission was complete when 1 000 μl 1⁻¹ of ethylene was used in the presence of 8 000 μl 1⁻¹ of 2,5-norbornadiene. When explants were exposed to 2 μl 1⁻¹ of ethylene, 2000 μl 1⁻¹ of 2,5-norbornadiene prevented abscission if applied up to 10 h after exposure to ethylene. After 18 h, applied 2,5-norbornadiene had little effect on abscission at 48 h. A Lineweaver-Burk plot gave a 1/2 maximum value of 0.12 μl 1⁻¹ for ethylene on abscission, 2,5-Norbornadiene gave competitive kinetics with respect to ethylene with a K₁ value of approximately 120 μl 1⁻¹ of 2,5-norbornadiene. The presence of 2,5norbornadiene stimulated ethylene production, which progressively increased as the 2,5-norbornadiene concentration was increased from 250 to 8 000 μl 1⁻¹ 2,5-Norbornadiene also suppressed the induction of cellulase and polygalacturonase by ethylene. Together, 2,5-norbornadiene and 2,4-dichlorophenoxyacetic acid were more effective than either alone in reducing abscission. 2,5-Norbornadiene also was effective in preventing the reduction of indole-3-acetic acid transport induced by ethylene.     

Action of Rice α-Glucosidase on Maltose and Starch

Rice seeds possess α-glucosidase I and II, and the action of the α-glucosidases on maltose and starch was studied. The activity on starch was increased 2.3~2.6 times in both enzymes at the concentration of 50 mM of potassium chloride. Such activation was also caused by mono and di-valent cations. The activity on maltose was not influenced by the cations. In mixed substrate experiments, liberation of ¹⁴C-glucose from ¹⁴C-maltose was not inhibited in the presence of starch, and this was also the case with that from ¹⁴C-starch in the existence of maltose. From these results, it was suggested that the α-glucosidases possess maltose-hydrolyzing site and starch-hydrolyzing site separately, and also probably regulatory. The α-glucosidases liberated only glucose from starch, and were presumed to complete hydrolysis of starch after longer incubation.     

Interaction of ethylene with indole-3-acetic acid in regulation of rooting in pea cuttings

Cuttings of pea (Pisum sativum L. cv Marma) were treated with 1-aminocyclopropane-l-carboxylic acid (ACC). This treatment caused increased ethylene production and reduction of root formation. The effect of 0.1 mM ACC on the level of endogenous indole-3-acetic acid (IAA) in the rooting zone and in the shoot apex was analyzed by gas chromatography-single ion monitoring mass spectrometry or by high pressure liquid chromatography with fluorimetric detection (HPLC). Concentrations of indole-3-acetylaspartic acid (IAAsp) in the stem bases were also determined using HPLC. The ACC treatment had little effect on the IAA level in the base measured after 24 h, but caused a considerable decrease during the 3 following days. IAAsp increased in the base on days 1, 2 and 3 and then declined. The build up of IAAsp in the base was not affected by ACC during the first two days of the treatment, but later this conjugate decreased more rapidly than in controls. No effect of the ACC treatment was found on the level of IAA in the apex. IAA (1 µM) applied to the cuttings during 24 h reduced the number of roots formed. The possibility that IAA-induced ethylene is involved in this response was investigated.Our results support earlier evidence that the inhibitory effect of ethylene on rooting in pea cuttings is due to decreased IAA levels in the rooting zone. The inhibitory effect of applied IAA is obtained if the internal IAA level is maintained high during the first 24 h, whereas stimulation of rooting occurs if the internal IAA level remains high during an extended period of time. Our results do not support the suggestion that ethylene mediates the inhibitory effect of applied IAA.     

The Effects of Growth Regulators on Flowering of Chenopodium murale Plants in vitro

In vitro culture of Chenopodium murale L. (ecotype 197) green and herbicide SAN 9789 - treated "white" plants was established and the effects of benzylaminopurine (BAP), indole-3-acetic acid (IAA) and gibberellic acid (GA₃) on growth and flowering were tested. Green plants did not flower on glucose free media, while 17 % of plants flowered on 5 % glucose-containing medium. SAN 9789 (10^–5 M) inhibited growth and flowering. BAP and IAA (0.1 – 5 mg dm^–3) also inhibited growth and flowering of green and "white" plants. GA₃ (10 mg dm^–3) stimulated leaf development in green plants, but had no significant effect on "white" plants, and stimulated flowering of green (41 %) and "white" (33 %) plants.     

Indole-3-acetic acid: A widespread physiological code in interactions of fungi with other organisms

Plants as well as microorganisms, including bacteria and fungi, produce indole-3-acetic acid (IAA). IAA is the most common plant hormone of the auxin class and it regulates various aspects of plant growth and development. Thus, research is underway globally to exploit the potential for developing IAA-producing fungi for promoting plant growth and protection for sustainable agriculture. Phylogenetic evidence suggests that IAA biosynthesis evolved independently in bacteria, microalgae, fungi, and plants. Present studies show that IAA regulates the physiological response and gene expression in these microorganisms. The convergent evolution of IAA production leads to the hypothesis that natural selection might have favored IAA as a widespread physiological code in these microorganisms and their interactions. We summarize recent studies of IAA biosynthetic pathways and discuss the role of IAA in fungal ecology.     

The effect of indole-3-acetylaspartic acid on adventitious root formation on bean cuttings

The influence of indole-3-acetylaspartic acid (IAAsp) on rooting of stem cuttings from bean plants (Phaseolus vulgaris L.) of different ages, cultivated at different temperatures (17°, 21° and 25°C) was studied and compared to that of indole-3-acetic acid (IAA). At a concentration of 10^–4 M, IAAsp only nonsignificantly stimulated adventitious root formation, approximately to the same level as IAA in all treatments. IAAsp at 5×10^–4 M further enhanced rooting, by up 200% of control values, with little influence of temperature conditions and stock plant age. This concentration of IAA usually stimulated rooting more than the conjugate. The largest differences between the effects of IAAsp and IAA occured at the highest cultivation temperature of 25°C where stock plant age also influenced the response. The number of roots produced in comparison with the control, was enhanced from 350% on cuttings from the youngest plants to more than 600% on cuttings from the oldest. In contrast to the conjugate, 5×10^–4 M IAA induced hypocotyl swelling and injury of the epidermis at the base of cuttings, in all treatments.     

An NMR method for the study of proton transport across phospholipid vesicles

We have identified [1-¹⁴C]-oxindole-3-acetic acid as a catabolic product of [1-¹⁴C]-indole-3-acetic acid metabolism in Zea mays seedlings. The isolation, and chemical and mass spectral characterization of oxindole-3-acetic acid from corn kernel tissue is described together with data suggesting oxindole-3-acetic acid to be a major catabolic product of indole-3-acetic acid.     

Identification of jasmonic acid in Mimosa pudica and its inhibitory effect on auxin- and light-induced opening of the pulvinules

Jasmonic acid was identified from Mimosa pudica L. plants by mass spectrometry, high performance liquid chromatography and thin layer chromatography. Effects of authentic jasmonic acid on pulvinule movement and transpiration of the pinnae were compared with those of abscisic acid. Jasmonic acid and abscisic acid each at 10⁻⁵ M inhibited both auxin- and light-induced opening of the pulvinules. A closure-inducing activity of jasmonic acid at 10⁻⁴ M was greater than that of abscisic acid at 10⁻⁴ M. Pinnae transpiration was reduced by 10⁻⁵ M abscisic acid but not by 10⁻⁴ M jasmonic acid.     

Regulation of cell wall synthesis by auxin and fusicoccin in different tissues of pea stem segments

Cell wall synthesis was studied by determining the incorporation of [¹⁴C]-glucose into epidermal and cortical cell walls of etiolated Pisum sativum L. cv. Alaska stem segments. Walls were fractionated into the matrix and cellulose components, and incorporation into these components assessed in terms of the total uptake of label into that tissue. When segments were allowed to elongate, the stimulation of total glucose uptake by indole-3-acetic acid (IAA) and fusicoccin (FC) was greater than their stimulation of incorporation. IAA and FC thus did not stimulate precursor incorporation in elongating segments. When elongation was inhibited by calcium, however, IAA and FC significantly promoted wall synthesis in the cortex and vasular tissue (which shows almost no growth or acidification response to auxin). In these tissues incorporation into matrix and cellulose was promoted approximately equally. In the epidermis (thought to be the tissue responsive to auxin in the control of growth), FC promoted a significant increase in wall synthesis, although less than that in the cortex, while there was some evidence of a similar promotion by IAA. Both IAA and FC had a greater effect on incorporation into the matrix component of the wall than into cellulose. The results that FC caused a substantial promotion of cell wall synthesis which was not due solely to elongation, and that the inner non-growth responsive cortical tissues can respond to IAA. Moreover, a comparison of the effects of IAA and FC on the different components of the wall suggests that the response in the epidermis differs from that in the other tissues.     

Chitosan and silver nanoparticles are attractive auxin carriers: A comparative study on the adventitious rooting of microcuttings in apple rootstocks

Nanocarriers for encapsulation and sustained release of agrochemicals such as auxins have emerged as an attractive strategy to provide enhanced bioavailability and efficacy for improved crop yields and nutrition quality. Here, a comparative study was conducted on the effectiveness of chitosan-as a biopolymeric nanocarrier- and silver-as a metallic nanocarrier- on in vitro adventitious rooting potential of microcuttings in apple rootstocks, for the first time. Auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) loaded silver (nAg) or chitosan nanoparticles (nChi) were synthesized. Scanning electron microscopy and transmission electron microscopy studies showed the spherical shape of the nanoparticles. The average particle size of IAA-nChi was 167.5 ± 0.1 nm while that of IBA-nChi was 123.2 ± 2.6 nm. The hydrodynamic diameter of the nAg-IAA and nAg-IBA particles were measured as 93.66 ± 5 nm and 71.41 ± 3 nm, respectively. Fourier transform infrared spectroscopy analyses confirmed the encapsulation of IAA or IBA in the chitosan nanoparticles. Meanwhile, the characteristic peaks of IAA or IBA were detected on silver nanoparticles. In-vitro adventitious rooting of microcuttings of Malling Merton 106 (MM 106) was significantly higher both in chitosan and silver nanoparticles loaded with IAA or IBA (91.7%–62.5%) compared to free IAA or IBA applications (50.0%–33.3%), except for 2.0 mg L^–1 IBA (66.7%). However, the application of 2 mg L^–1 IBA and IBA-nChi at all concentrations caused an undesirable large callus development.     

Levels of endogenous indole-3-acetic acid and indole-3-acetylaspartic acid during adventitious root formation in pea cuttings

Levels of endogenous indole-3-acetic acid (IAA) and indole-3-acetylaspartic acid (IAAsp) were monitored in various parts of leafy cuttings of pea ( Pisum sativum L. cv. Marma) during the course of adventitious root formation. IAA and IAAsp were identified by combined gas chromatography—mass spectrometry, and the quantitations were performed by means of high performance liquid chromatography with spectrofluorometric detection. IAA levels in the root forming tissue of the stem base, the upper part of the stem base (where no roots were formed), and the shoot apex remained constant during the period studied and were similar to levels occurring in the intact seedling. A reduction of the IAA level in the root regenerating zone, achieved by removing the shoot apex, resulted in almost complete inhibition of root formation. The IAAsp level in the shoot apex also remained constant, whereas in the stem base it increased 6-fold during the first 3 days. These results show that root initiation may occur without increased IAA levels in the root regenerating zone. It is concluded that the steady-state concentration is maintained by basipetal IAA transport from the shoot apex and by conjugation of excessive IAA with aspartic acid, thereby preventing accumulation of IAA in the tissue.     

Effect of light in the control of growth by auxin and its inhibitor(s) in the sunflower

Diffusates from the hypocotyl and leaves of sunflower seedlings ( Helianthus annuus L. cv. Mammoth) contain both auxin and inhibitor(s) of auxin-induced growth. The auxin activity has been evaluated with the conventional Avena coleoptile bioassay employing negative curvature, The inhibitor activity has been determined with a newly developed bioassay, measuring the positive curvature response of the Avena coleoptile. This bioassay has been standardized by the response to 2,3,5-triiodo-benzoic acid. Diffusates from plants in darkness have higher auxin activity and lower inhibitor activity than diffusates from plants in light. Irradiation at 730 nm promotes auxin synthesis in leaves, and! irradiation at 660 nm promotes synthesis of the inhibitor.     

Population variation and diurnal changes in the content of indole-3-acetic acid of pine seedlings (Pinus sylvestris L.) grown in a controlled environment

Pine seedlings ( Pinus sylvestris L.) were grown in a growth chamber under simulated summer conditions to an age of eight weeks after the beginning of seed germination. Single seedlings were analyzed for fresh weight, shoot and root lengths, and content of indole-3-acetic acid (IAA). The first three variables were normally distributed with standard deviations of 29%, 17% and 18%, respectively. The IAA content had a standard deviation of 39%, and this variable was not normally distributed. If this finding is of general significance, population variation must be considered when experiments involving IAA analyses are planned, and statistical methods based on a normally distributed population cannot be used to evaluate the result of such analyses unless samples of at least 20–30 individuals are analyzed. There were no correlations between the content of IAA and any of the three other variables. The content of IAA showed pronounced diurnal changes, rising from 15 ng g⁻¹ (fresh weight) in the morning to 42 ng g⁻¹ in the late evening. The initial rate of change was about 10% h⁻¹. Obviously, short-term fluctuations must be checked if long-term changes in IAA content are to be studied. IAA could also be released from the acidic buffer fraction by means of alkaline hydrolysis. This "bound alkali-hydrolysable" IAA did not show short-term fluctuations.     

Enhancement of Lipid Peroxidation by Indole-3-Acetic Acid and Derivatives: Substituent Effects

The peroxidation of liposomes by a haem peroxidase and hydrogen peroxide in the presence of indole-3-acetic acid and derivatives was investigated. It was found that these compounds can accelerate the lipid peroxidation up to 65 fold and this is attributed to the formation of peroxyl radicals that may react with the lipids, possibly by hydrogen abstraction. The peroxyl radicals are formed by peroxidase-catalyzed oxidation of the enhancers to radical cations which undergo cleavage of the carbon-carbon bond on the side-chain to yield CO2 and carbon-centred radicals that rapidly add oxygen. In competition with decarboxylation, the radical cations deprotonate reversibly from the Nl position. Rates of decarboxylation,pK_a values and rate of reaction with the peroxidase compound I indicate consistent substituent effects which, however, can not be quantitatively related to the usual Hammett or Brown parameters. Assuming that the rate of decarboxylation of the radical cations taken is a measure of the electron density of the molecule (or radical), it is found that the efficiency of these compounds as enhancers of lipid peroxidation increases with increasing electron density, suggesting that, at least in the model system, the oxidation of the substrates is the limiting step in causing lipid peroxidation.