Higher flower bud formation in haploid tobacco is connected with higher peroxidase/IAA-oxidase activity,lower IAA content and ethylene production

Haploid tobacco plants (cv. Samsun) form inflorescences with a larger number of flowers than diploid plants. Leaves of haploid plants were shown to have lower free IAA level (by 40 %), higher peroxidase (by 160 %) and IAA-oxidase (by 70 %) activities and produce less ethylene (by 25 %) than leaves of corresponding diploid plants. The increase of peroxidase activity in haploids was due to the increase in the activity of the cathodic isozyme which is known to have high IAA-oxidase activity. It is proposed that higher peroxidase/IAA-oxidase activity in haploid plants may take part in IAA catabolism, at least duringin vitro culture of haploid explants. Lowered IAA level and ethylene production may then be directly correlated with a larger number of flower buds; as a higher IAA level is generally considered to act as a background inhibitor of flowering.     

Effect of manganese toxicity on the indoleacetic Acid oxidase system of cotton

The effect of substrate manganese on tissue manganese levels and activity of the indoleacetic acid (IAA)-oxidase system of cotton (Gossypium hirsutum, L.) was investigated. A sand culture technique was used with 1, 3, 9, 27 and 81 mg manganese (MnSO₄) per liter nutrient solution applied in various experiments.

The following relationships held for both long-term (126 days) and short-term (12-14 days) exposures to manganese treatment: A) There was a direct relationship between substrate and tissue manganese. B) Only the 81 mg/liter Mn plants exhibited severe manganese toxicity symptoms. C) At the toxic level of manganese an increased IAA-oxidase activity and decreased IAA-oxidase inhibitor activity was observed. There was a direct relationship between degree of enzyme response and severity of visible symptoms. D) With the manganese toxicity plants, but none of the other treatments, extracts of the young leaves contained as much IAA-oxidase activity as extracts of much older leaves. E) Crude extracts from the plants grown with 81 mg manganese per liter solution, in contrast to those of other treatments, destroyed IAA without addition of MnCl₂ to the assay medium.

A hypothesis is advanced stating that manganese toxicity symptoms in cotton are expressions of auxin deficiency caused by IAA-oxidase activity increased by the abnormal tissue levels of manganese.

     

Influence of Substrate and Tissue Manganese on the IAA-Oxidase System in Cotton

Tissue manganese was found to influence the indoleacetic acid (IAA) system of cotton over a wide range of concentrations. The cofactor and inhibitor activities of the IAA-oxidase system were affected as the concentration of manganese in the tissue was varied. Maximum inhibitor activity was found in leaf extracts from the plants grown in 0.5 mg/l manganese (Hoagland's level). The inhibitor activity decreased in the leaf extracts of plants grown at concentrations of manganese either higher or lower than 0.5 mg/l. Abnormally high IAA-oxidase activity was found in the leaves of plants grown in deficient levels of manganese (<0.0005, 0.005 mg/l) and the extracts from plants in the <0.0005 mg/l Mn treatment showed IAA-oxidase cofactor activity.     

Simple phenolic glycosides as potential regulators of the IAA-oxidase system

The effect of phenols and simple phenolic glycosides on the activity of IAA-oxidase isolated from gherkin seedlings was studied in experimentsin vitro. Phenol stimulated the enzyme system activity, eugenol and quinol were proven as inhibitors. Simple phenolic glycosides (arbutin, gein and phenol glucoside) influenced IAA-oxidase activity only if β-glycosidase was present: rree phenols released from their bound form increased or decreased the IAA level. The potential fegulatory effect of simple phenolic glycosides on the IAA level in plants has been discussed; this effect is thought to be mediated by free phenols and by influence on the IAA-oxidase system.     

The Iron-Deficiency Induced Phenolics Accumulation May Involve in Regulation of Fe(III) Chelate Reductase in Red Clover

Although considerable researches have been conducted on the physiological responses to plant iron (Fe) deficiency stress in dicotyledonous plants, much still needs to be learned about the regulation of these processes. In the present research, red clover was used to investigate the role of root phenolics accumulation in regulating Fe-deficiency induced Fe(III) chelate reductase (FCR). The root FCR activity, IAA and phenolics accumulation, and also the phenolics secretion were greatly increased by the Fe deficiency treatment. The application of TIBA (2,3,5-triiodobenoic acid) to the stem, an IAA polar transport inhibitor, which could decrease IAA accumulation in root, significantly inhibited the FCR activity, but did not effect root phenolics accumulation and secretion, suggesting that IAA itself did not involve in root phenolics accumulation and secretion. In contrast, the Fe deficiency treatment significantly decreased the root IAA-oxidase activity. Interestingly the phenolics extracted from roots inhibited IAA-oxidase activity in vitro, and this inhibition was greater with phenolics extracted from roots of Fe deficient plants than that from Fe sufficient plants, indicating that the Fe deficiency-induced IAA-oxidase inhibition probably caused by the phenolics accumulation in Fe deficient roots. Based on these observations, we propose a model where under Fe deficiency stress in dicots, an increase in root phenolics concentrations plays a role in regulating root IAA levels through an inhibition of root IAA oxidase activity. This response, leads to, or at least partially leads to an increase in root IAA levels, which in turn help induce increased root FCR activity.Key Words: Fe deficiency, ferric chelate reductase, phenolics, Trifolium pretense     

IAA-oxidase in Impatiens balsamina Affected by GA3 and Tannic Acid

The activity of IAA-oxidase increased in the leaves of Impatiensbalsamina plants receiving inductive photoperiodic cycles andin plants receiving treatments with gibberellic acid (GA₃) and/ortannic acid (TA), even under non-inductive photoperiods; theactivity also increased in the stem receiving inductive photoperiodiccycles (8 h). Treatment with GA₃ and TA mimics the effect ofSD cycles in the development of some isoenzymes of IAA-oxidase.Thus a new isoenzyme at R_f 0.48 developed in the leaves andone at R_f 0.82 developed in both the stem and the leaves ofall plants receiving inductive treatments – photoperiodicor chemical – but not in water-treated controls undernon-inductive photoperiods. Another isoenzyme at R_f 0.68 developedonly in the stems. Flowering, gibberellic acid, IAA oxidase, Impatiens, phenols, photoperiod     

Effects of phenolic substances on metabolism of exogenous indole-3-acetic acid in maize stems

Four-day-old stem segments of Zea mays L. cv. Seneca 60 were treated sequentially with phenolic substances and indole-3-acetic [2-¹⁴C] acid ([2-¹⁴C]IAA). Formation of bound IAA was rapid, but a pretreatment with p-coumaric acid, ferulic acid or 4-methylumbelliferone decreased the level of bound IAA. The decrease is not likely related to the effect of the phenolics on enzymic oxidation of IAA, since the level of free IAA was not limiting and the activity of ferulic acid in enzymic oxidation of IAA is different from that of p-coumaric acid and 4-methylum-belliferone. Apparently these compounds inhibited the formation of bound IAA and consequently caused an accumulation of free IAA. In contrast, caffeic acid, protocatechuic acid and 2,3-dihydro-2, 2-dimethyl-7-benzofuranol had little effect. After the uptake of IAA there was a slow but steady incorporation of the radioactivity into the 80% ethanol-insoluble, 1 M NaOH-soluble fraction. Phenolic substances also affected this process. The compounds which are cofactors of IAA-oxidase increased the incorporation while those which are inhibitors of IAA-oxidase decreased the incorporation. Results suggest that the phenolics also affected the enzymic oxidation of IAA in vivo in the same way as in vitro.     

Distribution of an Indoleacetic Acid-oxidase-inhibitor in the Storage Root of Daucus carota

Indoleacetic acid (IAA)-oxidase from both secondary phloem and xylem was dependent on 2,4-dichlorophenol for activity, and was enhanced by addition of Mn²⁺. The pH optimum was 6.0 from both tissues. IAA-oxidase and its inhibitors were distributed differently in the secondary phloem and secondary xylem of carrot root. In the phloem a high IAA-oxidase activity was distributed uniformly along the radius but in the xylem a somewhat lower concentration decreased from the cambium. IAA-oxidase inhibitor in the phloem increased exponentially from a very low concentration near the cambium, whereas in the xylem an appreciable concentration was present near the cambium, decreasing linearly with distance from the cambium. Longitudinal gradients in the xylem parallel studies by other workers with the greatest IAA-destroying capacity present in older tissues. In the xylem inhibitor decreased and IAA-oxidase increased from the root apex. In the phloem IAA-oxidase was uniform, whereas the inhibitor increased in older tissue.

The IAA-oxidase inhibitors in phloem and xylem may be different. In the xylem the IAA-oxidase inhibitor may be a lignin precursor present in young cells which disappears as lignification proceeds. In the phloem IAA-oxidase reacting with endogenous IAA appears to form a physiologically active product.

     

Relationship of IAA-oxidase Activity to Gibberellinand IAA-induced Elongation of Light-grown Cucumber Seedlings

The growth and IAA-oxidase activity of light-grown cucumber seedlings (cv. Aonagajibae) were investigated in response to GA₃ and IAA. Both GA₃ and IAA induced significant elongation of the hypocotyl. The fresh and dry weights of the hypocotyl increased due to GA₃ or IAA treatment, whereas no significant change was observed in the cotyledons of GA₃-treated seedlings as compared with the controls. The fresh and dry weights of IAA-treated cotyledons were both lower than those of controls. Treatment with GA₃ or IAA resulted in retardation of IAA-oxidase activity in the hypocotyl and cotyledons. The degree of retardation was less in the cotyledons than in the hypocotyl. An inverse relationship was recognized between GA₃- or IAA-induced elongation and IAA-oxidase activity in the hypocotyl. The auxin-mediated mechanism for gibberellin action was discussed.     

THE EXTRACTABLE AND DIFFUSIBLE AUXIN-AUXIN INHIBITOR LEVEL IN NICOTIANA GLAUCA,NICOTIANA LANGSDORFFII AND THEIR AMPHIDIPLOID HYBRID

The auxin-auxin inhibitor (effective auxin content—EAC)² level of stems and leaves of normal Nicotiana plants (N. glauca, N. langsdorffii and of their spontaneously tumor-producing hybrid) was investigated by the use of the diffusion method into agar blocks and by the wet-ether and wet-alcohol extraction method. In all series, the EAC was tested by the Avena curvature test. Although the Avena curvature produced from extracts and diffusates in these tobacco plants is low, in general there is a consistently greater curvature in the parents (on an average for N. glauca, 6.7° in the diffusion and 6.2° in the extraction tests; for N. langsdorffii, 2.7° in the diffusion and 4.6° in the extraction tests) than in the hybrid (on an average of 0.7° in the diffusion and 2.6° in the extraction tests). This suggests that the question “Why do tumors form in the hybrid?” cannot be answered simply by suggesting that it is a function of excessive auxin. It is known that IAA must be added in high concentrations to aseptic cultures of N. langsdorffii and N. glauca whereas cultures of hybrid tissues do not require such an exogenous auxin supply for growth. This might indicate differences in the availability of IAA for growth of these tissues.     

Effect of gibberellic acid and salicylic acid on the activity and electrophoretic pattern of IAA-oxidase during floral induction inImpatiens balsamina

IAA-oxidase activity increased in the stem as well as in the leaves of plants treated with GA₃, SA and GA₃ + SA during the early stages under inductive and non-inductive photoperiods, the activity being the highest in GA₃ + SA-treated plants. An isoenzyme of IAA-oxidase with Rm 0.15 developed in the stem as well as in the leaves subsequent to 1 or 2 inductive treatments. As this band persisted till the end of the experiment, it may be associated with the initiation as well as development of floral buds. Another band (Rm 0.30) appears to be associated with the phenol (SA) as it developed in the stem as well as in the leaves of SA- and GA₃ + SA-treated plants under both photoperiods. A band with Rm 0.60 developed in the leaves but not in the stem of GA₃-, SA- and GA₃ + SA-treated plants under both photoperiods.     

The masking of peroxidase-catalysed oxidation of IAA in Vigna

Partially purified enzyme preparations of extracts of Vigna seedlings exhibited guaiacol-oxidase activity but not IAA-oxidase activity. However, by ageing the enzyme preparations, or by treating them with H₂O₂, it was possible to unmask IAA-oxidase activity. Gel filtration of Vigna extracts on Sepharose yielded separate peaks for IAA-oxidase, guaiacol-oxidase and auxin protectors. The appearance of a separate IAA-oxidase peak reflected the overlap of peroxidase and protector; the apparent difference in the migration rate of IAA-oxidase and guaiacol-oxidase activity proved to be an artifact. The data imply that previous reports of differences between peroxidase and IAA oxidase need to be reinvestigated to rule out the possible effect of contamination by endogenous, high MW auxin protectors. A rapid method for removing most of the auxin protectors and thereby unmasking IAA-oxidase activity is described.     

Peroxidase and IAA Oxidase Activity and Isoenzyme Patterns in Cucumber Plants, as Affected by Sex Expression and Ethephon

Peroxidase and IAA-oxidase activity, electrophoretic patterns of total proteins and isoenzymes and the effect of Ethephon (= Ethrel, 2-chloroethane phosphonic acid) on these patterns were compared in extracts of monoecious and gynoecious cucumber plants. The activity of both peroxidase and IAA oxidase was greater in gynoecious than in monoecious plants. Ethephon treatments given at the 2-leaf stage increased peroxidase activity, especially in monoecious plants, and decreased IAA-oxidase activity. Ethephon treatment did not affect total protein or isoenzyme patterns, but increased band intensity, especially that of one band in monoecious plant extracts. No differences between monoecious and gynoecious plants were found in total protein, peroxidase and IAA-oxidase isoenzyme patterns. Peroxidase and IAA-oxidase activity sites on the gel were similar.     

Decarboxylative metabolism of [1′-14C]indole-3-acetic acid by tomato pericarp discs during ripening

The rate of decarboxylation of [1′-¹⁴C]indole-3-acetic acid (IAA) infiltrated into tomato (Lycopersicon esculentum Mill.) pericarp discs was much more rapid in green than in breaker and pink tissues. Studies were carried out in order to determine whether the decarboxylative catabolism occurring in the green pericarp discs was associated with ripening or was a consequence of wound-induced peroxidase activity and/or ethylene production. After a 2-h lag, the decarboxylative capacity of the green pericarp discs increased exponentially during a 24-h incubation period. This increase was accompanied by increases in IAA-oxidase activity in cell-free preparations from the intercellular space and cut surface of the discs. Although higher IAA-oxidase activity was detected in extracts from the tissue residue, which comprises mainly intracellular peroxidases, this activity did not increase during the 24-h incubation period. Analysis of the cell-free preparations by isoelectric focusing revealed the major component in all samples was a highly anionic peroxidase (pI=3.5) the levels of which did not increase during incubation. However, the intercellular and cut-surface preparations contained additional anionic and cationic peroxidases which increased in parallel with the increases in both the IAA-oxidase activity of the preparations and the decarboxylative capacity of the green pericarp discs from which they were derived. Treatment of green discs with the ethylene-biosynthesis inhibitors aminooxyacetic acid and CoCl₂, inhibited the development of an enhanced capacity to decarboxylate [1′-¹⁴C]IAA but the inhibition was not counteracted by exogenous ethylene. Another ethylene-biosynthesis inhibitor, aminoethoxyvinyl glycine, also reduced ethylene levels but did not affect IAA decarboxylation, indicating that the decarboxylation was not a consequence of wound-induced ethylene production. The data obtained thus demonstrate that the enhanced capacity to decarboxylate [1′-¹⁴C]IAA that develops in green tomato pericarp discs following excision is not associated with ripening but instead is attributable to a wound-induced increase in anionic and cationic peroxidase activity in the intercellular fluid and at the cut surface of the excised tissues.     

Light and the transport and metabolism of indoleacetic acid in normal and albino dwarf pea seedlings

Summary The patterns of transport and metabolism of IAA-2-¹⁴C applied to the apices of intact normal and albino dwarf pea seedlings were essentially similar under given light conditions. Light greatly reduced the decarboxylation of the applied IAA and stimulated the synthesis of indoleaspartic acid (IAAsp) in both normal and albino plants.In light considerably more ¹⁴C was exported from the apices of normal than albino plants; this result was attributed to the reduced capacity of the transport system in the latter.The specific activity of ¹⁴C in the stem decreased logarithmically with increasing distance from the treated apex. Light increased the steepness of the logarithmic profile. These results are discussed in relation to the rate of immobilization of IAA along the transport pathway by conversion to IAAsp.No evidence was found to support a previous suggestion (Pilet and Phipps, 1968) that IAA-oxidase activity and chlorophyll levels were causally linked.     

Temperature determines the occurrence of CAM or C<Subscript>3</Subscript> photosynthesis in pineapple plantlets grown <Emphasis Type="Italic">in vitro</Emphasis>

Summary Ananas comosus (L.) Merr. var. Smooth Cayenne plants when grown in vitro under different temperature regimes developed as CAM or as C₃ plants. The plants used in this study were developed from the lateral buds of the nodal etiolated stem explants cultured on Murashige and Skoog medium for 3 mo. The cultures were maintained under a 16-h photoperiod for different thermoperiods. With 28°C light/15°C dark thermoperiod, as compared with constant 28°C light and dark, pineapple plants had a succulence index two times greater, and also a greater nocturnal titratable acidity and phosphoenolpyruvate carboxylase (PEPCase) activity, indicating CAM-type photosynthesis. The highest abscisic acid (ABA) level occurred during the light period, 8 h prior to maximum PEPCase activity, while the indole-3-acetic acid (IAA) peak was found during the dark period, coinciding with the time of highest PEPCase activity. These plants were also smaller with thicker leaves and fewer roots, but had greater dry weight. Their leaves showed histological characteristics of CAM plants, such as the presence of greater quantities of chlorenchyma and hypoderm. In addition, their vascular system was more conspicuous. In contrast, under constant temperature (28°C light/dark) plants showed little succulence in the leaves. There was no significant acid oscillation and diurnal variation in PEPCase activity in these plants, suggesting the occurrence of C₃ photosynthesis. Also, no diurnal variation in ABA and IAA contents was observed. The results of this study clearly indicate a role for temperature in determining the type of carbon fixation pathway in in vitro grown pineapple. Evidence that ABA and IAA participate in CAM signaling is provided.     

Role of IAA-Oxidase in Abscission Control in Cotton

The potential role of indoleactic acid (IAA)-oxidase as an in vivo abscission regulating system in the cotton (Gossypium hirsutum L.) cotyledonary explant was investigated. Phenols (usually monophenols), which are cofactors of cotton IAA-oxidase in vitro, accelerated abscission. Phenols (usually orthodihydroxyphenols), which inhibit cotton IAA-oxidase in vitro, inhibited abscission. Inhibition or stimulation of abscission was accomplished by phenols both with and without IAA. Results were similar when treatments were applied as lanolin pastes to the cut petiole ends or as solutions in which explants were submerged. An abscission accelerating phenol stimulated the decarboxylation of IAA-1-¹⁴C by explants and an abscission inhibiting phenol inhibited the decarboxylation of IAA-1-¹⁴C.     

2,4-D Hyper Accumulation Induced Cellular Responses of <i>Azolla pinnata</i> R. Br. to Sustain Herbicidal Stress

In the present experiment with ongoing concentration (0 µM, 100 µM, 250 µM, 500 µM and 1000 µM) of 2,4-D, the responses of Azolla pinnata R.Br. was evaluated based on cellular functions. Initially, plants were significantly tolerated up to 1000 µM of 2,4-D with its survival. This was accompanied by a steady decline of indole acetic acid (IAA) concentration in tissues with 78.8% over the control. Membrane bound H⁺ -ATPase activity was over expressed within a range of 1.14 to 1.25 folds with activator (KCl) and decreased within a range of 57.3 to 74.6% in response to inhibitor (Vanadate) application. With regards to IAA metabolism, plants recorded a linear increase with wall bound oxidase activity up to maximum concentration of 2,4-D. The variations were more moderated when wall bound IAA-oxidase recorded a linear increase proportionate to the 2,4-D concentrations. This was more extended with the presence of different isoforms of IAA-oxidase which was much more pronounced with distinct polymorphisms of expressed proteins, however, not independent to the 2,4-D concentrations. Polyamines like spermine, spermidine and putrescine (spm, spd and put) were not consistent in concentration with the dosages of 2,4-D. Besides these, plants were induced to apoplastic NAD(P)H oxidase activity maximally by 1.6 folds under 500 µM 2,4-D over control. Still, putrescine responded more or less consistently and recorded maximally 11.9 folds at 500 µM 2,4-D as compared to the control. NAD(P)H oxidase activity recorded maximally 1.6 folds against control and remain consistent throughout the concentrations of 2,4-D. GPX along with APX were more linear in responses through the concentration of 2,4-D except CAT as compared to control. On enzymatic antioxidative activity, peroxidases (GPX and APX) were overexpresed in a similar manner except for catalase with a non-significant rise. In stabilization of cellular redox, glutathione reductase attended maximum value by 2.45 folds at 1000 µM evidenced with significant variations in protein polymorphism. The sensitivity of 2,4- D also appeared in Azolla with a maximum loss of nucleic acids as documented by the comet assay. Moreover, the Azolla might have some DNA damage protective activity as evident using frond extract with plasmid nick assay. Therefore, Azolla plants with its cellular responses is evident to sustain against the 2,4-D herbicidal stress and may be granted in bio remediation process for the contaminated soil.     

A cytogenetic and phenotypic characterization of somatic hybrid plants obtained after fusion of two different dihaploid clones of potato (Solatium tuberosum L.)

Summary Somatic hybrid plants of various ploidy levels obtained after chemical fusion between two dihaploid clones of potato Solanum tuberosum L. have been analysed by cytological, morphological and molecular methods. The hybrid nature of tetraploid and hexaploid plants and the genome dosage in hexaploid hybrids were confirmed by Giemsa C-banding. Tetraploid and hexaploid hybrids showed numerical as well as structural chromosome mutations. The latter occurred mainly in the nuclear organizing chromosome. The tetraploid hybrids were more vigorous than the dihaploid parents as demonstrated by an increase in height, enlargement of leaves, increase in the number of internodes, restored potential for flowering and increased tuber yield. The grouping of tetraploid somatic hybrids into various classes on the basis of leaf morphology revealed that plants with a full chromosome complement were more uniform than aneuploids. Many hexaploid somatic hybrids were also more vigorous than the dihaploid parents and could be grouped into two different classes on the basis of floral colour and tuber characteristics, the differences being due to their different dosage of parental genomes. Most of the tetraploid somatic hybrids showed pollen development halted at the tetrad stage as one of the parental clones contained a S. Stoloniferum cytoplasm. However, one tetraploid plant produced pollen grains with high viability. The chloroplast genome in the hybrid plants was determined by RFLP analysis. All of the hybrids had a cpDNA pattern identical to one parent, which contained either S. Tuberosum or S. Stoloniferum cpDNA. A slight preference for S. Tuberosum plastids were observed in hybrid plants. No correlation between pollen development and plastid type could be detected.     

The influence of kinetin on the endogenous content of indoleacetic acid in swelling seeds of Phaseolus, Zea and Pinus and young plants of Phaseolus

Treatment of different plant materials, seeds of Phaseolus vulgaris, Zea mays and Pinus silvestris and young plants of Phaseolus, with kinetin increased the level of extractable IAA. For seeds this increase was most pronounced in bean seeds, which contained the lowest amount of endogenous IAA and cytokinins, and lower in maize seeds with high endogenous content of IAA and cytokinins. – For young bean plants the kinetin treatment significantly increased the extractable amounts of IAA from all parts of the plant, hypocotyls, cotyledons, epicotyls and primary leaves, when the cut plants were placed for 24 h in kinetin solution. For plants sprayed with kinetin solution only the primary leaves showed a significantly higher level of extractable IAA, which could be explained by the fact that the plants were growing very close together, so that the primary leaves received most of the kinetin during spraying.