Comparison of ripening processes in intact tomato fruit and excised pericarp discs

Physiological processes characteristic of ripening in tissues of intact tomato fruit (Lycopersicon esculentum Mill.) were examined in excised pericarp discs. Pericarp discs were prepared from mature-green tomato fruit and stored in 24-well culture plates, in which individual discs could be monitored for color change, ethylene biosynthesis, and respiration, and selected for cell wall analysis. Within the context of these preparation and handling procedures, most whole fruit ripening processes were maintained in pericarp discs. Pericarp discs and matched intact fruit passed through the same skin color stages at similar rates, as expressed in the L^*a^*b^* color space, changing from green (a^* < −5) to red (a^* > 15) in about 6 days. Individual tissues of the pericarp discs changed color in the same sequence seen in intact fruit (exocarp, endocarp, then vascular parenchyma). Discs from different areas changed in the same spatial sequence seen in intact fruit (bottom, middle, top). Pericarp discs exhibited climacteric increases in ethylene biosynthesis and CO₂ production comparable with those seen in intact fruit, but these were more tightly linked to rate of color change, reaching a peak around a^* = 5. Tomato pericarp discs decreased in firmness as color changed. Cell wall carbohydrate composition changed with color as in intact fruit: the quantity of water-soluble pectin eluted from the starch-free alcohol insoluble substances steadily increased and more tightly bound, water-insoluble, pectin decreased in inverse relationship. The cell wall content of the neutral sugars arabinose, rhamnose, and galactose steadily decreased as color changed. The extractable activity of specific cell wall hydrolases changed as in intact fruit: polygalacturonase activity, not detectable in green discs (a^* = −5), appeared as discs turned yellow-red (a^* = 5), and increased another eight-fold as discs became full red (a^* value +20). Carboxymethyl-cellulase activity, low in extracts from green discs, increased about six-fold as discs changed from yellow (a^* = 0) to red.     

Cell Wall Metabolism in Ripening Fruit (VII. Biologically Active Pectin Oligomers in Ripening Tomato (Lycopersicon esculentum Mill.) Fruits)

A water-soluble, ethanol-insoluble extract of autolytically inactive tomato (Lycopersicon esculentum Mill.) pericarp tissue contains a series of galacturonic acid-containing (pectic) oligosaccharides that will elicit a transient increase in ethylene biosynthesis when applied to pericarp discs cut from mature green fruit. The concentration of these oligosaccharides in extracts (2.2 [mu]g/g fresh weight) is in excess of that required to promote ethylene synthesis. Oligomers in extracts of ripening fruits were partially purified by preparative high-performance liquid chromatography, and their compositions are described. Pectins were extracted from cell walls prepared from mature green fruit using chelator and Na2CO3 solutions. These pectins are not active in eliciting ethylene synthesis. However, treatment of the Na2CO3-soluble, but not the chelator-soluble, pectin with pure tomato polygalacturonase 1 generates oligomers that are similar to those extracted from ripening fruit (according to high-performance liquid chromatography analysis) and are active as elicitors. The possibility that pectin-derived oligomers are endogenous regulators of ripening is discussed.     

Fruit-localized phytochromes regulate lycopene accumulation independently of ethylene production in tomato

We show that phytochromes modulate differentially various facets of light-induced ripening of tomato fruit (Solanum lycopersicum L.). Northern analysis demonstrated that phytochrome A mRNA in fruit accumulates 11.4-fold during ripening. Spectroradiometric measurement of pericarp tissues revealed that the red to far-red ratio increases 4-fold in pericarp tissues during ripening from the immature-green to the red-ripe stage. Brief red-light treatment of harvested mature-green fruit stimulated lycopene accumulation 2. 3-fold during fruit development. This red-light-induced lycopene accumulation was reversed by subsequent treatment with far-red light, establishing that light-induced accumulation of lycopene in tomato is regulated by fruit-localized phytochromes. Red-light and red-light/far-red-light treatments during ripening did not influence ethylene production, indicating that the biosynthesis of this ripening hormone in these tissues is not regulated by fruit-localized phytochromes. Compression analysis of fruit treated with red light or red/far-red light indicated that phytochromes do not regulate the rate or extent of pericarp softening during ripening. Moreover, treatments with red or red/far-red light did not alter the concentrations of citrate, malate, fructose, glucose, or sucrose in fruit. These results are consistent with two conclusions: (a) fruit-localized phytochromes regulate light-induced lycopene accumulation independently of ethylene biosynthesis; and (b) fruit-localized phytochromes are not global regulators of ripening, but instead regulate one or more specific components of this developmental process.     

Effect of alcohols and their interaction with ethylene on the ripening of epidermal pericarp discs of tomato fruit

Ethanol concentrations that were induced in pericarp discs of mature-green tomato fruit (Lycopersicon esculentum Mill, cv Castlemart) either by anaerobic metabolism or by exposure to ethanol vapor inhibited ripening without increasing the rate of ion leakage. Inhibition of ripening (i.e. lycopene synthesis) of excised tomato pericarp tissue by ethanol vapor was reversed by increasing concentrations of the plant hormone ethylene. A Lineweaver-Burk plot indicated noncompetitive interaction between ethanol and ethylene. Methanol and n-propanol also inhibited lycopene synthesis without significantly increasing ion leakage. The similar inhibitory effects of methanol, ethanol, and n-propanol at concentrations which did not stimulate ion leakage, and the relationship between activity and lipophilia of the alcohols suggest that their mode of action was through disruption of membranes associated with ethylene action.     

Glucose Inhibits ACC Oxidase Activity and Ethylene Biosynthesis in Ripening Tomato Fruit

Experiments were carried out to evaluate the effect of glucose on ripening and ethylene biosynthesis in tomato fruit (Lycopersicon esculentum Mill.). Fruit at the light-red stage were vacuum infiltrated with glucose solutions post-harvest and changes in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC, ACC oxidase, and ethylene production monitored over time. ACC oxidase activity was also measured in pericarp discs from the same fruits that were treated either with glucose, fructose, mannose, or galactose. While control fruit displayed a typical peak of ethylene production, fruit treated with glucose did not. Glucose appeared to exert its effect on ethylene biosynthesis by suppressing ACC oxidase activity. Fructose, mannose, and galactose did not inhibit ACC oxidase activity in tomato pericarp discs. Glucose treatment inhibited ripening-associated colour development in whole fruit. The extent of inhibition of colour development was dependent upon the concentration of glucose. These results indicate that glucose may play an important role in ethylene-associated regulation of fruit ripening.     

The Role of Polygalacturonase (PG) in Tomato Fruit Ripening and Effects of Divalent Metal Ions and Ethylene on PG Activity

It has been reported that PG is a key enzyme related to the tomato fruit ripening. In this study tomato fruits were harvested at the mature-green stage and stored at room temperature. The cell ultrastructure of pericarp tissue was observed at different ripening stages, and the effects of treatments with ethylene and calcium on PG activity and fruit ripening were examined. The object of this study is to elucidate the role of PG in regulation of tomato fruit ripening by ethylene and calcium. PG activity, was undetectable at mature-green stage, but it rose rapidly as fruif ripening. The rise in PG activity was coincided with the dechnmg of fruit firmness during ripening of tomato fruits. The observation of cell ultrastructure showed that the most of grana in chloroplast were lost and the mitochondrial cristae decreased as fruit ripening. Striking changes of cell wall structure was most noted, beginning with dissolution of the middle lamella and eventual disruption of primary cell wall. A similar pattern of changes of cell wall and chloroplast have been observed in pericarp tissue treated with PG extract. In fruits treated with calcium and other divalent metal ions atmature-green stage, the lycopene content and PG activity decreased dramatically. Ethylene application enhanced the formation of lycopene and PG activity. The inhibition of Ca2+ on PG ac ivity was removed by ethylene. Based on the above results, it was demonstrated that PG played a major role in ripening of tomato fruits, and suggested that the regulation of fruit ripening by ethylene and Ca2+ was all mediated by PG. PG induced the hydrolysis of cell wall and released the other hydrolytic enzymes, then effected the ripening processes follow up.     

PG在番茄果实成熟中的作用及二价金属离子与乙烯对PG活性的影响

对采后番茄果实的电镜观察表明:当果实成熟衰老时,叶绿体数量减少,多数基粒结构丧失;成熟果实胞壁中胶层水解成中空的电子透明区,初生壁的纤丝也发生一定程度的水解,相邻细胞分离;外源 PG(多聚半乳糖醛酸酶)提取物处理绿熟期果实组织,也可引起胞壁结构和叶绿体发生与正常衰老相同的变化。Ca~(2+)、Mg~(2+)、Co~(2+)二价金属离子处理果实,可明显降低番茄红素含量和 PG 活性,延缓果实软化。外源乙烯处理果实,可促进番茄红素的形成,提高 PG活性,并能解除钙对 PG 活性的抑制。本文也对 PG 在乙烯和 Ca~(2+)调节果实成熟中的作用进行了讨论。     

Acetaldehyde Is a Causal Agent Responsible for Ethanol-Induced Ripening Inhibition in Tomato Fruit

Inhibition of tomato (Lycopersicon esculentum Mill.) fruit ripening by exogenously applied ethanol was shown to be caused by elevated endogenous levels of acetaldehyde (AA). Exposure of excised pericarp discs of mature-green tomato fruit to ethanol or AA vapors produced elevated levels of both compounds in the tissue, but only the levels of AA were associated with ripening inhibition. Ripening inhibition was dependent on both the applied concentration and the duration of exposure. Discs treated with inhibitory levels of AA had levels of ethanol that were elevated but below that associated with inhibition of ripening. The in vivo activity of alcohol dehydrogenase was inhibited 40 to 60% by 4-methylpyrazole (4-MP), a competitive inhibitor of this enzyme. The inhibitory effect of ethanol on ripening was reduced by the simultaneous application of 4-MP. Tissue treated with 4-MP plus AA vapors had higher endogenous levels of AA and ripening was inhibited longer than in tissue without 4-MP. The tissue AA level resulting from ethanol or AA application appears to be the critical determinant of ripening inhibition.     

Wound ethylene and 1-aminocyclopropane-1-carboxylate synthase in ripening tomato fruit

Ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) levels and ACC-synthase activity were compared in intact and wounded tomato fruits (Lycopersicon esculentum Mill.) at different ripening stages. Freshly cut and wounded pericarp discs produced relatively little ethylene and had low levels of ACC and of ACC-synthase activity. The rate of ethylene synthesis, the level of ACC and the activity of ACC synthase all increased manyfold within 2 h after wounding. The rate of wound-ethylene formation and the activity of wound-induced ACC synthase were positively correlated with the rate of ethylene production in the intact fruit. When pericarp discs were incubated overnight, wound ethylene synthesis subsided, but the activity of ACC synthase remained high, and ACC accumulated, especially in discs from ripe fruits. In freshly harvested tomato fruits, the level of ACC and the activity of ACC synthase were higher in the inside parts of the fruit than in the pericarp. When wounded pericarp tissue of green tomato fruits was treated with cycloheximide, the activity of ACC synthase declined with an apparent half life of 30–40 in. The activity of ACC synthase in cycloheximide-treated, wounded pericarp of ripening tomatoes declined more slowly.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid     

5'-Methylthioadenosine nucleosidase and 5-methylthioribose kinase activities in relation to stress-induced ethylene biosynthesis

The activities of 5'-methylthioadenosine (MTA) nucleosidase (EC 2.2.2.28) and 5-methylthioribose (MTR) kinase (EC 2.7.1.100) were related to changes in ethylene biosynthesis in tomato ( Lycopersicon esculentum Mill. cv. Rutgers) and cucumber ( Cucumis sativus Mill. cv. Poinsett 76) fruit following wounding and chemically induced stresses. Stress ethylene formation in wounded tomato and cucumber tissue continued to increase after wounding, reached its peak by 3h, and then declined. The activities of MTA nucleosidase and MTR kinase increased parallel to stress ethylene in both tissues. At peak ethylene formation, MTA and MTR kinase activities were 2- to 4-fold higher in wounded than in intact tissue. Wounded, mature-green tomato tissue treated with specific inhibitors of MTA nucleosidase and MTR kinase showed a significant reduction in the activities of these enzymes, which was concomitant with a decline in stress ethylene biosynthesis. When mature-green tomato discs were infiltrated with [¹⁴CH₃] MTA and wounded, radioactive MTR and methionine were formed. Incubation of mature-green tomato discs with Cu²⁺ and Li⁺ in the presence of kinetin increased ethylene biosynthesis. MTA nucleosidase activity was higher than that of the control in the presence of Cu²⁺ but not in the presence of Li⁺, while MTR kinase activity was lower than that of the control in both Cu²⁺ and Li⁺ treatments. Data indicate that MTA nucleosidase and MTR kinase are required for wound-induced ethylene biosynthesis but not for chemical stress-induced ethylene by Cu²⁺ or Li⁺ treatments.     

Transient increase in locular pressure and occlusion of endocarpic apertures in ripening tomato fruit

Locular pressure was monitored during ripening of tomato (Lycopersicon esculentum Mill.) fruit and the anatomy of the endocarp surface examined using scanning electron microscopy. The manometric pressure of the locule tissue increased from 0 in mature-green fruit to 10 to 50 Pa at the turning or pink stages, and then subsided in ripe fruit. Nonclimacteric fruit containing the ripening inhibitor (rin) mutation showed a similar pattern of internal pressure accumulation during senescence. Build-up of locular tissue pressure occurred in fruit ripening, on or off the plant, as well as in fruit with different susceptibility to cuticle cracking. Apertures ranging from 18-31 μm in width and 33-41 μm in length, with densities ranging from 6.7 to 47.9 apertures · mm⁻² were observed in the endocarp of mature-green fruit. These apertures were progressively occluded during early ripening and were absent in late ripening fruit. Aperture occlusion might result in reduced gas exchange between the locule and external fruit atmosphere, resulting in modification of the locular gas composition.     

Gibberellic acid,synthetic auxins,and ethylene differentially modulate alpha-L-Arabinofuranosidase activities in antisense 1-aminocyclopropane-1-carboxylic acid synthase tomato pericarp discs

Alpha-L-Arabinofuranosidases (alpha-Afs) are plant enzymes capable of releasing terminal arabinofuranosyl residues from cell wall matrix polymers, as well as from different glycoconjugates. Three different alpha-Af isoforms were distinguished by size exclusion chromatography of protein extracts from control tomatoes (Lycopersicon esculentum) and an ethylene synthesis-suppressed (ESS) line expressing an antisense 1-aminocyclopropane-1-carboxylic synthase transgene. alpha-Af I and II are active throughout fruit ontogeny. alpha-Af I is the first Zn-dependent cell wall enzyme isolated from tomato pericarp tissues, thus suggesting the involvement of zinc in fruit cell wall metabolism. This isoform is inhibited by 1,10-phenanthroline, but remains stable in the presence of NaCl and sucrose. alpha-Af II activity accounts for over 80% of the total alpha-Af activity in 10-d-old fruit, but activity drops during ripening. In contrast, alpha-Af III is ethylene dependent and specifically active during ripening. alpha-Af I released monosaccharide arabinose from KOH-soluble polysaccharides from tomato cell walls, whereas alpha-Af II and III acted on Na(2)CO(3)-soluble pectins. Different alpha-Af isoform responses to gibberellic acid, synthetic auxins, and ethylene were followed by using a novel ESS mature-green tomato pericarp disc system. alpha-Af I and II activity increased when gibberellic acid or 2,4-dichlorophenoxyacetic acid was applied, whereas ethylene treatment enhanced only alpha-Af III activity. Results suggest that tomato alpha-Afs are encoded by a gene family under differential hormonal controls, and probably have different in vivo functions. The ESS pericarp explant system allows comprehensive studies involving effects of physiological levels of different growth regulators on gene expression and enzyme activity with negligible wound-induced ethylene production.     

Induction and regulation of ethylene biosynthesis by pectic oligomers in cultured pear cells

Pectic oligomers induced a rapid, transient increase in ethylene biosynthesis when added to pear cells in suspension culture. The rate of ethylene biosynthesis increased within 30 to 40 minutes after oligomer addition, reached a maximum between 90 and 120 minutes after addition, and then decreased to basal rates of synthesis. Both the rapid increase and decrease in biosynthesis appear to be precisely regulated components of the ethylene response to oligomers. Induction of ethylene biosynthesis by pectic oligomers resulted in a reduced sensitivity of cells to further ethylene induction. This reduction in sensitivity occurred within 90 minutes after an oligomer treatment, slightly preceding the decline in ethylene synthesis. The degree of insensitivity induced was proportional to the concentration of oligomer in the first treatment. Induced insensitivity to elicitors appears to represent a novel mechanism which may limit continued ethylene biosynthesis after ethylene induction. Ethylene was produced by pear cells throughout the cell growth cycle, as cells increased in density over a 6 day period. Endogenous ethylene biosynthesis was at a maximum during the first 4 days of rapid cell growth, then declined to half the peak rate through day 10. Pectic oligomers could induce an increase in ethylene biosynthesis above this background rate only after day 5, as endogenous biosynthesis declined. Changes in sensitivity to added oligomer during the growth cycle may result from insensitivity to elicitors induced by growth processes.     

Effect of pectic oligomers on physiological responses of chilling injury in discs excised from zucchini (Cucurbita pepo L.).

The effect of pectic oligomers (OG) on ethylene biosynthesis, electrolyte leakage (EL), and CO(2) production was studied in discs excised from zucchini fruit (Cucurbita pepo L.) and stored at 20 or 2.5 degrees C. At 20 degrees C, OG enhanced ethylene biosynthesis and had a transient effect on decreasing EL, but showed little effect on respiratory rate; both the amount and size of the oligomer were important in changing both ethylene synthesis and EL. At 2.5 degrees C, OG increased both ethylene biosynthesis and respiratory rate with a maximum effect at 100 microg of oligomer and peaking at 6 h; shorter oligomers demonstrated an even greater effect on ethylene biosynthesis, but differences were smaller in respiratory rate. EL at 2.5 degrees C was affected most by 1 microg of OG and by monomeric galacturonic acid, with transient increases that peaked at 8 h. We suggest a signaling role for OG in the early steps of cold acclimation or chilling injury.     

Effect of silver ions on ethylene biosynthesis by tomato fruit tissue

Mature-green tomato fruit (Lycopersicon esculentum Mill.) were treated asymmetrically with 2 millimolar silver thiosulfate (STS) through a cut portion of the peduncle while still attached to the plant. One-half of the fruit received silver and remained green while the other half ripened normally and was silver-free (less than 0.01 parts per billion). Harvested mature-green fruit were also treated with STS through the cut pedicel. Green tissue from silver-treated fruit had levels of 1-aminocyclopropane-1-carboxylic acid (ACC, the immediate ethylene precursor) slightly less or similar to that of turning or red-ripe tissue from the same fruit, and similar to that of mature-green tissue from control fruit. Ethylene production was higher in green tissue from silver-treated fruit than from either red tissue from the same fruit, or mature-green tissue from control fruit. By inhibiting ACC synthesis with aminoethoxyvinyl glycine, and by applying ACC ± silver to excised disks of pericarp tissue from control or silver-treated tomatoes, we showed that short-term silver treatment did not affect the biological conversion of ACC to ethylene, while long-term treatment stimulated both the conversion of ACC to ethylene and the synthesis of ACC.     

Methyl bromide inhibits ripening and ethylene production in tomato (lycopersicon esculentum mill.) fruit

Tomato fruit ripening and ethylene production were inhibited following treatment with methyl bromide (MB). Methyl bromide significantly delayed ripening initiation in mature-green (MG) fruit and retarded the rate of ripening of turning (T) fruit as measured by color development and flesh softening. Treatment with MB caused an initial transient burst of ethylene production, but the subsequent ripening-associated increase in ethylene was delayed. Ethylene treatment partially overcame MB inhibition in MG fruit but had no affect on T fruit. The inhibition of ethylene production by MB appears to be due to lack of formation of 1-aminocycloprone-1-carboxylic acid (ACC) in MG fruit, whereas in T fruit lack of conversion of ACC to ethylene is indicated. A key feature of MB inhibition of ripening in tomato appears to be reduced sensitivity to ethylene.     

Apoplastic pH and inorganic ion levels in tomato fruit: A potential means for regulation of cell wall metabolism during ripening

Apoplastic pH and ionic conditions exert strong influence on cell wall metabolism of many plant tissues; however, the nature of the apoplastic environment of ripening fruit has been the subject of relatively few studies. In this report, a pressure-bomb technique was used to extract apoplastic fluid from tomato fruit ( Lycopersicon esculentum Mill.) pericarp at several developmental stages. pH and the levels of K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl⁻ and P were determined and compared with the values for the bulk pericarp and locule tissues. The pH of the apoplastic fluid from pericarp tissue decreased from 6.7 in immature and mature-green fruits to 4.4 in fully-ripe fruit. During the same period, the K⁺ concentration increased from 13 to 37 m M . The levels of Na⁺ and divalent cations did not change, whereas the anions P and Cl⁻ increased in ripe fruit. Ca²⁺ levels remained relatively constant during ripening at 4–5 m M , concentrations that effectively limit pectin solubilization. The electrical conductivity of the apoplastic liquid increased 3-fold during ripening, whereas osmotically active solutes increased 2-fold. Pressure-treated fruit retained the capacity to ripen. The decline in apoplastic pH and increase in ionic strength during tomato fruit ripening may regulate the activity of cell wall hydrolases. The potential role of apoplastic changes in fruit ripening and softening is discussed.     

Application of CP-MAS and liquid-like solid-state NMR experiments for the study of the ripening-associated cell wall changes in tomato

¹³C and ¹H NMR spectra of an ethanol insoluble material (EIM) prepared from the pericarp of mature-green (MG) and red-ripe (RR) tomato fruits were acquired in ‘liquid-like’ and cross-polarisation with dipolar decoupling and magic angle spinning (CPMAS) conditions using the same triple resonance probe. Such a strategy allowed acquisitions of various NMR experiments aimed at detecting compositional differences as well as distinguishing differences in molecular mobility for various constituent polysaccharides related with the two ripening stages. Increase of the proton dipolar decoupling power levels from 3 to 50–55 kHz during single pulse ¹³C acquisition, led to more intense signals for pectic and hemicellulosic polysaccharides. This behaviour was interpreted as reflecting motional restrictions of these polysaccharides inside the porous cell wall network. Measurements of the proton rotating frame relaxation times T_1ρ in the ‘liquid-like’ conditions and of the proton transverse relaxation times T₂ from CPMAS spectra, revealed changes in mobilities for some pectic polysaccharides in relation with ripening, particularly for the H1 and H5 protons of -1,5 arabinan (Ara) side chains of rhamnogalacturonans. These data are discussed in relation with known pectic modifications occurring during ripening and associated with the tomato fruit softening.     

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.