Integrated relationships of biochemical and physiological peroxidase activities

Proceedings Chapter

Reference

Integrated relationships of biochemical and physiological peroxidase activities

GASPAR, Thomas

Abstract

For a plant physiologist who is not initiated in the field, the term "peroxidases" still represents a true morass. The points which cast a certain doubt as to peroxidases really playing a causal role in developmental physiological processes are reviewed. These points are being argued taking into consideration some recent developments in the knowledge of the properties of these particular enzymes. A place and a role for some peroxidases in a general sequence of reactions which, at different degrees, would be operating in the physiology of plant growth and development, are proposed.

GASPAR, Thomas. Integrated relationships of biochemical and physiological peroxidase

activities. In: Hubert Greppin, Claude Penel & Thomas Gaspar. Molecular and physiological aspects of plant peroxidases . Genève : Université de Genève, Centre de botanique, 1986. p.

455-468

Available at:

http://archive-ouverte.unige.ch/unige:100798

Disclaimer: layout of this document may differ from the published version.

1 / 1

Th.GASPAR

Hormonologie fondamentale et appliquee,

Institut de Botanique B22, Universite de Liege

Sart tilman, B-4000 Liege, Belgium, and University of Geneve, Switzerland

Abstract

For a plant physiologist who is not initiated in the field, the term "peroxidases" still represents a true morass. The points which cast a certain doubt as to peroxidases really playing a causal role in developmental physiological processes are reviewed. These points are being argued taking into consideration some recent developments in the knowledge of the properties of these particular enzymes. A place and a role for some peroxidases in a general sequence of reactions which, at different degrees, would be operating in the physiology of plant growth and development, are proposed.

Introduction

"It is probably true to say that among all the enzymes few have attracted more attention than has peroxidase. This widely distributed enzyme has claimed the attention of physical chemists, organic chemists, biochemists and physiologists for many years. Several thousand papers have been published on the nature of the enzyme, its mode of action and probable function, and each year the addition to this literature concerning peroxidase and related haematoporphyrin catalysts is considerable. It may be said with justification that

our present knowledge is very imperfect in many substantial ways and in this state of uncertainty it may well be argued that this is not yet the time for a review on peroxidase''.

These preface sentences are from Saunders, Holmes-Siedle and Stark, the authors of the book "PEROXIDASE" (a versatile enzyme) published in 1964 (19). The authors, however, took the opposite view namely that an overall assessment of the state of knowledge might serve a useful purpose at that time.

Twenty years later, through this symposium, we exactly had the same attitude.

It remains that for a botanist, e.g.

initiated in the field, the term plant physiologist, non­

"(iso)peroxidases" still represents a true morass for some main reasons:

the large and variable number of peroxixase depending on both the plant material and the techniques used as well;

isoforms, separation - their apparent localization in any part and organelle of the cell;

- the incredible number of "substrates": if the oxidant is usually hydrogen peroxide, in certain circumstances, other oxidizing agents may be employed. Although most typical peroxidases catalyse the oxidations of a fairly restricted number of classes of organic compounds including phenols; aromatic primary, secondary and tertiary amines; leuco-dyes; certain heterocyclic compounds (e.g. ascorbic acid and indole); and certain inorganic ions, particularly the iodide ion, the range of hydrogen donors capable of undergoing oxidation in the presence of a peroxidase system becomes especially large if coupled oxidations are also considered;

the variation of peroxidases (number, activity) with physiological processes examined and their response to any change in the chemical, physical or biological factors of the plant environment;

- the existence of numerous forms (compounds I, II, III, ... ) of peroxidase and the different mechanisms of action (11).

It is clear that peroxidase, although apparently primarily somewhat specific in its initial reactions, can promote a large variety of consequent reactions and therefore can exhibit a degree of versatility not surpassed by any other single enzyme.

Up to recently, these points really cast a certain doubt as to peroxidases really playing a causal role in developmental physiological processes. Some of them are argued as simply as possible here below. A general sequence of reactions indicating a place and a role for some peroxidases is also proposed.

Preliminary advertisement

Before going through the various steps in the control of the

level of peroxidase activity in plants, it must be stated that the simple measure of peroxidase activity in crude extracts often is a misleading reflection of the actual number of peroxidase molecules really present in the tissue before extraction. Plant cells, indeed, contain several substances (ions, phenolics and others) which modify the abil�ty of peroxidases to oxidize the electron donors generally used to detect their activity. These compounds, which probably are separated from peroxidases in whole cells as a result of different compartmentalization, are mixed with the enzymes when cells are broken and interact with the enzymes during the assay.

A variation of peroxidase activity, extract, thus has no longer any following precisions.

measured in a single crude significance without the - Does the variation result from

= an inactivation or activation of pre-existing molecules?

a change in the rate of synthesis of the enzymes, including a post-translational control?

= the transfer of molecules from one cell compartment to another one?

= a simple change of substrate specificity?

- Which isoforms are concerned?

Which is the kinetics of the variation in the course of the physiological process or how late does it occur after a given stimulus?

At which place does the peroxidase variation occur (in the signal percepting organ or elsewhere at some distance) and which kind of cells are concerned, ... ?

Checking the significance of this enzyme variation of course requires the measure of the variation of the concerned endogenous natural substrates and / or the expected products formed.

Answering these questions is not mind the recent development of structure, biosynthesis, isoenzymes, activity.

possible without having in our knowledge on peroxidase localization and control of

Chemical structure, biosynthesis and transport

Complete amino acid sequence for some plant peroxidases has been determined (23). Besides the hemin prosthetic group, the analysis of HRP C indicates 2 ca²⁺ and 308 amino acid residues, including 4 disulfide bridges, in a polypeptide chain that carries 8 neutral carbohydrate side-chains for a native molecular weight close to 44,000. Calcium particularly contributes to maintaining the structural conformation of the protein. Free calcium readily exchanges with some isoenzymes. Some isoenzymes would not contain carbohydrate.

Our knowledge of the biosynthesis steps of peroxidase has been

reviewed (11, 22). The studies of peroxidase synthesis at the transcriptional level in their actual development do not lead to definitive conclusions. It appears however that the life of messenger RNA would be extremely variable (from 1.5 hours to days or months). Synthesis of the peroxidase protein moiety is classically cycloheximide dependent. An additional key factor in the control of peroxidase synthesis could be the availability of heme which itself could be dependent upon porphyrin metabolism and the synthesis of other porphyrin molecules, such as chlorophyll and cytochromes.

Post-translational controls of activity of peroxidase molecules are likely to involve several cations (among which calcium certainly plays the main role) and incorporation of the sugar residue, for some isoperoxidases at least.

Finally, the transport of peroxidases towards their actual site of action may also be considered as a control of peroxidase activity (18). Cytochemical studies have shown that the final locations of peroxidases are likely to be either cell wall and intercellular free spaces or vacuoles.

As in animal cells, peroxidase activity is found in cisternal space of rough endoplasmic reticulum and in stocked Golgi cisternae. It is also present in transport vesicles which probably are issued from the endoplasmic reticulum and fused with Golgi cisternae. There are numerous tubular connections between the cisternae of the endoplasmic reticulum and the Golgi apparatus. Therefore, the structure also exists for a direct transport between these two membranous systems, without the migration of vesicles. Release of peroxidase occurs by exocytosis. Since peroxidase reaction product remains membrane - bound in all compartments participating in the secretory process until its release by exocytosis, an alternative way of transport of this enzyme could well be in association with the migration of membranes from endoplasmic reticulum towards Golgi apparatus and then to plasmamembrane. This membrane flow implies a progressive biochemical transformation of membranes from endoplasmic reticulum-like to plasmamembrane - like (18). Vesicles from the Golgi apparatus are partially differentiated into plasmalemma before they fuse with the plasmamembrane. By this way, the secretory products enclosed in vesicles or bound to their membranes are moved to the cell exterior.

Although the migration of proteins synthesized in endoplasmic reticulum, through Golgi towards the exterior of cell after exocytosis is likely to occur, discharge by direct fusion of endoplasmic reticulum to plasmalemma or through vesicles directly derived from endoplasmic reticulum, and direct transfer of cytoplasmic enzymes across plasmalemma as well cannot be excluded (18).

Isoenzymes and control of activity

Electrophoresis, ion exchange chromatography and isoelectric focusing of enzymatic extracts from different higher plants source materials separate two groups of peroxidase isoenzymes:

the basic and acid ones. Although the number of isoenzymes obtained is dependent largely on the separation technique used and notwithstanding the number of artefacts which cast a certain amount of doubt as to them being all native molecules, the chemical constitution and properties of peroxidases of these two groups clearly appear to be different (11 ) .

Inhibitors of protein synthesis suppress the physiological responses to different stimuli (11 ) , but generally fail to prevent the rapid increase in activity of the basic peroxidases.

Furthermore, these enzymes do not incorporate labelled protein precursors in response to short radioactive pulse that readily label other types of proteins and enzymes (10). The rapid increase in basic peroxidase activity would thus not result from de novo protein synthesis but from the activation of enzymes already present in the tissues. Experiments done on wounded and mechanically irritated materials indicate that basic peroxidases, not revealed in crude extracts were, nevertheless, present in the cells and in the free space already before treatment, although they were masked in vitro by unknown compounds. The enhanced activity of these basic peroxidases in the crude extract, as a result of the treatments, appears as a demasking effect due to a rapidly decreased level of the masking substance. Some phenolic compounds might act as masking substances in crude extracts since their removal by PVP considerably enhances basic peroxidase activity, and since a decreased level of phenolics follows immediately after the stressing treatments. Moreover, the level of phenolics compounds and the activity of basic peroxidases show an inverse relationship in the course of various processes (10 ) . However, extracellular peroxidase activity does increase after the appropriate stimuli, independently of the presence of masking substances. The in vivo regulation of peroxidase activity by phenols, however, will remain an open question until the sub­

cellular localization of both types of substances and their possible connections are elucidated.

Rapid increase of basic peroxidase activity in the free space of differently treated materials indicates a possible stimulus control of active cellular secretion as shown in cell suspension cultures (10 ) . This rapid activation of a secretory process has now been confirmed and shown to be under the control of the intracellular ca²⁺ content, in experiments using ca²⁺, chelators, and ionophores (17). The ca²⁺ - controlled activation of basic peroxidase secretion could be initiated by a primary, stimulus­

induced release of K⁺. The Li⁺ inhibition of several stress-

mediated physiological responses and its are in agreement with this hypothesis.

intracellular Ca²⁺ level or its ratio to K⁺ factor is as yet unknown.

reversal by potassium Whether the absolute is the determining ca²⁺ also promotes the binding of basic peroxidases to membranes and this ca²⁺_mediated binding might be related to the secretion of these enzymes (17, 18). In addition, some elements exist for substantiating the hypothesis that phytohormones regulate protein secretion through the mediation of Ca - ATPase and a second messenger such as c - AMP or ca²⁺ / calmodulin.

However, the existence of an exchange mechanism between H⁺ and ca²+ suggests that plant growth regulators especially auxin could modify the secretory processes by a modification of the distribution of protons which indirectly affect ca²+ compartmentation. It is furthermore interesting to note that secretion of basic peroxidases into the free space as a result of ozone treatment may be accompanied by a simultaneous release of at least one of its natural substrates (e.g. ascorbic acid in Sedum), and that these basic peroxidases exhibit a much higher affinity towards the substrate released (up to 6 times) than the acidic isoenzymes (3, 10). It should be determined to what extent the external stimuli that trigger peroxidase secretion might modulate this affinity (see below for acid peroxidases). IAA is an other substrate whose secretion is under the control of calcium (6). Another possible type of control of peroxidase activity could be through a direct effectory action of ca²+ ( 17). ca²+ would principally activate basic peroxidases, but an effect on acid isoenzymes is not excluded. Actually ca²⁺ is necessary for the correct conformation of the peroxidase apoprotein (23).

In contrast to the basic peroxidases, the acidic ones rapidly incorporate labelled amino acids, indicating a higher turnover rate. The level of incorporation is increased in response to factors favouring enhanced activity of the acidic peroxidases.

There is thus a good probability that genetically regulated protein synthesis is involved in their control. The extent of the secretion of acidic peroxidases would be more dependent on the nature of the physiological process than this of basic

peroxidases which seems to be generalized.

Roles of basic and acidic peroxidases

Arguments for the roles of basic and acidic peroxidases, viz.

in auxin catabolism and lignification, respectively, were developed in a previous review (11). More recent experimental data allow further insights into these roles and the mechanisms involved. The evidence cited above concerning the controlled secretion of basic peroxidases into the free space raises the

question of the specificity of this compartment as a preferential site for interaction between these isoenzymes and IAA, since a main pathway for transport/circulation of the hormone would precisely be the free space (10). The question whether the basic peroxidase-mediated auxin catabolism occurs solely outside the protoplasts or not remains to be answered.

One should remember that peroxides are the true substrates for peroxidases so that the simultaneous release of basic peroxidases and ascorbic acid into the free space in response to pollutants (3), which induce the formation of superoxide ions and different perhydroxylic radicals might be interpreted as a detoxifying role played by the enzymes.

Without specification of its nature, a role for peroxidase as ACC-oxidase in the conversion of ACC to ethylene has recently been critically examined without a final decision (1).

Notwithstanding the question of the stereochemical reliability of cell-free ethylene-forming systems, there is a good correlation between the activity of membrane-bound peroxidases and ethylene production. Data from in vitro conversion of ACC to ethylene using membranes to which basic or acidic peroxidases were previously bound, indicate the participation of the basic isoenzymes only (unpublished results). Association of these basic peroxidases to membrane structures and the need for manganese as an activating ion seem to be prerequisites for obtaining an ACC­

oxidase activity.

The possible roles for peroxidases in cell wall genesis have been reviewed (4, 15). One may consider that peroxidases play a key role in the overall process of cell wall edification and lignin formation namely by generating HzOz necessary for oxidation and polymerization of cinnamyl alcohols, oxidizing cinnamyl alcohols to phenoxy radicals with the rapid formation of oligomers, converting ferulic to diferulic acid, which can act as a hemicellulose cross link, · binding cinnamic acid to wall proteins or carbohydrates and polymerizing cinnamyl alcohols in walls. The variation in the level of acidic peroxidases is correlated with lignification as shown by the use of syringaldazine as a specific substrate for lignifying peroxidases (4). Basic and acidic peroxidases are found in soluble, membrane, wall-ionic and wall-covalent cell fractions, but in each fraction the acidic ones show the highest affinity to syringaldazine.

Acidic peroxidases are secreted and activated by ca²⁺ in the free space, where they would rapidly meet their substrates since enzyme exocytosis is paralleled by an efflux of phenolics which could be the lignin precursors. Tissue cicatrisation after wounding and suberization would also be achieved by acidic peroxidases.

Fig. 1.

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VITRIFICATION

Hypothetical sequence of reactions leading to t^ritrification.

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physiological processes basic and acidic peroxidases A cascade variation of

invariably observed while relation to chemically and

basic and acidic peroxidases studying isoperoxidase changes physically controlled growth

in

was in developmental processes in our laboratories and in others (2, 3, and 8, 10, 12, 16). For instance, the thigmomorphogenetic response (response to mechanical stimulus such as rubbing, brushing) is typically a rapid reduction of growth due to inhibition of cell division and elongation and stimulation of radial growth with a progressive lignification of the whole tissue (2). Such growth inhibition and lignification processes have been observed in response to polluting agents (3). In both examples, an increase in activity of only the basic isoperoxidases is measured minutes after the treatment, followed a few hours later by a progressive increase of activity of the acidic ones.

Conversely, hyperhydric malformations in the so-called vitrified tissues apparently result from a deficiency of cell wall rigidification and of lignification (13). The process is preceded by an increased activity of basic isoperoxidases fol lowed by a decreased activity of the acidic ones (12, 13).

Root formation in cuttings or explants from different sources occurs as the result of successive inductive and initiative phases (8) characterized first by increased and subsequently by decreased activity of basic peroxidases, whereas the activity of acidic peroxidases regularly increased during both periods. Xylem formation starts with initiation of root primordia at the beginning of the initiative phase.

These examples illustrate the two-step sequential response of basic and acidic peroxidases irrespective of the chemical and/or physical stimulus. They also indicate that an increased basic peroxidase activity is not aut�atically followed by an increased acidic peroxidase activity or the reverse but that enhanced rigidification of parenchymatous or xylem cells occurs upon enhanced activity of the acidic peroxidases.

A common pathway for different physiological processes?

Plant reactions, in which the two-step control of basic and acidic peroxidases were observed, involved processes of growth and development. A common feature of these physiological processes is lignification, which is enhanced except in vitrification. Normal developmental processes such as rhizogenesis necessarily demand lignification for xylem formation. Growth processes also

lignin plays an important role through rigidification. It is

include cell wall changes where in addition to cellulose, mainly also likely that peroxidases are

responsible for the assembly of polymers (lignin, polysaccharides and proteins ) in the cell wall and, therefore, are involved in the differentiation processes (4, 15).

In all of these processes the intermediate role of auxin is generally acknowledged. Although recognized later as a hormone, ethylene is also assumed to play a determining role in the same phenomena. Auxin-peroxidase--ethylene mediated perturbation of the normal lignification process is involved in the formation of local necrotic lesions, growth and morphogenetic changes in response to various physical, chemical (in vitro rooting after auxin application might be considered as a response to a chemical stress) and biological stresses. In a manner quite similar to that used for the study of the thigmomorphogenetic process (1, 2, 5), the phenomenon of vitrification of tissues cultured in vitro was investigated in detail (9, 12, 13, 14). The kinetical analysis of the results led to propose the sequence of reactions of Figure 1.

Vitrification is considered as a morphological response to non-wounding stress conditions, for instance waterlogging and an excess of some mineral ions or cytokinins. Such stresses are known to mediate rapid bursts in ACC, ACC-oxidase, and ethylene together with an inhibition of elongation and with lateral expansion. The gas would subsequently, by feedback inhibition, reduce its own synthesis by repressing the formation of ACC­

synthase and by stimulation of the conversion of ACC to malonyl­

ACC. The decreased activities of acidic peroxidases and of PAL would be a consequence of decreased ethylene production and would explain the reduced cell wall rigidification and lignification in vitreous plants, which would allow a higher turgescence of the cells. The early burst in ACC level would result from the stress­

mediated rapid increase in basic peroxidases functioning as IAA­

oxidases. The resulting reduction in !AA content could enhance the ACC-synthase system and hence the ACC-level. Membrane-bound basic peroxidases and/or the soluble ones might in parallel function as ACC-oxidases which again would result in enhanced ethylene release. A rapid demasking and activation of basic peroxidases would thus initiate the process of vitrification in the soluble part of the cytoplasm and the membrane without intervention of nucleic acids. The subsequent increase in activity of ACC-synthase, acidic peroxidases and PAL probably is the result of de novo protein synthesis in response to the stresses induced in culture.

This sequence of reactions might be a general one since it has been found in response to different types of physical and chemical stimuli (10 ) . Figure 2 shows that these stimuli would cause an immediate redistribution of electrochemical potentials at the membrane level and generate different kinds of free radicals, for instance peroxide radicals, which in turn initiate

lipid peroxidation. Ethane production is a marker of this process. The degradation of lipoprotein cell membranes by lipid peroxidation induced by free radical reactions may bring about changes in ionic status and fluxes at the plasmalemma level and allow the passage of solutes such as phenolics, ascorbic acid, and even IAA (see above), most of which are electron donors for peroxidase. Release of K⁺ in particular, results in a modified endogenous ca²⁺ relative level, which regulates both the secretary process and the binding of basic peroxidases to membranes. Fatty acids may contribute both to peroxidase activation and to conformational changes resulting in additional strong binding sites. Using free peroxide

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Fig. 2. Suggested common pathway of reactions in some cell compartments in response to different physical and chemical stimuli (vertical arrow). It shoh'S the two-step control of basic and acidic peroxidases and their interdependent roles in cell wall rigidification through the mediation of ethylene. Arrow along bottom of fig·ure indicates the time spans. (Repri11ted from paper 10 with the permission of Physiologia Plantarum).

radicals,

and among the basic peroxidases might attack the electron donors them IAA and/or ACC. As ACC-oxidases, the membrane- bound peroxidases would directly regulate ethylene production,

the limiting factor being the immediately available ACC pool, although ca²+ may also have a stimulating effect on ACC production (7). The free IAA level resulting from the so-called IAA-oxidase activity of peroxidases (whatever the mechanism involved, see 11) would also regulate the ACC level through a mediated control of the conversion of SAM to ACC. Ethylene, in turn, might be a feedback controller of its own synthesis and of the peroxidases involved in IAA degradation, judging from how peroxidase changes under the effect of ethylene. However, the main role of ethylene would be to regulate PAL and acidic peroxidases thereby controlling also the lignification process (20) . The indirect but central role of Ca²+ in these processes is indirectly supported by the stress-protective effect of polyamines that inhibit the binding of peroxidases to membranes by ca²+. Polyamines have indeed been shown to induce release of ca²⁺ from whole cells (10).

In other words, growth reaction in response to any stress involves a rapid activation of basic peroxidases as a first step.

The changes brought about in auxin and ethylene metabolisms generally induce enhanced acidic peroxidase synthesis in a second later step. But by doing so, the basic peroxidases act as detoxifying agents for the peroxides formed in a sort of homeostatic reaction. We must wonder whether this is not the true role of peroxidases in the presence of peroxide or peroxide radicals, that is their natural substrates. IAA and ACC could have been destroyed by accident. Growth and developmental processes in response to stimuli would finally be the consequence of the detoxication effect of peroxidases against the peroxides formed.

So peroxidases appear to be

of the whole plants or key molecules in a rapid adaptation of some of their organs separately to changes in their environment.

The metabolism presented in the scheme may, therefore, be involved in growth processes in general. It actualizes the well­

known role of auxin in cell differentiation, showing its indirect way of action through ethylene. Auxin and ethylene are not implied as circulating hormones but also as regulatory intermediates at any point in the plant. This falls within the scope of the actual reconsideration of their hormonal status

( 21) .

References

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2 BOYER N, MO DESB IEZ, M HOF INGER, Th GASPAR 1983 Effects of lithium on thigmomorphogenesis in Bryonia dioica.

Ethylene production and sensitivity. Plant Physiol 72:

522-525

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British Plant Growth Regulator Group, Wantage, pp 39-49 9 GASPAR Th, C KEVERS, P DEBERGH, L MAENE, M PAQUES, Pb BOXUS

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cance for growth and development. Physiol Plant 64:

418-423

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gical roles in higher plants. Universite de Geneve­

Centre de Botanique

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69-74

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dases, phenylalanine ammonia-lyase, and lignin changes in relation to vitrification of carnation tissue cultured in vitro. J Plant Physiol 118: 41-48

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585-657

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Plant Cell Environ 4: 203-228

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Th Gaspar, eds, Molecular and Physiological Aspects of Plant Peroxidases. Universite de Geneve, pp 125-129 23 WEL INDER KG, L NORSKOV-LAURITSEN 1986 Structure of plant

peroxidases. Preliminary fitting into the molecular model of yeast cytochrome £ peroxidase. In H Greppin, C Penel, Th Gaspar, eds, Molecular and Physiological Aspects of Plant Peroxidases. Universite de Geneve, pp 61-69