Soluble peroxidase gradients in lupin hypocotyls and the control of the level of polarly transported indole-3yl-acetic acid

The distribution of basic soluble isoperoxidases along the growth gradient of lupin hypocotyl was studied in order to establish the role of these isoenzymes in controlling polarly transported indole-3yl-acetic acid (IAA) levels. The observation that the levels of basic isoperoxidases, which diminish from the young (vascular differentiating) to the older (vascular differentiated) tissues, are related with previously reported IAA oxidation rates in decapitated plants, suggests that these isoenzymes can play a role in the oxidation of IAA during polar transport. The fact that the level of basic isoperoxidases is controlled by IAA in hypocotyl sections harvested from different growth zones is in accordance with the previously described adaptative activation of basic isoperoxidases to IAA content. This adaptative activation of basic isoperoxidases might constitute the basic characteristic of a system of subcellular oscillators, coupled at the cellular level, necessary to generate the supracellular auxinwave associated with auxin transport.     

The vacuolar localization of basic isoperoxidases in grapevine suspension cell cultures and its significance in indole-3-acetic acid catabolism

The study of the subcellular localization of the basic isoperoxidases in grapevines was carried out by using cells cultured in suspension as a model system. Results from subcellular fractionation, isoenzyme analysis, enzyme binding and cytochemical probes suggest that basic isoperoxidases are localized mainly in the vacuolar sap of the suspension cultured cells, probably in equilibrium with a pool of the same basic isoperoxidases bound to the internal face of tonoplast membranes through a Ca²⁺-saline bridge. This vacuolar location of basic isoperoxidases raised the question of their function, since indole-3-acetic acid (IAA) oxidase activity of these isoperoxidases is almost totally inhibited by vacuolar anthocyanins in the in vivo concentration range of these compounds. Thus, a central role is proposed for these isoenzymes in the H₂O₂-dependent oxidative phenol metabolism which occurs in grapevines, discarding therefore a possible role of these isoperoxidases in the control of IAA levels during the later stages of development of anthocyanin-rich grapes.     

The effect of 4-chlororesorcinol on the endogenous levels of IAA,ABA and oxidative enzymes in cuttings

Treatment of bean cuttings with 4-chlororesorcinol (4-CR), known to increase the number of roots and extend their distribution, prevented the accumulation of free indol-3-yl-acetic acid (IAA) in the hypocotyls within 24 h after cutting preparation. In mung bean there was no change in the distribution (upper half vs. 1 ower half of the hypocotyl) of IAA within the hypocotyl as a result of the treatment. In bean cuttings the treatment with 4-CR prevented the accumulation of IAA in the bottom of the cutting. Oxidation of IAA as a measure of IAA oxidase activity in bean was enhanced appreciably by 4-chlororesorcinol. The level of abscisic acid in mung bean, on the other hand, remained 3–4 fold higher than in the control, yet still about 50% lower than the zero time level. In untreated mung bean cuttings the activity of peroxidase increased after cutting preparation. In contrast, the activity of peroxidase in 4-Cr-treated cuttings was consistently lower. In order to relate to the effect of exogenously applied auxin the level of peroxidase was measured also in indol-3-yl-butyric acid-treated cuttings. The overall peroxidase activity in IBA-treated cuttings was not affected. However, when assaying for the different isozymes the drop in peroxidase activity was most evident in the inducible basic isoperoxidases both in 4-CR and IBA treatments. It appears that the exposure to 4-CR exerts an effect that is similar to that of exogenously applied auxin, affecting the activity of basic peroxidases and enhancing the oxidation of endogenous IAA, thus allowing the organization of the primordia.Abbreviations ABA - abscisic acid - 4-CR - 4-chlororesorcinol - IAA - indol-3-yl-acetic acid - IBA - indol-3-yl-butyric acid     

Changes in the concentration of indole-3-acetic acid during the growth of etiolated lupin hypocotyls

The longitudinal distribution of unaltered radioactive indole-3-acetic acid (IAA), after application of [5-³H]-IAA to decapitated etiolated lupin hypocotyls. exhibited a wave-like pattern similar to that obtained with endogenous IAA. Waves of radioactive IAA were localizated both in the elongation zone and in the non-growing basal region of the hypocotyl. These IAA waves were transient because of basipetal polar transport and metabolism of IAA.
The level of endogenous IAA in different zones of the hypocotyl varied with age, following a wave-like pattern. During the elongation period of each zone, IAA was parallel to the bell-shaped curve of the growth rate. In addition, a role in secondary cell wall deposition is suggested for the other IAA wave that appeared after the cell elongation period, since an electron microscopic morphometric analysis of the cell wall showed that the cell wall thickness increased once the cell elongation ceased.
As the oscillation of endogenous IAA level occured in both space (distribution along the hypocotyl) and time (variation with age), it is suggested that the level of IAA really depended on the growth status of the cells. The response of the cells to the positional information submitted by the auxin waves as regards the growth status of the cell is discussed.     

Indole-3-methanol is the main product of the oxidation of indole-3-acetic acid catalyzed by two cytosolic basic isoperoxidases from Lupinus

The nature of the products of the auxin catabolism mediated by both basic and acidic isoperoxidases has been studied. While indole-3-methanol is only a minor product of the oxidation of indole-3-acetic acid catalyzed by extracellular acidic isoperoxidases, it is the only product of the oxidation of indole-3-acetic acid catalyzed by two cytosolic basic isoperoxidases (EC 1.11.1.7) from lupin (Lupinus albus L.) hypocotyls. The putative indole-3-methanol formed by these latter isoperoxidases was isolated and then characterized by mass spectrometry and ¹H-nuclear magnetic resonance spectrometry. These results are discussed with respect to the diversity and compartmentation of the catabolism of indole-3-acetic acid in plant tissues.Abbreviations DCP 2,4-dichlorophenol - IAA indole-3-acetic acid - IM indole-3-methanol     

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.     

The rib1 mutant of Arabidopsis has alterations in indole-3-butyric acid transport, hypocotyl elongation, and root architecture

Polar transport of the auxin indole-3-butyric acid (IBA) has recently been shown to occur in Arabidopsis (Arabidopis thaliana) seedlings, yet the physiological importance of this process has yet to be fully resolved. Here we describe the first demonstration of altered IBA transport in an Arabidopsis mutant, and show that the resistant to IBA (rib1) mutation results in alterations in growth, development, and response to exogenous auxin consistent with an important physiological role for IBA transport. Both hypocotyl and root IBA basipetal transport are decreased in rib1 and root acropetal IBA transport is increased. While indole-3-acetic acid (IAA) transport levels are not different in rib1 compared to wild type, root acropetal IAA transport is insensitive to the IAA efflux inhibitor naphthylphthalamic acid in rib1, as is the dependent physiological process of lateral root formation. These observed changes in IBA transport are accompanied by altered rib1 phenotypes. Previously, rib1 roots were shown to be less sensitive to growth inhibition by IBA, but to have a wild-type response to IAA in root elongation. rib1 is also less sensitive to IBA in stimulation of lateral root formation and in hypocotyl elongation under most, but not all, light and sucrose conditions. rib1 has wild-type responses to IAA, except under one set of conditions, low light and 1.5% sucrose, in which both hypocotyl elongation and lateral root formation show altered IAA response. Taken together, our results support a model in which endogenous IBA influences wild-type seedling morphology. Modifications in IBA distribution in seedlings affect hypocotyl and root elongation, as well as lateral root formation.     

Development of isoperoxidases along the growth gradient in the mung bean hypocotyl

Cytoplasmic and wall bound peroxidases were extracted from successive segments of decreasing growth potential along the mung bean hypocotyl. Active wall bound peroxidases were present in the epidermis and external parenchyma layers at the end of the elongation phase. Two fast migrating anionic isoperoxidases covalently bound to the cell walls increased when the cell walls lost their plasticity. These isoenzymes were characterized by a high affinity for several peroxidase substrates and high thermal stability.     

Low-fluence red light increases the transport and biosynthesis of auxin

In plants, light is an important environmental signal that induces photomorphogenesis and interacts with endogenous signals, including hormones. We found that light increased polar auxin transport in dark-grown Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) hypocotyls. In tomato, this increase was induced by low-fluence red or blue light followed by 1 d of darkness. It was reduced in phyA, phyB1, and phyB2 tomato mutants and was reversed by far-red light applied immediately after the red or blue light exposure, suggesting that phytochrome is involved in this response. We further found that the free indole-3-acetic acid (IAA) level in hypocotyl regions below the hook was increased by red light, while the level of conjugated IAA was unchanged. Analysis of IAA synthesized from [13C]indole or [13C]tryptophan (Trp) revealed that both Trp-dependent and Trp-independent IAA biosynthesis were increased by low-fluence red light in the top section (meristem, cotyledons, and hook), and the Trp-independent pathway appears to become the primary route for IAA biosynthesis after red light exposure. IAA biosynthesis in tissues below the top section was not affected by red light, suggesting that the increase of free IAA in this region was due to increased transport of IAA from above. Our study provides a comprehensive view of light effects on the transport and biosynthesis of IAA, showing that red light increases both IAA biosynthesis in the top section and polar auxin transport in hypocotyls, leading to unchanged free IAA levels in the top section and increased free IAA levels in the lower hypocotyl regions.     

The decrease in auxin polar transport down the lupin hypocotyl could produce the indole-3-acetic Acid distribution responsible for the elongation growth pattern

The variation of indole-3-acetic acid (IAA) transport along Lupinus albus L. hypocotyls was studied using decapitated seedlings and excised sections. To confirm that the mobile species was IAA and not IAA metabolites, dual isotope-labeled IAAs, [5-³H]IAA + [1-¹⁴C]IAA, were used. After apical application to decapitated seedlings, the longitudinal distribution of both isotopes at different transport periods showed that the velocity of IAA transport was higher in the apical, elongating region than in the basal, non-growing region. This variation in velocity was not a traumatic consequence of decapitation because after application of IAA to the basal region of decapitated seedlings, both the velocity and intensity of IAA transport were lower than in the apical treatment. The variation in IAA transport down the hypocotyl was confirmed when it was measured in excised sections located at different positions along the hypocotyl. The velocity and, to a greater extent, the intensity of IAA transport decreased from the apical to the basal sections. Consequently, if the amount of IAA reaching the apical zones of lupin hypocotyl were higher than the IAA transport capacity in the basal zones, accumulation of mobile IAA might be expected in zones located above the basal region. In fact, an IAA accumulation occurred in the elongating region during the first 4-h period of transport after apical treatment with IAA. It is proposed that the fall in IAA transport along the hypocotyl might be responsible for the IAA distribution and, consequently, for the growth distribution reported in this organ. An indirect proof of this was obtained from experiments that showed that the excision of the slowly transporting basal zones strongly reduced the growth in the remaining part of the organ, whereas excision of the root caused no significant modification in growth during a 20-h period.     

Influence des régulateurs de croissance sur la prolifération,l'activité peroxydasique et les isoperoxydases de la suspension cellulaire deSilene alba

Growth ofSilene alba (MILLER) E. H. L. KRAUSE cells, as well as their peroxidase pattern and activity are studied. Cells were grown in the presence and absence either of IAA, or NAA or 2,4-D. The subculture is dependent upon the growth regulator used to sustain the growth of cells. For 14 days' passages, subculture is possible with 2,4-D (5 x 10^-7M) or NAA (10^-5M) but impossible with IAA or without any growth regulators. Cells grown using 2,4-D or NAA in the medium contain a smaller number of isoperoxidases and have lower activities than those grown using IAA or no growth regulator. The nature of growth substances does not affect the compartimontation of the peroxidase; in fact the bulk of the peroxidaso activity is always liable to the ionic wall bound fractions. Tho electrophorotic mobilities of peroxidase isoenzymes detected in the modium are not the same as those of tho eytoplasmic isoenzymes. Cell cultures grown with and without growth regulators show different patterns of modium peroxidase activities. Some forms are present both in cells and media and some other only in the media; this may indicate that there is some selection made in tho cells for retention of particulars forms; the others could be secreted as exoenzymes shortly after they are synthesized in the cells. The nature of the growth regulator used could act on the release of certain isoperoxidases. These results are discussed from the viewpoint of the correlation of isoperoxidase patterns with the possibility of subculture.     

Isoperoxidase Patterns in Tobacco Pith and their Alteration Following Tissue Excision

Previous work has shown that young tobacco pith cells containlow peroxidase activity resolvable into two anodic iso-zymesby electrophoresis at pH 8.3 on starch gel. During normal cellenlargement and ageing in situ, these two isoenzymes increaseslowly in activity. By contrast, within 24 h after excisionof pith, high activities appear in several apparently new anodicand cathodic isoperoxidases. This development of new isoperoxidasesis prevented by physiological levels of IAA and by inhibitorsof RNA and protein synthesis. The present work describes additional quantitative isoperoxidasegradients, both vertical and horizontal, in the pith. Severaladditional isoenzymes, some of which are excreted into the mediumduring incubation, have been detected by improved electrophoretictechniques. Enzyme leakage can be prevented by incubation inmoist chambers and by auxin application through terminally placedagar blocks. With such techniques, new peroxidases form preferentiallynear cut surfaces within 12 h under aerobic conditions. Tissuesperipheral to the pith retard the development of new peroxidaseactivity. IAA in agar blocks applied terminally to a cut pithcylinder retards peroxidase formation mainly at the base. Theregulation of the quantity and quality of isoperoxidases seemsto be correlated with certain morphogenetic phenomena.     

The interaction of light irradiance with auxin in regulating growth of <Emphasis Type="Italic">Helianthus annuus</Emphasis> shoots

Plants growing under canopy shade or in near-neighboring proximity of taller vegetation are the receivers of shade light conditions. The effect of light irradiance (photosynthetically active radiation [PAR]), one of the main components of shade light, on the growth of various tissues of sunflower seedlings and the possible role of auxin were investigated. Gradual reductions in PAR irradiance level from near-normal to low and very low result in significant and gradual increases in sunflower hypocotyl growth and endogenous auxin content. Similar reductions in PAR level resulted in significant and gradual decreases in sunflower cotyledon and leaf growth, and endogenous auxin content. Exogenously applied auxin increased hypocotyl elongation under near-normal PAR, where IAA levels are below optimum, but decreased elongation under very low PAR, where IAA levels are already at optimum. These results suggests that auxin acts as positive growth regulator of sunflower hypocotyls subjected to low light irradiance stress. This is further supported by the transfer experiments where seedlings transferred, for example, from near-normal PAR to very low PAR showed increased elongation associated with increased IAA levels. Therefore, it is reasonable to conclude that light irradiance-mediated changes in hypocotyl elongation of young sunflower seedlings are regulated by endogenous auxin levels.     

Indole-3-Acetic Acid Control on Acidic Oat Cell Wall Peroxidases

Incubation of oat coleoptile segments with 40 μm indoleacetic acid (IAA) induced a decrease of 35–60% in peroxidase activity at the cell wall compartment. Treatment with IAA also produced a similar decrease in the oxidation of NADH and IAA at the cell wall. Isoelectric focusing of ionic, covalent, and intercellular wall peroxidase fractions showed that acidic isoforms (pI 4.0–5.5) were reduced preferentially by IAA treatment. Marked differences were found between acidic and basic wall isoperoxidases in relation to their efficacy in the oxidation of IAA. A peroxidase fraction containing acidic isoforms oxidized IAA with a V _max/s_0.5 value of 2.4 × 10⁻² min⁻¹· g fw⁻¹, 4.0 times higher than that obtained for basic peroxidase isoforms (0.6 × 10⁻² min⁻¹· g fw⁻¹). In contrast, basic isoforms were more efficient than acidic isoperoxidases in the oxidation of coniferyl alcohol or ferulic acid with H₂O₂ (5.6 and 2.1 times, respectively). The levels of diferulate and lignin in the walls of oat coleoptile segments were not altered by treatment with IAA. The decrease in cell wall peroxidase activity by IAA was related more to reduced oxidative degradation of the hormone than to covalent cell wall cross-linking. Received November 1, 1998; accepted December 14, 1998     

Effect of maleic hydrazide on peroxidase isoenzymes in relation to rooting hypocotyl cuttings ofPhaseolus mungo

A time course study of changes in the pattern of peroxidase isoenzymes shows that two new isoenzymesa andb appeared in hypocotyl cuttings cultured either in indolyl-3 acetic acid (IAA) + sucrose or in water but not in maleic hydrazide (MH). Roots were also initiated in the former two cases but not in the last case. That these isoenzymes may be associated with root initiation is also evident from the fact that these appeared when hypocotyl cuttings were transferred from MH to IAA + sucrose. The time of appearance of these isoenzymes in different cultures also synchronized with the time of microscopic root initiation. While the isoenzymea disappeared,b persisted in cuttings transferred from IAA + sucrose to MH. The number of roots produced on cuttings transferred to MH was very low, though their development was normal. It is suggested that isoenzymesa andb may be associated with root initiation andc andd with root development.     

Synergistic interaction between coumarin 1,2-benzopyrone and indole-3-butyric acid in stimulating adventitious root formation in Vigna radiata (L.) Wilczek cuttings: I. Endogenous free and conjugated IAA and basic isoperoxidases

Cuttings from 7-day-old Vigna radiata seedlings were treated for 24 h with various concentrations of coumarin and/or indole-3-butyric acid (IBA), applied either alone or in combination, in order to stimulate adventitious root formation (ARF). The effects of treatment on endogenous free and conjugated indole-3-acetic acid (IAA), basic peroxidase (basic PER) activity and its isoperoxidases analysis and their relation to ARF were then investigated at the potential rooting sites during the first 96 h after application. Simultaneously, combined treatments acted synergistically in inducing more adventitious roots in treated cuttings than in those treated with coumarin or IBA individually, as compared with the control. Endogenous free IAA increased transiently in treated cuttings as compared with the control and the maximum increase occurred with the combined treatment. This suggests that coumarin and IBA may act synergistically in increasing the endogenous free IAA level during the induction phase of rooting to initiate more roots. Likewise, higher level of conjugated IAA was also found in treated cuttings than in untreated ones, during the primary events of ARF, with the maximum level occurring in the combined treatment. Comparison of the dynamics of conjugated IAA and activity of basic PERs led to conclusion that the former but not the latter is responsible for downregulation of endogenous IAA levels significantly during the primary events of ARF. A sharp increases in basic PERs occurred during the secondary events of ARF, suggesting their role in root initiation and development rather than root induction.     

Indole acetic acid distribution coincides with vascular differentiation pattern during Arabidopsis leaf ontogeny

We used an anti-indole acetic acid (IAA or auxin) monoclonal antibody-based immunocytochemical procedure to monitor IAA level in Arabidopsis tissues. Using immunocytochemistry and the IAA-driven beta-glucuronidase (GUS) activity of Aux/IAA promoter::GUS constructs to detect IAA distribution, we investigated the role of polar auxin transport in vascular differentiation during leaf development in Arabidopsis. We found that shoot apical cells contain high levels of IAA and that IAA decreases as leaf primordia expand. However, seedlings grown in the presence of IAA transport inhibitors showed very low IAA signal in the shoot apical meristem (SAM) and the youngest pair of leaf primordia. Older leaf primordia accumulate IAA in the leaf tip in the presence or absence of IAA transport inhibition. We propose that the IAA in the SAM and the youngest pair of leaf primordia is transported from outside sources, perhaps the cotyledons, which accumulate more IAA in the presence than in the absence of transport inhibition. The temporal and spatial pattern of IAA localization in the shoot apex indicates a change in IAA source during leaf ontogeny that would influence flow direction and, consequently, the direction of vascular differentiation. The IAA production and transport pattern suggested by our results could explain the venation pattern, and the vascular hypertrophy caused by IAA transport inhibition. An outside IAA source for the SAM supports the notion that IAA transport and procambium differentiation dictate phyllotaxy and organogenesis.     

Transport of the two natural auxins, indole-3-butyric acid and indole-3-acetic acid, in Arabidopsis

Polar transport of the natural auxin indole-3-acetic acid (IAA) is important in a number of plant developmental processes. However, few studies have investigated the polar transport of other endogenous auxins, such as indole-3-butyric acid (IBA), in Arabidopsis. This study details the similarities and differences between IBA and IAA transport in several tissues of Arabidopsis. In the inflorescence axis, no significant IBA movement was detected, whereas IAA is transported in a basipetal direction from the meristem tip. In young seedlings, both IBA and IAA were transported only in a basipetal direction in the hypocotyl. In roots, both auxins moved in two distinct polarities and in specific tissues. The kinetics of IBA and IAA transport appear similar, with transport rates of 8 to 10 mm per hour. In addition, IBA transport, like IAA transport, is saturable at high concentrations of auxin, suggesting that IBA transport is protein mediated. Interestingly, IAA efflux inhibitors and mutations in genes encoding putative IAA transport proteins reduce IAA transport but do not alter IBA movement, suggesting that different auxin transport protein complexes are likely to mediate IBA and IAA transport. Finally, the physiological effects of IBA and IAA on hypocotyl elongation under several light conditions were examined and analyzed in the context of the differences in IBA and IAA transport. Together, these results present a detailed picture of IBA transport and provide the basis for a better understanding of the transport of these two endogenous auxins.     

Homology of Plant Peroxidases: AN IMMUNOCHEMICAL APPROACH

Antisera specific for the basic peroxidase from horseradish (Amoracea rusticana) were used to examine homology among horseradish peroxidase isoenzymes and among basic peroxidases from root plants. The antisera cross-reacted with all tested isoperoxidases when measured by both agar diffusion and quantitative precipitin reactions. Precipitin analyses provided quantitative measurements of homology among these plant peroxidases. The basic radish (Raphanus sativus L. cv. Cherry Belle) peroxidase had a high degree of homology (73 to 81%) with the basic peroxidase from horseradish. Turnip (Brassica rapa L. cv. Purple White Top Globe) and carrot (Daucus carota L. cv. Danvers) basic peroxidases showed less cross-reaction (49 to 54% and 41 to 46%, respectively). However, the cross-reactions of antisera with basic peroxidases from different plants were greater than were those observed with acidic horseradish isoenzymes (30 to 35%). These experiments suggest that basic peroxidase isoenzymes are strongly conserved during evolution and may indicate that the basic peroxidases catalyze reactions involved in specialized cellular functions. Anticatalytic assays were poor indicators of homology. Even though homology among isoperoxidases was detected by other immunological methods, antibodies inhibited only the catalytic activity of the basic peroxidase from radish.     

Lupin peroxidases. II. Binding of acidic isoperoxidases to cell walls

Extracellular acidic isoperoxidases (EC 1.11.1.7), isolated from both the cell walls and intercellular spaces of lupin ( Lupinus albus L. cv. multolupa) hypocotyls, bound to water-insoluble pectins of wall fragments also isolated from the hypocotyls. The binding was sáturable by increasing the isoenzyme concentration in the assay medium and it was dependent on the pH; neutral pH (6.0–7.0) favoured release, while acidic pH (4.0–5.0) favoured the attachment to the cell wall. Binding of acidic isoperoxidases to wall fractions was correlated with the in vitro acid-induced growth of hypocotyl segments, and both were modulated in the same direction by the Ca²⁺/H⁺ ratio in the incubation media, although the two responses were clearly separated when the Ca²⁺/H⁺ ratio varied. Binding of acidic isoperoxidases of cell walls could operate as a fine control of the activity of these cell wall enzymes, although its physiological role in the cell wall stiffening remains unclear. Some aspects of Ca²⁺ on the control of peroxidase activity at this level are also discussed.