Stimulation of the oxidative decarboxylation of indole-3-acetic acid in citrus tissues by ethylene

Ethylene has been shown to stimulate the degradation of indole-3-acetic acid (IAA) in citrus leaf tissues via the oxidative decarboxylation pathway, resulting in the accumulation of indole-3-carboxylic acid (ICA). Preliminary data indicated that ethylene stimulates only the first step of this pathway, i.e. the decarboxylation of IAA which leads to the formation of indole-3-methanol. The effect of ethylene seems to be a specific one since 2,5-norbornadiene, an ethylene action inhibitor, significantly inhibited the stimulation of IAA decarboxylation by ethylene. It has long been suggested that peroxidase or a specific form of the peroxidase complex (`IAA oxidase') catalyse this step. However, we did not observe a clear effect of ethylene on the peroxidase system. An alternative possibility, that the stimulatory effect of ethylene on IAA catabolism results from increased formation of hydrogen peroxide (H₂O₂), a co-factor for peroxidase activity, was verified by direct measurements of H₂O₂ in the tissues or by assaying the activity of gluthathione reductase, which has been shown to be induced by oxygen species. This possibility is further supported by the observations showing that IAA decarboxylation in control tissues was enhanced to the level detected in ethylene-treated tissues by application of H₂O₂.     

Hydroxyproline and Peroxidases in Cell Walls of Pisum sativum: Regulation by Ethylene

Purified cell-wall preparations from the epicotyl of etiolatedPisum sativum contain covalently bound peroxidases and hydroxyproline-richproteins. Towards the end of cell elongation there is a largerise in these wall components and thereafter a continuing slowrise which is associated with increasing age of tissue. Ethyleneat concentrations of 0.1 ppm or more increases both peroxidaseactivity and hydroxyproline levels in the walls, the greatestresponse occurring in immature tissue including the apical hook.Growth of these tissues is highly sensitive to ethylene whichcauses an inhibition of elongation in extending cells and anenhanced lateral cell expansion. We suggest that the effectsof ethylene on wall-bound peroxidase and hydroxyproline areimplicated in the ethylene regulation of cell growth. The covalently bound wall peroxidase was found to be extremelystable and to contain unique isoenzymes which do not occur ineither the cytoplasm or in the peroxidase which is ionicallybound to walls. Ethylene increases peroxidase activity in boththe cytoplasmic and the ionically bound wall fractions, butthere is little or no increase in their hydroxyproline content.The possible relationships between covalently bound wall peroxidaseand hydroxyproline are discussed and we speculate that thisperoxidase may be involved in the hydroxylation of proline inthe walls.     

Binding of ferulic acid to cell walls by peroxidases of Pinus elliottii

Lignin is formed abundantly in the maturing walls of slash pine cambial cells, but very little in slash pine callus cell walls. Peroxidases removed from the cytoplasm of callus or cambial cells with phosphate buffer (soluble peroxidase), from the walls with NACl (ionically bound peroxidase), and from the walls with cellulase (covalently bound peroxidase) differed in their capacity to catalyze bond formation between carbohydrate and ferulic acid or its condensation products. Bond formation per unit of enzyme was highest in the peroxidases of cambium, especially in those attached ionically or covalently to the cell walls. The wall-bound peroxidases also catalyzed the strongest linkages between lignin monomers and carbohydrates as estimated by their resistance to hydrolysis by NaOH.     

Salicylic acid and ethylene pathways are differentially activated in melon cotyledons by active or heat-denatured cellulase from Trichoderma longibrachiatum

Infiltration of cellulase (EC 3.2.1.4) from Trichoderma longibrachiatum into melon (Cucumis melo) cotyledons induced several key defense mechanisms and hypersensitive reaction-like symptoms. An oxidative burst was observed 3 hours after treatment and was followed by activation of ethylene and salicylic acid (SA) signaling pathways leading to marked induction of peroxidase and chitinase activities. The treatment of cotyledons by heat-denatured cellulase also led to some induction of peroxidase and chitinase activities, but the oxidative burst and SA production were not observed. Co-infiltration of aminoethoxyvinil-glycine (an ethylene inhibitor) with the active cellulase did not affect the high increase of peroxidase and chitinase activities. In contrast, co-infiltration of aminoethoxyvinil-glycine with the denatured enzyme blocked peroxidase and chitinase activities. Our data suggest that the SA pathway (induced by the cellulase activity) and ethylene pathway (induced by heat-denatured and active protein) together coordinate the activation of defense mechanisms. We found a partial interaction between both signaling pathways since SA caused an inhibition of the ethylene production and a decrease in peroxidase activity when co-infiltrated with denatured cellulase. Treatments with active or denatured cellulase caused a reduction in powdery mildew (Sphaerotheca fuliginea) disease.     

Effect of Gibberellic Acid and Ethylene on Peroxidase in Pea Internodes

Ethylene at 5–80 µl l^–1 inhibited elongationand induced swelling in internodes of light-grown normal anddwarf pea plants; GA₃ did not prevent swelling in response toethylene. GA₃ neither inhibited nor enhanced the activity of isoperoxidasesin the internodes, regardless of its effect on their elongation.Ethylene at 80 µl l^–1 enhanced peroxidase in GA₃-untreatedand treated normal and dwarf plants. At 5 µl l^–1,ethylene had only a weak effect on peroxidase activity or none.The enzyme enhancement by ethylene was not related to its effecton cell expansion and seems do be due, at least in part, tochemical injury. Electron microscopy revealed peroxidase activity in the roughER and cell walls, including intercellular spaces. Stainingof walls in ethylene-treated tissues was more pronounced thanin untreated ones. Golgi vesicles did not seem to be involvedin the assembly of the enzyme carbohydrate moiety in ethylene-treatedcells. The peroxidase fraction extracted with 20 mM phosphate buffer,pH 6, and that extracted from wall debris with 1 M NaCl accountedfor 98% of total enzyme activity. Both fractions contained thesame six cathodic isoforms which comprised 85–90% of theiractivity. Electrophoresis did not reveal differences in thequalitative isoenzyme patterns in relation to variety, age,GA₃, or ethylene. The only observed quantitative differenceswere age-dependent. Procedural artefacts during separation of protoplast and wallionically bound peroxidase fractions are discussed.     

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.     

Expression of a Trichoderma reesei β-1,4 endo-xylanase in tall fescue modifies cell wall structure and digestibility and elicits pathogen defence responses

An endo-xylanase from Trichoderma reesei (xyn2) has been expressed in tall fescue targeted to the vacuole, apoplast or Golgi, constitutively under the control of the rice actin promoter, and to the apoplast under the control of a senescence enhanced gene promoter. Constitutive xylanase expression in the vacuole, apoplast, and golgi, resulted in only a small number of plants with low enzyme activities and in reduced plant growth in apoplast, and golgi targeted plants. Constitutive expression in the apoplast also resulted in increased levels of cell wall bound hydroxycinnamic acid monomers and dimers, but no significant effect on cell wall xylose or arabinose content. In situ constitutive xylanase expression in the Golgi also resulted in increased ferulate dimers. However, senescence induced xylanase expression in the apoplast was considerably higher and did not affect plant growth or the level of monomeric hydroxycinnamic acids or lignin in the cell walls. These plants also showed increased levels of ferulate dimers, and decreased levels of xylose with increased levels of arabinose in their cell walls. While the release of cell wall hydroxycinnamic acids on self digestion was enhanced in these plants in the presence of exogenously applied ferulic acid esterase, changes in cell wall composition resulted in decreases in both tissue digestibility and cellulase mediated sugar release. In situ detection of H₂O₂ production mediated by ethylene release in leaves of plants expressing apoplast xylanase could be leading to increased dimerisation. High-level xylanase expression in the apoplast also resulted in necrotic lesions on the leaves. Together these results indicate that xylanase expression in tall fescue may be triggering plant defence responses analogous to foliar pathogen attack mediated by ethylene and H₂O₂.     

Production of Pectinase and Cellulase by Cladosporium cucumerinum with Dissolved Carbohydrates and Isolated Cell Walls of Cucumber as Carbon Sources

The scab fungus Cladosporium cucumerinum can use pectins and polygalacturonic acid as sole sources of carbon. Cellulose and Ca-polygalacturonate are not available carbon sources for the fungus. When growing on sucrose or pectin, pectinase is produced. In these cases the production of cellulase is insignificant. On a mixture of pectin and carboxymethylcellulose also cellulase is produced. Both pectinase and cellulase are released into the culture filtrate when the fungus grows on cell walls without ionic proteins, whereas only cellulase is released when cell walls with ionic proteins are the carbon source. Pectinase produced by the pathogen can bind to isolated cell walls. The bound pectinase can be extracted with 1 M NaCl from cell walls without ionic proteins, but not from cell walls with ionic proteins. A water-extract or 1 M NaCl-extract of cucumber hypocotyls with visible disease symptoms contains cellulase but no pectinase activity. Lack of pectinase activity in the 1 M NaCl-extract may be due to inhibition by a component that could be extracted by NaCl from the cucumber cell walls.     

Massive synthesis of ribonucleic Acid and cellulase in the pea epicotyl in response to indoleacetic Acid, with and without concurrent cell division

Measurements were made over a 4-day period of the effect of added indoleacetic acid (IAA), puromycin, actinomycin D and 5-fluorodeoxyuridine (FUdR) on growth and the levels of total DNA, RNA, protein and cellulase in segments of tissue at the apex of decapitated etiolated epicotyls of Pisum sativum, L. var. Alaska.

The hormone induced swelling of parenchyma cells and cell division. By 3 days after IAA application, the amounts of DNA and protein were approximately double, RNA triple and cellulase 12 to 16 times the levels in controls. All of these changes were prevented by both puromycin and actinomycin D. FUdR prevented DNA synthesis and cell division but not swelling or synthesis of RNA, protein and cellulase.

It is concluded that IAA-induced RNA synthesis is required for cellulase synthesis and lateral cell expansion, whether or not cell division takes place.

     

Peroxidase isozyme patterns in the skin of maturing tomato fruit

The cessation of tomato fruit growth is thought to be induced by an increase in the activity of enzymes which rigidify cell walls in the fruit skin. Peroxidase could catalyse such wall‐stiffening reactions, and marked rises in peroxidase activity were recently reported in skin cell walls towards fruit maturity. Peroxidase isoforms in the fruit are here analysed using native gel electrophoresis. New isoforms of apparent M_r 44, 48 and 53 kDa are shown to appear in cell walls of the fruit skin at around the time of cessation of growth. It is inferred that these isozymes are present in the cell wall in vivo. Fruit from a range of non‐ripening mutants were also examined. Some of these do not soften or ripen for many weeks after achieving their final size. The new isozymes were found in skin cell walls of mature fruit in each of these mutants, as in the wild‐type and commercial varieties. It is concluded that the late‐appearing isozymes are not associated with fruit ripening or softening, and are probably not ethylene‐induced. They may act to control fruit growth by cross‐linking wall polymers within the fruit skin, thus mechanically stiffening the walls and terminating growth.     

甘露醇和6—BA处理对水稻细胞过氧化物酶及IAA氧化酶活性的影响

研究了甘露醇和６０ＢＡ处理对水稻服浮细胞再分化、过氧化物酶及ＩＡＡ氧化酶的影响。结果表明,甘露醇处理能延迟水稻细胞衰老,提高细胞再分化能力,降低细胞过氧化物酶和ＩＡＡ氧化酶活性,６－ＢＡ（２ｍｇ／Ｌ）虽然明显降低细胞过氧化物酶活性,但对ＩＡＡ氧化酶及细胞衰老无明显影响,讨论了过氧化物酶及ＩＡＡ氧化酶在水稻胚性细胞形成上的可能作用。     

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.     

Inactivity of Oxidation Products of Indole-3-acetic Acid on Ethylene Production in Mung Bean Hypocotyls

The suggestion that indole-3-acetic acid (IAA)-stimulated ethylene production is associated with oxidative degradation of IAA and is mediated by 3-methyleneoxindole (MOI) has been tested in mung bean (Phaseolus aureus Roxb.) hypocotyl segments. While IAA actively stimulated ethylene production, MOI and indole-3-aldehyde, the major products of IAA oxidation, were inactive. Tissues treated with a mixture of intermediates of IAA oxidation, obtained from a 1-hour incubation of IAA with peroxidase, failed to stimulate ethylene production. Furthermore, chlorogenic acid and p-coumaric acid, which are known to interfere with the enzymic oxidation of IAA to MOI, had no effect on IAA-stimulated ethylene production. Other oxidation products of IAA, including oxindole-3-acetic acid, indole-3-carboxylic acid, (2-sulfoindole)-3-acetic acid, and dioxindole-3-acetic acid, were all inactive. 1-Naphthaleneacetic acid was as active as IAA in stimulating ethylene production but was decarboxylated at a much lower rate than IAA, suggesting that oxidative decarboxylation of auxins is not linked to ethylene production. These results demonstrate that IAA-stimulated ethylene production in mung bean hypocotyl tissue is not mediated by MOI or other associated oxidative products of IAA.     

Optimal and Spatial Analysis of Hormones,Degrading Enzymes and Isozyme Profiles in Tomato Pedicel Explants During Ethylene-Induced Abscission

Activities of degrading enzymes, hormones concentration and zymogram patterns were investigated during control and ethylene-induced abscission of tomato pedicel explants. Exogenous ethylene accelerated abscission of pedicel explants. It was showed that IAA concentration in abscission zone tended to decline at first and then was reduced before separation in control and ethylene-treatment. Moreover, IAA (indole acetic acid) and ABA (abscise acid) concentrations were elevated in each segment when exposing to ethylene, but GA_{1 + 3} (gibberellin1 + gibberellin3) concentration was decreased in abscission zone and the proximal side. Activities of cellulase, polygalacturonase and pectinesterase in the explants were induced in the separating process and strengthened by ethylene. However, comparing with the proximal side, cellulase and polygalacturonase activities in abscission zone and distal side were higher. Electrophoresis of isozymes revealed that at least three peroxidase and three superoxidase isozymes appeared in the explants, respectively. One peroxidase isozyme exhibited differentially among the three positions in control and ethylene-treatment. One esterase isozyme weakened or disappeared in the following hours, but three novel esterase isozymes were detectable from beginning of the process. The data presented support the hypothesis that the distal side, together with abscission zone of explants plays a more important role in separation than does the proximal side. The possible roles of degrading enzymes, hormones and isozymes in three segments during ethylene-induced abscission of tomato pedicel explants are discussed.     

Force extension analysis of Avena coleoptile cell walls

Summary A method is described for measuring the cell wall mechanical properties of Avena coleoptiles in the absence of turgor stress or influences of a living protoplast. Forceextension curves obtained with a constant-rate-of-extension instrument and standard fiber-testing techniques demonstrate the permanence of cell wall loosening effects of prior indoleacetic acid (IAA) treatment of living tissue and provide evidence that these changes involve interactions between cell wall polymers. By this method various chemical and enzymatic modifications of cell walls can be evaluated in terms of altered mechanical properties. Thus, it was possible to remove over 97% of the cell nitrogen (including some hydroxyproline-containing protein) by hot methanol followed by enzymatic treatment and not change the extensibility properties of the tissue. In contrast, coleoptile mechanical properties were markedly influenced by chemical acetylation procedures or cellulase treatment.With 3 Figures in the Text     

Brassinosteroids affect ethylene production in the primary roots of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

To better understand the physiological roles of brassinosteroids (BRs) in the primary roots of maize, we examined their effect on ethylene production. Exogenously applied brassinolide (BL; 10^-9 to 10^-7 M) incrementally increased the level ethylene in a dose-dependent manner. This BL-induced production was enhanced in the presence of IAA, thereby implying a synergistic effect between BR and IAA. At 10^-7 M BL, the level of free ACC was increased, but that of conjugated ACC was diminished. Moreover, greater concentrations of BL proportionally increased ACC oxidase activity. In contrast, higher levels of IAA increased the endogenous content of conjugated ACC as well as ACC synthase activity. Based on these results, we conclude that BR activates ethylene production mainly via ACC oxidase, and interacts with IAA to produce ethylene. However, the functional site for ethylene production is different for each hormone.     

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 Auxin on Autolysis of Cell Walls in Azuki Bean Epicotyls

Native cell walls of azuki bean epicotyls incubated in bufferautolytically released neutral sugars, abundant in galactose,and uronic acids. Treatment with 10^–5 M IAA of subapicalor basal epicotyl segments for 3 h did not influence the amountof total neutral sugars released from the cell walls duringautolysis. However, the amount of glucose and xylose releasedfrom subapical cell walls was increased by IAA. Pretreatmentwith IAA of subapical epicotyl segments enhanced the solubilizationof neutral sugars from pectinase-treated cell walls during incubationin buffer at pH 5 to 6. The amount of fucose, xylose, and glucosereleased was specifically increased by IAA. Of the sugar fractionsreleased from pectinase-treated cell walls during autolysisand subsequently separated by gel filtration on a ToyopearlHW-40S column, IAA promoted the release of oligosaccharides,consisting mainly of glucose and xylose. These results suggestthat autolytic degradation of xyloglucans is closely relatedto IAA-induced growth of azuki bean epicotyls. (Received May 19, 1989; Accepted January 5, 1990)     

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 [1-¹⁴C]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.     

Changes in IAA,Phenolic Compounds,Peroxidase, IAA Oxidase,and Polyphenol Oxidase in Relation to Flower Formation in Crocus sativus

Changes in levels of IAA, phenolic compounds, peroxidase, polyphenol oxidase, and IAA oxidase activities in the corm and the apical bud of Crocus sativusL. during bud growth and development, with special emphasis on the flowering stage, were studied. In the bud, flower formation was accompanied by enhanced activities of peroxidase, polyphenol oxidase, IAA oxidase, and higher contents of phenolic compounds as well as lower levels of IAA. In the corm, during the flower formation, these enzymes showed an opposite behavior. Moreover, the contents of phenolics and IAA in the corm tissues during flower formation and growth were higher than at the other developmental stages. It may be concluded that the transition of saffron plants to flowering is correlated with peroxidase, polyphenol oxidase, and IAA oxidase. Furthermore, these enzymes might exert their roles in the regulation of flowering through their participation in IAA catabolism. The hypothesis of regulation of bud development by an interaction between phenolics and the enzymes involved in IAA catabolism is discussed.