IAA Oxidase Research Articles

Behaviour of Polyphenol Oxidase, IAA Oxidase and Catalase in Germinating Rice in Relation to Salt Tolerance

Activities of the enzymes polyphenol oxidase, indole-3-acetic acid (IAA) oxidase and catalase were compared from endosperms and embryoaxes of germinating rice (Oryza sativa) seeds of two sets of cultivars differing in salt tolerance, raised under increasing levels of NaCl salinity. Salt sensitive cvs. Ratna and Jaya were characterized by higher polyphenol oxidase activity in embryoaxes under salinisation as compared to salt tolerant cvs. CSR-1 and CSR-3. In endosperms of salt sensitive cultivars the enzyme activity decreased under salinisation whereas in tolerants it increased due to salinity. In vitro studies using desalted enzyme extracts showed a continuous increase in polyphenol oxidase activity with increasing concentration of NaCl (0–1000 mM) in the reaction medium. IAA oxidase activity increased due to salinity in embryoaxes of both sets of cultivars, the effect was greater in sensitive cultivars than tolerants. NaCl when incorporated in the reaction medium caused decrease in IAA oxidase activity. Activity of catalase decreased with increase in salinity with greater decrease in sensitive cultivars than tolerants. Results suggest possible different behaviours of oxidative enzymes in germinating seeds of rice cultivars differing in salt tolerance and that salt tolerance ability is associated with lower polyphenol oxidase and IAA oxidase activities and higher catalase activity in embryoaxes under salinisation.

Some Biochemical and Ultrastructural Aspects of Peach Fruit Development

`Redhaven' peach [Prunus persica (L.) Batsch.] fruit growth, expressed as cheek diameter, displayed a double-sigmoid pattern in which four stages were defined (SI, SII, SIII, SIV). Free IAA concentration, as determined by polyclonal antibodies (PcAb) enzyme-linked immunosorbent assay (ELISA), paralleled fruit growth rate, peaking at 30 and 85 days after full bloom (AFB), concurrently with the exponential phases of growth. The highest peroxidase (EC 1.11.1.7) (POD) and IAA oxidase (IAAox) activities occurred during endocarp lignification. The main structural events described were mesocarp cell division within the first 2 weeks AFB and, later, cell enlargement, modifications of the epicarp cells, lignification of the endocarp, differentiation of the chloroplasts, and changes in their starch content. Chemical name used: indole-3-acetic acid (IAA).

Peroxidase and IAA oxidase activities and peroxidase isoenzymes in the pericarp of seeded and seedless “Redhaven” peach fruit

Parthenocarpic peach fruit (Prunus persica L. Batsch., cv. Redhaven) were induced with 1-(3-chlorophthalimide)-cyclohexane carboxamide (AC 94377). The activities of soluble, and ionically and covalently bound peroxidase and indole-3-acetic acid (IAA) oxidase in the pericarp of both seeded and parthenocarpic fruit were determined from 21–43 days after anthesis. Seedless fruit grew faster during early stage I and ceased growth earlier than seeded fruit. Total peroxidase and IAA oxidase activities increased with development on both types of fruit, but higher values were found in seedless fruit. The ionic fraction showed the greatest increase for both enzyme activities. Isoperoxidase profile showed new cationic isoenzymes and higher levels of the less anionic isoenzymes in the pericarp of seedless fruit, whereas the seeded fruit contained higher levels of the more acidic isoperoxidases.

Growth Stimulation by Catecholamines in Plant Tissue/Organ Cultures

Addition of catecholamines at micromolar concentrations caused a dramatic stimulation of growth of tobacco (Nicotiana tabacum) thin cell layers (TCLs) and Acmella oppositifolia "hairy" root cultures. A threefold increase in the rate of ethylene evolution was observed in the catecholamine-treated explants. Aminooxyacetic acid and silver thiosulfate, inhibitors of ethylene biosynthesis and action, respectively, reduced the growth-promoting effect of dopamine. However, these compounds alone could also inhibit the growth of the TCL explants. When ethylene in the culture vessel was depleted by trapping with mercuric perchlorate, dopamine-stimulated growth was still obtained, suggesting that ethylene does not mediate the dopamine effect. Dopamine potentiated the growth of TCLs grown in Murashige and Skoog medium supplemented with indoleacetic acid (IAA) and kinetin. When IAA was replaced by 2,4-dichlorophenoxyacetic acid, dopamine addition showed no growth-promoting effect. Instead, 2,4-dichlorophenoxyacetic acid stimulated the growth of TCL explants to the same extent as that obtained with IAA plus dopamine. Because synthetic auxins do not appear to be substrates for IAA oxidizing enzymes, we hypothesized that catecholamines exert their effect by preventing IAA oxidation. Consistent with this explanation, dopamine (25 micromolar) inhibited IAA oxidase activity by 60 to 100% in crude enzyme extracts from tobacco roots and etiolated corn coleoptiles, but had no effect on peroxidase activity in the same extracts. Furthermore, addition of dopamine to TCL cultures resulted in a fourfold reduction in the oxidative degradation of [1-(14)C]IAA fed to the explants. Because the growth enhancement by catecholamines is observed in both IAA-requiring and IAA-independent cultures, we suggest that these aromatic amines may have a role in the regulation of IAA levels in vivo.

ウンシュウミカン果実のクロロフィリドａ分解酵素（クロロフィリドａペルオキシダーゼ）の精製と酵素化学的性質について

A chlorophyllide a breaching enzyme extracted from degreening Citrus unshiu fruits was purified some 1, 000-fold and characterized.1. The enzyme catalyses chlorophyllide a degradation in the presence of H2O2 and the phenols, 2, 4-dichlorophenol or p-coumaric acid. It behaves as a protein with a MW of 42, 000 on Sephacryl S-200 gel filtration but it does not give a single band on polyacrylamide gel electrophoresis (PAGE), SDS-PAGE, and upon staining with guaiacol in the presence of H2O2. The Michaelis constant, Km, for chlorophyllide a was 13.2 μM.2. The enzyme exhibited IAA oxidase activity in the presence of Mn++; it was inhibited by hydroquinone, Tiron, potassium cyanide (KCN), diethyldithiocarbamate (DIECA) but not by iodoacetate, p-chloromercuribenzoate (PCMB), N-ethylmaleimide, histidine, D-mannose, dimethylfuran, triethylenediamine, ethylenediaminetetraacetic acid (EDTA), NaN3 and thiourea.The possible in vivo participation of this in vitro enzyme system as a H2O2-scavenging system in the dark is discussed.

Evaluating quantitative screening methods for manganese toxicity in cotton genotypes1

Abstract Identification of cotton genotypes more tolerant of toxic concentrations of soil solution manganese (Mn2+) would integrate well with soil ameliorations of that problem. Several quantitative and semi‐quantitative methods to determine the amount of Mn toxicity were evaluated on three genotypes of Gossypium hirsutum (LaDSIS 12513, LaDASS 5175, and Coker gl 79–501) and one genotype of Gossypium barbadense (Pitnas S‐5). Specific leaf weight (SLW) and the semi‐quantitative, ‘percentage of leaves that were damaged’ (PLD) correlated the least with other methods of Mn toxicity determination. Neither SLW or PLD provided more separation between genotypes than area/leaf (AL), peroxidase (POD) activity, and indole‐3‐acetic acid oxidase (IAAO) activity. Similar genotype separations occurred for AL, POD, and IAAO at 10 mg/L Mn in solution, but POD and IAAO produced more genotype separations than AL at 5 mg/L of Mn. There were differences in enzyme activity between genotypes at control (0.25 mg/L) Mn solution co...

IAA oxidase, peroxidase, and barrier zone formation in red maple

AbstractWounding the cambium of red maple (Acer rubrum L.) altered peroxidase isoenzymes and enhanced the activities of peroxidase and indole‐3‐acetic acid oxidase in homogenates of adjacent cambial tissue. Four separate experiments were conducted, in May and July in each of two successive years. Wounds made in July caused greater increases in enzyme activity than those made in May. Alterations in peroxidase isoenzyme pattern were consistent for all experiments. These changes are intrepretable as aspects of the molecular regulation of barrier zone formation.

Indole‐3‐acetic acid oxidase and syringaldazine oxidase activities of peroxidase isozymes in soybean root nodules

Many isoperoxidases with indole‐3‐acetic acid oxidase (IAA) and syringaldazine oxidase activities were detected by polyacrylamide gel electrophoresis in soybean root nodules [Glycine max (L.) Merrill, cv. Asgrow], detached at the onset of flowering. The kinetics of the two activities were studied with some of the isoperoxidases partially purified by ion exchange chromatography. IAA oxidase activity of the cationic isoforms showed a sigmoidal kinetic behaviour and a higher substrate affinity than the anionic ones, whereas typical saturation kinetics were found with an anionic fraction that contained leghemoglobins. So, nodule IAA oxidase activity may mainly be displayed by the cationic isoforms. These cationic isoperoxidases had high affinity towards syringaldazine and they also may be associated with cell wall rigidification.

Changes in the content of phenolic compounds and IAA-oxidase activity during the growth of tobacco crown gall suspension culture

Phenolic content and IAA-oxidase (IAA-o) activity have been assayed in cells and medium of tobacco crown gall suspension culture in several stages of culture cycle. The highest content of total phenolics in the cells were found prior to cell division and in the middle stage of intensive growth. The beginning of intensive growth is accompanied by temporary reduction in phenolic level in the cells as well as their intensive secretion to the medium. In the second part of the culture cycle, when the phenolic production was weaker, the majority of these compounds were maintained in the cells. The highest activity of IAA-o in the cells was detected in the middle stage of intensive growth, simultaneously with high phenolic content; following it a considerable decrease of IAA-o activity is correlated with maximum of chlorogenic acid (ChA) content (at reduced amount of total phenolics). IAA-o activity increased again at the end of the stage of intensive growth when the level of phenolics was low including ChA. These data suggest that IAA-o in relation to phenolic level determines cell growth in the culture. In the culture medium — fairly distinct negative correlation between IAA-o activity and phenolic content suggests that the latter participates in enzyme activity regulation. During intensive growth IAA-o activity is strongly inhibited. The results prove that phenolic level. IAA-o activity and auxin level are closely correlated and may constitute essential elements of a mechanism of regulation crown gall cell growth in culture.

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by <italic>Agrobacterium tumefaciens</italic> III. Formation of IAA Conjugates in Cultured Carrot Tissues Transformed with Wild-type and Mutant Ti Plasmids

Inactivation of IAA was examined in cultured carrot tissues transformed with Agrobacterium tumefaciens that harbored wild-type, aux− or cyt− Ti plasmids. IAA oxidase activities were similar among the three types of transformed tissue. Metabolites of IAA were examined by following the fate of 3-indolyl-[l-l4C]IAA. IAA conjugates were detected in all transformed tissues as well as in hypocotyl segments of non-transformed carrot seedlings. The rate of formation of IAA conjugates was ten times higher in the tissues transformed with wild-type or cyt− Ti plasmids than in the tissues transformed with aux− Ti plasmids. When the tissues transformed with aux− Ti plasmids were cultured on medium that contained IAA for 6 h, the rate of formation of IAA conjugates in these tissues became as high as that in tissues transformed with wild-type or cyt− Ti plasmids. The tissues transformed with wild-type or cyt− Ti plasmids not only synthesize larger amounts of IAA but also convert a larger amount of free IAA to conjugated IAA than do non-transformed and aux− transformed tissues. Presumably, in carrot, the formation of IAA conjugates decreases the amount of free IAA in the transformed tissues that synthesize large amounts of IAA and, consequently, the level of free IAA can be maintained fairly constant.

Carbon Dioxide-induced Brown Stain Development as Related to Phenolic Metabolism in Iceberg Lettuce

Abstract Controlled atmospheres containing air + 11% CO2 caused tissue injury and induced phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity in iceberg lettuce (Lactuca sativa L.) midrib tissue. Injury symptoms included brown stain (browning of epidermal tissue) and sunken epidermal areas (pitting) a few millimeters in diameter. Pitting occurred in high-CO2 atmospheres at 5C with no increase in phenolic content, but browning did not develop until the tissue had been transferred to air at 25C. Browning developed within several hours of transfer to air and the degree of browning was correlated with the soluble phenolic content. The oxidation of soluble phenolic compounds to brown substances by polyphenol oxidase (PPO, EC 1.10.3.2) could account for tissue browning. Lignification was associated with cell wall thickening in discolored tissue and was accompanied by an increase in ionically bound and soluble peroxidase (POD, EC 1.11.1.7) activities. Exposure of tissue to elevated CO2 increased ionically bound indoleacetic acid (IAA) oxidase activity, but reduced soluble IAA oxidase activity. Application of an aqueous solution of 1.0 mm IAA to the tissue before treatment did not significantly reduce browning. Lettuce tissue exposed to 1.5% O2 + 11% CO2 had reduced PAL activity and lower soluble phenolic content than lettuce exposed to air + 11% CO2. Depending on the sensitivity of the lettuce tissue to CO2 injury, low-O2 atmospheres either reduced or slightly retarded browning induced by 11% CO2.

Regulation of Russet Spotting, Phenolic Metabolism, and IAA Oxidase by Low Oxygen in Iceberg Lettuce

Abstract A 1.5% O2 atmosphere, relative to normal air, dramatically inhibited ethylene-induced russet spot development, PAL, and ionically bound POD, and ionicaliy bound IAA oxidase activities and reduced soluble phenolic content in stored iceberg lettuce (Lactuca sativa L.). Low O2 also inhibited eythylene production and respiration. Polyphenol oxidase activity was slightly inhibited by low O2. The results suggest that low O2 inhibition of ethylene action and attendant effects on phenolic metabolism and IAA oxidase activity may be responsible for inhibition of russet spotting by 1.5% O2. Chemical names used: indole-3-acetic acid (IAA); phenylalanine ammonia-lyase (PAL); peroxidase (POD); polyphenol oxidase (PPO).

Developmental Control of Russet Spotting, Phenolic Enzymes, and IAA Oxidase in Cultivars of Iceberg Lettuce

Abstract Leaf midrib tissue from six cultivars of iceberg lettuce (Lactuca sativa L.) was excised from 20- to 100-day-old plants and tested at 5C for russet spotting (RS) susceptibility under 10 μl ethylene/liter. There was no RS development in tissue excised from 20-day-old plants from any of the six cultivars. RS first developed in tissue from 50-day-old plants and tended to be more severe in tissue excised from older plants. ‘Winter Haven’ and ‘Salinas’ were most susceptible, while ‘Calmar’ was most resistant to RS; ‘Climax’, ‘El Toro’, and ‘Sea Green’ were moderately susceptible. Phenylalanine ammonia-lyase activity in the six cultivars at different developmental stages correlated with their degree of RS development. For ‘Winter Haven’, the increase in ionically bound POD and IAA oxidase activity, but not PPO activity, was associated with the increase in RS score during plant development. ‘Winter Haven’ tissue had much higher ionically bound peroxidase and IAA oxidase activities than ‘Calmar’ tissue from 100-day-old plants. Chemical names used: indole-3-acetic acid (IAA), phenylalanine ammonia-lyase (PAL), peroxidase (POD), polyphenol oxidase (PPO).

Changes in Acidic and Basic Peroxidase Activities during Tomato Fruit Ripening

Abstract Activities of the acidic and basic peroxidases from tomato fruit (Lycopersicon esculentum Mill. cv. Flora Dade) were determined at six ripening stages, from green to red-ripe fruits. Both the acidic and basic peroxidases reached a maximum during the climacteric, at the pink stage, but the relative increase in basic peroxidase activity was much more pronounced. Changes in the peroxidase, IAA oxidase, and ACC oxidase activities of the basic peroxidases paralleled the changes in ethylene production. However, in the presence of calcium, the degree of activation of peroxidase was constant throughout ripening, whereas the IAA and ACC oxidase activities of the basic peroxidases were only activated at the pink stage.

Interactive Effect of NaCl Salinity and Putrescine on Shoot Growth and Activity of IAA oxidase, Invertase and Amylase of Rice (Oryza sativa L. var. GR-3)

Summary Interactive effect of NaCl salinity and putrescine on shoot growth, activity of IAA oxidase, acid invertase, amylase and the level of sucrose, starch and protein of rice (Oryza sativa L. var. GR-3) was studied. NaCl salinity (12 dS/m) markedly reduced the extension growth as well as dry weight of shoot system. The activity of acid invertase and amylase was found decreased whereas that of IAA oxidase increased as a result of soil salinity. Sucrose concentration in the shoot system of plants exposed to salinity was higher while starch and protein registered lower levels compared to the control. Exogenous application of putrescine (10−5 M) enhanced the extension growth, dry matter accumulation and the activity of acid invertase and amylase as well as the contents of starch and protein of saltstressed plants. On the other hand, activity of IAA oxidase and the level of sucrose were appreciably decreased in the shoot system of plants grown under saline condition in response to putrescine administration. These results are discussed in relation to the proposed functions of polyamines in the regulation of plant growth.

Phenols and bud dormancy

summaryQualitative analyses of the endogenous phenols of Fagus sylvatica L. were determined separately in leaf primordia and bud scales of dormant buds (apical, subapical and basal) of Fagus sylvatica L. until bud burst.Qualitative analyses in all leaf primordia showed the presence of phenols which are potential cofactors of IAA oxidase (vanillic acid, 2′4,4′‐trihydroxychalcone), uncoupling agents of oxidative phosphorylation (norwogonin, orobol 7‐O‐rhamnoglucoside, baicalin, 2′4,4′‐trihydroxychalcone and chlorogenic acid), a synergic stimulator of IAA (chlorogenic acid) and others. In all bud scales the qualitative phenol composition includes a mixture of potential IAA oxidase cofactors (vanillic acid and 2′,4,4′‐trihydroxychalcone), uncoupling agents (baicalin and 2′,4,4′‐trihydroxychalcone) and other compounds.In both leaf primordia and bud scales, the qualitative phenol composition does not change significantly during dormancy. These data do not indicate correlations between phenolic matabolism in bud scales and leaf primordia or between single phenolic compounds and the inception and maintenance of dormancy.

Plant Hormone Interaction and Phenolic Metabolism in the Regulation of Russet Spotting in Iceberg Lettuce

Russet spotting (RS) is a physiological disorder induced in iceberg lettuce (Lactuca sativa L.) by exposure to parts per million levels of ethylene at 5 +/- 2 degrees C. Ethylene induced phenylalanine ammonia-lyase and ionically bound peroxidase activities that correlated with development of RS symptoms. The ethylene-treated tissue had significantly higher lignin content than air control tissue with lignification localized in walls of RS-affected cells. Ethylene also caused the accumulation of the flavonoids (+)catechin and (-)epicatechin and the chlorogenic acid derivatives 3-caffeoyl-quinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid. These soluble phenolic compounds were readily oxidized to brown substances by polyphenol oxidase isolated from RS tissue. Ethylene substantially increased ionically bound indole-3-acetic acid (IAA) oxidase activity, while IAA application greatly reduced ethylene-induced phenylalanine ammonia-lyase, peroxidase, and IAA oxidase activities, soluble phenolic content, and RS development.

Changes in levels of peroxidases and phenolics during root formation in Vitis cultured in vitro

Root formation in vine (Vitis vinifera L. cv. Albariño) was accompanied by at first a rise and then a fall in total peroxidase (EC 1.11.1.7) activity in the explant. These variations ran parallel to similar changes in the amount of the three cathodic isope‐roxidases detected, the most abundant of which, C2, also exhibited IAA oxidase activity. The anodic isoperoxidase bands detected were very weak and underwent no variation during rooting. Changes in the opposite direction (a fall followed by a rise) were shown by certain endogenous phenolics, including monoferuloyl, monocaffeoyl and mono‐ρ‐coumaroyl tartaric acids, some of which may act as auxin protectors.

Glyphosate‐increased levels of indole‐3‐acetic acid in yellow nutsedge leaves correlate with gentisic acid levels

The effects of glyphosate [N‐(phosphonomethyl)glycine] on endogenous in‐dole‐3‐acetic acid (IAA) level, IAA oxidase activity, and possible interactions with alterations in phenolic metabolism have been examined in yellow nutsedge (Cyperus esculentus L.) plants. IAA was quantified by flame ionization detector gas‐chroma‐tography, phenols were quantified by high‐performance liquid chromatography and the auxin protection and the IAA oxidase activities were determined spectrophoto‐metrically and/or polarographically. A significant increase in IAA content was recorded after glyphosate treatment. No IAA oxidase activity was detected in control and treated tissues. Auxin protection activity and gentisic acid were present in all extracts assayed, and their concentrations increased as the rate of glyphosate application increased. Addition of gentisic acid to an extract of control plants increased the auxin protection detected. These findings indicate that the high levels of free IAA in yellow nutsedge leaves after glyphosate application are due to the inhibition of the IAA oxidase activity by increased levels of the IAA‐protecting phenol gentisic acid.

The Effect of Pseudomonas putida Colonization on Root Surface Peroxidase

Increased activities of peroxidase and indole 3-acetic acid (IAA) oxidase were detected on root surfaces of bean (Phaseolus vulgaris) seedlings colonized with a soil saprophytic bacterium, Pseudomonas putida. IAA oxidase activity increased over 250-fold and peroxidase 8-fold. Enhancement was greater for 6-day-old than for 4- or 8-day-old inoculated plants No IAA oxidase or peroxidase activities were associated with the bacterial cells. Native polyacrylamide gel electrophoresis demonstrated that washes of P. putida-inoculated roots contained two zones of peroxidase activity. Only the more anodic bands were detected in washes from noninoculated roots. Ion exchange and molecular sizing gel chromatography of washes from P. putida-colonized roots separated two fractions of peroxidase activity. One fraction corresponded to the anodic bands detected in washes of P. putida inoculated and in noninoculated roots. A second fraction corresponded to the less anodic zone of peroxidase, which was characteristic of P. putida-inoculated plants. This peroxidase had a higher IAA oxidase to peroxidase ratio than the more anodic, common enzyme. The changes in root surface peroxidases caused by colonization by a saprophytic bacterium are discussed with reference to plant-pathogen interactions.