Ethylene-induced Isocoumarin Formation in Carrot Root Tissue

Sweet potato (Ipomoea batatas) root parenchyma and tobacco (Nicotiana tabacum) stem pith, both known to increase peroxidase activity after excision, differed from each other in their isoperoxidase patterns and in the isoperoxidase responses to injury and exogenous ethylene.In potato root sections, the injury-dependent peroxidase increase was due to an induction of two isoenzymes, as well as to a promotion of some constitutive ones. In tobacco pith, this increase was entirely due to seven isoperoxidases not detectable, or detectable only in traces, immediately after excision. Actinomycin D did not inhibit the development of any isoperoxidases in the potato root sections and strongly repressed the development of all injury-induced isoenzymes in tobacco pith. Cycloheximide totally inhibited the development of all isoperoxidases in both species, with the exception of two injuryenhanced isoenzymes in root parenchyma.In root sections, indoleacetic acid had a weak inhibitory effect on one injury-induced isoperoxidase only, whereas in tobacco pith it inhibited the development of the injury-induced, as well as the constitutive, isoperoxidases.Exogenous ethylene did not induce, enhance, or significantly suppress any of the tobacco pith isoenzymes, whereas in potato root sections, it suppressed slightly the development of the injury-induced, had no effect on some of the injuryenhanced, and greatly promoted some of the injury-unaffected or-enhanced isoperoxidases. Removal of ethylene stopped the ethylene-dependent peroxidase increase without affecting the injury-induced increase. When applied to intact potato roots, ethylene did not induce any new isoperoxidases and promoted the same constitutive isoenzymes as it did in root sections.

Section: Discussionmentioning

confidence: 61%

Comparative Studies on Tobacco Pith and Sweet Potato Root Isoperoxidases in Relation to Injury, Indoleacetic Acid, and Ethylene Effects

Birecka¹,

Briber²,

Catalfamo³

1973

“…1, Table I (2). Since these concentrations of ethylene induced low activity in carrot root discs (Fig.…”

Section: Resultsmentioning

confidence: 99%

Ethylene-induced Phenylalanine Ammonia-Lyase Activity in Carrot Roots

Chalutz

1973

Ethylene enhanced the activity of phenylalanine ammonialyase in carrot (Daucus carota L., var. "Nautv") root tissue. Slight increase in enzyme activity was exhibited by root discs incubated in ethylene-free air. It was probably due to the ethylene formed within the sliced tissue. Addition of ethylene to the air stream increased phenylalanine ammonia-lyase activity and the total protein content of the discs until maximum activity was reached after 36 to 48 hours of incubation. (4,8,10,15,16 'Abbreviation: PAL: phenylalanine ammonia-lyase.MATERIALS AND METHODS Fresh carrot (Daucus carota L., var. "Nanty") roots of uniform size were washed in detergent and water and surface sterilized with 70% ethanol. Uniform discs (1 cm thick and 1.5 cm in diameter) were cut from the roots and placed on moist filter paper in open Petri plates. About 50 discs were incubated at 20 C in the dark, inside 20-liter glass jars through which air, ethylene-air, and other gaseous mixtures saturated with water were continuously passed at a rate of 100 ml/min.For nucleic acid and protein synthesis inhibitor studies, discs were dipped for 1 min into the inhibitor solution, then placed on filter paper in Petri plates which contained 2 ml of the inhibitor solution.Acetone powders were prepared from the root discs by cutting the tissue into small pieces and placing 50-g tissue in 500 ml of acetone previously chilled to -18 C. The acetone was changed twice within a period of 1 hr. The tissue was then homogenized for 3 min, filtered through a Whatman No. 1 filter paper in a Buchner funnel, and rinsed with two 500-ml portions of chilled acetone. Mats were dried for 1 hr at room temperature, then kept overnight under vacuum, and stored at -18 C.PAL activity in acetone powders was assayed by the method of Rahe et al. (14) with slight modifications. PAL was extracted by suspending 0.5 g of powder in 10 ml of cold 0.1 M borate buffer, pH 8.8. The suspension was kept in an ice bath for 1 hr with occasional stirring and centrifuged at 40,000g for 20 min at 1 C. The supernatant served as the enzyme preparation. For the assay, 1.5 ml of the supernatant was added to a test tube containing 1.0 ml of 0.05 M L-phenylalanine and 2.5 ml of 0.1 borate buffer, pH 8.8. The assay mixture was incubated in a water bath at 40 C for 1 hr, the reaction was stopped by the addition of 0.1 ml of 5 N HCl, and the acidified mixture was extracted with 7 ml of ether. Two and one-half ml of the ether phase were evaporated to dryness, and the residue was dissolved in 2.5 ml of 0.05 N NaOH. The concentration of cinnamic acid was determined by absorbance at 269 nm, with 0.05 N NaOH as reference. Two controls were run in each assay; a no-substrate assay in which 1.0 ml of deionized water was substituted in the reaction mixture for the L-phenylalanine solution and a zero time stop reaction assay mixture acidified at zero time of incubation. Both controls were assayed as described above. Values obtained from the zero time incubation were applied in the determination of PAL ac...

“…Intact and sliced carrot roots are known to show increases in isocoumarin synthesis (8) or phenylalanine ammonialyase activity (7) when treated with ethylene. The lack of peroxidase or phenolase reaction to ethylene in carrot, parsnip, or swede roots has been related to the very low level of phenolic compounds in these species (29,36).…”

Section: Discussionmentioning

confidence: 99%

Cell Wall and Protoplast Isoperoxidases in Relation to Injury, Indoleacetic Acid, and Ethylene Effects

Birecka¹,

Miller²

1974

In tobacco (Nicotiana Tabacum) pith, sweet potato (Ipomnoea batatas), and carrot (Daucus carota) storage roots, differences were found between cell wall and protoplast peroxidases in their isoenzyme patterns, activity, and reaction to tissue injury.In the pith of elongating tobacco internodes, 90% of total activity was associated with the walls, 80% of which was due to the ionically and covalently bound fractions. With senescence, the increase in activity occurred mainly in the protoplast and the free fraction in the walls. The major protoplast isoperoxidases formed the free wail fraction, and the minor ones formed the wail bound fractions. The minor isoperoxidases were the only ones whose activities were affected by cut injury. Neither senescence nor injury caused qualitative changes in the cell isoperoxidase pattern. In the potato root cells, the isoperoxidases were also distinctive in their distribution and in their reactions to injury as well as to ethylene.In potato and carrot roots, the wall fractions contributed 30 and 95 %, respectively, to total peroxidase activity. The injury-induced increase in peroxidase activity occurred mainly in the protoplast of tobacco pith and potato roots and almost entirely in the ionically bound fraction of the walls in carrot roots. Ethylene stimulated peroxidase development in potato roots, especially in the protoplast, but had no effect in either tobacco pith or carrot roots. Ethylene did not affect the inhibiting action of indoleacetic acid on peroxidase activity in tobacco pith, nor did indoleacetic acid significantly affect the stimulating action of ethylene in potato roots.Actinomycin D inhibited the injury-dependent development of peroxidase in tobacco pith, stimulated the injury-and ethylene-dependent peroxidase development in potato root, and had no effect on peroxidase development in carrot root. Cycloheximide prevented the increase in peroxidase activity in tobacco pith and potato root, but not in the carrot root. Thus, the enhancement of peroxidase activity resulted from either enzyme activation exclusively or front enzyme synthesis. RNA synthesis seems to be only indirectly related to isoperoxidase synthesis caused bv injury or ethylene.Previous investigation (5) (3,11,25,31) and its possible involvement in cell wall lignification and extensibility has been discussed in detail (10,20,32,35). Therefore, we extended our comparative studies to include the cell wall isoperoxidase fractions. This report deals with the interaction between injury and IAA, cycloheximide, or actinomycin D in the development of the soluble and cell wall bound isoperoxidases in the presence or absence of added ethylene or in the presence of mercuric perchlorate used to remove endogenous ethylene. The reported lack of reaction of carrot root-soluble peroxidase to added ethylene, in contrast to sweet potato root (36), prompted us to include the carrot root in this study. Special attention has been paid to the free peroxidase fraction in tobacco pith cell walls, since a significant r...