PHYSIOLOGICAL ROLE OF PHENOLICS

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ZESZYTY PROBLEMOWE POSTĘPÓW NAUK ROLNICZYCH 2008 z. 524: 23-44

PHYSIOLOGICAL ROLE OF PHENOLICS

Agnieszka PłaŜek¹, Franciszek Dubert²

1 Department of Plant Physiology, Agricultural University, Kraków

2 Institute of Plant Physiology Polish Academy of Sciences, Kraków

Characteristics and classification of phenolics

Phenolics are secondary metabolites that contain phenol ring and at least one hydroxyl group. Moreover, the phenol ring is joined by other function groups such as carboxyl (-COOH) or methoxyl (CH₃O-). These substituents, mainly carboxyl and hydroxyl confer to phenolics polar (hydrophilic) character, and often water solubility [STRZAŁKA 2002].

In the literature one can find various classifications of these group compounds.

Apart from the above mentioned definition, chemical term phenylpropanoids exists based on the path of their biosynthesis [HAHLBROCK, SHEEL 1989]. Practically all phenolics belong to phenylpropanoids, however there are some exceptions: cinnamic acid is not a phenolic, but is included in phenylpropanoids, on the other hand, salicylic acid is a phenolic, while its chemical structure exludes it from phenylpropanoid group.

Taking into account that majority of phenolics belong to phenylpropanoids according to authors’ opinion, it is not big mistake to use both terms interchangeably. In the work term „phenylpropanoids” is consistently used.

Biosynthesis of phenylpropanoids in plants starts from the reaction of erytrose-4- phosphate with pyruvic acid and formation of dehydrochinoic acid. From this metabolite as a result of dehydration, the dehydroshikimic acid and finally the shikimic acids are synthesized. The last compound is the precursor of aromatic amino acids:

phenylalanine and tyrosine (p-hydroxyphenylalanine), (Fig. 1). Tyrosine structure permits to include this amino acid among phenolics. Both amino acids can initiate the phenylpropanoid pathway. The first reaction in both cases is a deamination catalyzed by ammonia lyase of phlenylalanine (PAL) or tyrosine (TAL) respectively, hence the first products of the deamination of both amino acids are cinnamic and and p-coumaric acids. All phenolics, which are descended from cinnamic acid are called phenylpropanoids, because of their molecular structure, where phenyl ring is substituted with the rest of the propanoic acid (Fig. 2).

Plant phenylpropanoids are classified into two groups: simple phenylpropanoids and their derivatives, and composed phenylpropanoids, which include tannins, flavonoids and isoflavonoids [STRZAŁKA 2002]. However, according to IUPAC (International Union of Pure and Applied Chemistry) based on HAHLBROCK andSCHEEL

[1989], phenylpropanoids can be classified into: cinnamic acids, cinnamaldehydes, monolignols, coumarins, flavonoids, stilbenoids, furanocoumarins, lignans, lignins and suberins. This classification was performed on the basis of biosynthetic pathways common for particular groups of metabolites. We will describe several phenylpropanoids according to the classification usually presented in the cited literature

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A. PłaŜek, F. Dubert 24

[STRZAŁKA 2002], and we hope, that the readers, especially biochemists will forgive us this inaccuracy.

shikimic acid kwas szikimowy

phenylalanine tyrosine

Fig. 1. Shikimic acid is a precursor of aromatic amino acids

Rys. 1. Kwas szikimowy jest prekursorem aminokwasów aromatycznych

fenyloalanina

tyrozyna

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PHYSIOLOGICAL ROLE OF PHENOLICS 25

phenylalanine fenyloalanina

cinnamic acid kwas cynamonowy PAL

-NH₃

phenylpropanoids fenylopropanoidy

Fig. 2. Desamination of phenylalanine; PAL - phenylalanine ammonia-lyase Rys. 2. Dezaminacja fenyloalaniny; PAL - amoniakoliaza fenyloalaniny

Simple phenylpropanoids

Phenolic acids and alcohols play a main role among simple phenylpropanoids.

The simplest phenolic is salicylic acid (o-hydroxybenzoic acid) (SA). According to DIXON and PAIVA [1995] SA is synthesized directly from cinnamic (via benzoic acid as the intermediate) and ends this short metabolic path. However, METRAUX [2002]

demonstrates the alternative pathway of SA biosynthesis performed mainly in plastids.

This path is more complicated and leads from chorismatic and isochorismatic acid (derived from shikimic acid), (Fig. 3). The main precursor of phenolic acids is cinnamic acid (according to IUPAC classification, the whole group of metabolites derived from cinnamic acid is called „cinnamic acids”). From cinnamic acid p-coumaric acid is synthesized, and as mentioned above this compound can also be the product of tyrosine deamination. Coumaric acid is the substrate for synthesis of all phenolic acids, aldehydes and alcohols. Among the most frequently found phenolic acids one can include sinapic, conipheryl, ferrulic, caffeic, quinic and chlorogenic acids. The last one is synthesized from caffeic acid and quinic acid (in the reaction caffeic acid is the donor of carboxyl group, while quinic acid is the donor of hydroxyl group). The originated specific compound is called „depside” (Fig. 4) [KOHLMÜNZER 2003].

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chorismatic acid benzoic acid

kwas choryzmowy kwas benzoesowy

salicylic acid kwas salicylowy

Fig. 3. The salicylic acid pathway Rys. 3. Szlak syntezy kwasu salicylowego

Phenolic alcohols are produced by the reduction of respective acids via aldehydes. In that way sinapic, conipheryl and ferrulic alcohols can be obtained. These compound are substrates in lignin biosynthesis.

Some alkaloids like morphine and codeine also belong to the group of simple phenylpropanoids. They are products of tyrosine metabolism gained from immature fruit of poppy (Papaver somniferum). It is worth to stress that morphine possess the structure typical for phenolics, while codeine as the methylether of morphine formally is not a phenolic.

pyruvic acid kwas pirogronowy

COOH OH

COOH

OH

O COOH

CH

2

COOH

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caffeic acid kwas kawowy quinic acid

kwas chinowy

+

chlorogenic acid

kwas chlorogenowy + H₂O

depside depsyd

Fig. 4. Scheme of chlorogenic acid synthesis

Rys. 4. Schemat syntezy kwasu chlorogenowego, (depsydu)

Another simple phenylpropanoid is vanillin, which is obtained from the hydrolysis of odourless glucovanillin. However, the characteristic aroma of vanilla is composed of more than 300 compounds evaporated from vanilla pods undergoing the fermentation process.

Coumarins also belong to simple phenylpropanoids. Coumarin is formed during the drying-up of some plants like Melilothus officinalis, that contain o-coumaric lactone.

This volatile substance confers the characteristic aroma of dry hay. However, in insufficiently drying-up hay, fungi like Penicillium or Mucor develop and transform coumarol into dicoumarol. The last compound blocks the blood coagulation, which results in internal hemorrhage in animals fed fodder infected by these fungi. Scopoletin (6-methoxy, 7-hydroxycoumarin) can be found in the seed cover of caraway (Carum carvi), where it brakes the germination process. It is worth to mention that this compound also demonstrates the antifungal activity [STRZAŁKA 2002; KITAMURA et al.

2005]. A derivative of coumarin - psoralen (furanocoumarin) excitated by the UV radiation, forms stable bonds with DNA pyrimidine bases, leading to disfunction of translation and transcription.

Aflatoxins (difurancoumarins) belong to the numerous group of coumarin derivatives, which are dangerous for people and animals. These compounds are produced by specific fungi developing on plants stored in the unfavorable conditions.

They may be present in forage and food products infected by fungi of the kinds of Aspergillus (A. flavus, A. niger or A. parasitians) and Penicillium (P. expansum or P.

digitatum) [MÉNDEZ-ALBORES et al. 2007]. This is supposed that aflatoxins were responsible for unexpected deaths of a few archeologists - discoverers of ancient tombs of Tutenchamon in Egipt and Kazimierz Jagiellończyk in Poland. Spores of the fungi, mentioned above, raising in the atmosphere of the tombs may penetrate human lung and brain and while developing they may secrete aflatoxins, resulting in a stroke or coronary thrombosis [ŚWIĘCH 2003].

Composed phenylpropanoids (tannins, flavonoids and isoflavonoids)

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Tannins

The name was introduced by a French chemist Prost taking into account their ability to tan (fix) crude animal skin to produce „industrial” leather. Tanning process is based on bounding of the tannins to skin proteins resulting in protein denaturation. At present the name tannins means Nitrogen-free natural substances of big molecular weight (500-3000 Da), water soluble and containing numerous hydroxyl substituents.

Tannins are divided into two groups:

- hydrolyzing, being the composition of numerous molecules of gallic acid or other phenolic acids and sugars (usually glucose), or

- non-hydrolyzing if the main monomer is catechin (catechol) or leucoanto- cyanidines - the product of catechin oxidation. This group of tannins is also called proanthocyanidins or condensed tannins [HASLAM 1989].

Tannins are found in numerous plant species, for example in the bark of oak, rhizome of Potentilla erecta, Polygonum bistorta and others. They are also the components of cocoa, tea or wine. Their characteristic traits are the decomposition of free radicals, bounding of heavy metal ions and UV absorbtion. Tannins give plants specific tart, unpleasant taste, hence they can frighten some animals. Proanthocyanidins are the precursors of anthocyanidins. For example, the polymers of catechin and epicatechin are the source of cyanidine, the polymers of gallocatechin and epigallocatechin are the precursors of delphinidine, while the polymers of flavan-3-ol are the source of pelargonidine.

Flavonoids

They form the most numerous group of compounds, which break down into smaller subgroups like anthocyanins, flavons, flavonols, flavanons, flavanonols, flavans, flavanols, chalkons, lignans or aurons. The classification of flavonoids slightly differs in some books. According to IUPAC they are divided into: flavonoids, derived from 2-phenylchromen-4-one (2-phenyl-1,4-benzopyrone) structure, isoflavonoids, derived from 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) structure and neoflavonoids, derived from 4-phenylcoumarine (4-phenyl-1,2- benzopyrone) structure.

In this paper the most frequently found classification and description of flavonoids is presented. The basic flavonoid structure is phenyl ring (marked A on Fig.

5) originating from acetyl-CoA via malonyl-CoA, and the structure of phenylpropane (B and C₃) synthesized from shikimic acid (on the phenylpropanoid pathway). The three-carbon-atom chain may cycle forming the third heterocyclic ring (C). The names of particular flavonoid group originate from their molecular structure. The compound basic for all flavonoids is flavan (Fig. 6). After adding an oxygen atom to the 4^th carbon atom flavanon originates. Hydroxylation of the 3^rd carbon atom makes flavanonol.

Double bond between the 2^ndand 3^rd makes flavon, and finally flavon bearing hydroxyl group attached to the 3^rd carbon transfers into flavonol. Majority of flavonoids occur as glycosides.

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Fig. 5. Basic structure of flavonoids: benzene ring A, and phenylopropane (ring B + C₃).

Three-carbon chain can create new heterocyclic ring C

Rys. 5. Podstawowa struktura flawonoidów: pierścień benzenu A i fenylopropan (pierścień B + łańcuch trójwęglowy). Łańcuch trójwęglowy moŜe utworzyć nowy pierścień heterocykliczny C

Fig. 6. Flavan Rys. 6. Flawan Anthocyanins

They are synthesized from malonyl-CoA and p-coumaroil-CoA via interme- diating chalcons, flavanons, dihydroflavanols and flavanols, while to precede the last stage of the synthesis (between flawan-3,4-diole and anthocyanins) sun light is necessary [LEWIS et al. 1998]. Anthocyanins form cations, usually chlorides. The basic structure of anthocyanidins is the flavylium ion with seven specific places bounding substituents such as hydrogen atom, hydroxyl or methoxyl group (Fig. 7). Anthocyanins have hydroxyl groups, which permit them to form glycoside bounds with sugars, hence anthocyanins are easily dissolved in water. The aglicon rest of anthocyanins is called as anthocyanidine.

--C--C--C--

A B

A C

B

O

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Fig. 7. Flavylium ion Rys. 7. Jon flawyliowy

Anthocyanidine molecule possesses eight coupled double bonds, however, they are formed by 15 electrons only, instead of 16 required. As the result some of oxygen or carbon atoms are temporarily positively charged. The structures originated that way transform one into another being in mutual equilibrium though the most frequent is the structure bearing a positive charge at the oxygen atom.

Dissociation of the anthocyanidine salts is related to pH: in acidic solution anthocyanidine forms cations, while anions are formed in basic solution (dissociate all hydroxyl groups), (Fig. 8). This process changes the configuration of double coupled bonds and modifies compound colour. The colour of anthocyanins depends mainly on the type and number of substituents joined to the flavylium ion. The colour evolves from red in strongly acidic solution, via pink in weakly acidic, blue in weakly basic and finally to green with the high pH. The diversity of anthocyanidin colour increases as a result of bounding of glycone with various acids (i.e. acetic, pyruvic, caffeic acid). The example of such complex is cyanidine 3-acetylglucosid found in the orange fruits.

Despite of pH the colour of anthocyanins is also influenced by bonding with metal ions (Fe³⁺, Sn²⁺, Al³⁺, Mg²⁺, Ca²⁺) and tannins, the presence of yellow flavonoids, concentration of anthocyanidine and others. All these factors result from the shift of the maximum of light absorption by anthocyanidins. Figure 9 presents the examples of anthocyanidines: orange-red pelargonidine, purple cyanidine and violet-blue delphinidine. As it can be seen if more hydroxyl groups are joined to the rings the anthocyanidine colour becomes strongly blue.

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Fig. 8. Charge change of flavylium ion in acidic and alkalic solution

Rys. 8. Zmiana ładunku elektrycznego jonu flawyliowego w środowisku kwaśnym i zasadowym

The example of flavons is luteolin, one of the most common flavonoids, while the example of flavonols are quercetin and kempherol. These yellowish or cream compounds are found together with anthocyanins, changing the shade of their colour and the UV absorbtion. Every compounds mentioned above are found in green parts of Chelidonium majus, while luteolin in Achillea millefolium, in flowers of plants belonging to the Asteraceae family and in leaves of Digitalis purpurea. Kempherol, as the complex of quercetin with disaccharide composed of glucose and rhamnose is contained in the tea tree (Camelia sinensis) leaves or in the flowers of Prunus spinosa.

O

^-

O

^-

O

^-

O

^-

O

^-

OH OH

OH HO

OH O

⁺

acidic conditions środowisko kwaśne

alkali conditions środowisko zasadowe

O

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blue-violet delphinidyne niebieskofioletowa delfinidyna orange-red pelargonidyne

pomarańczowoczerwona pelargonidyna

crimson cyanidyne karmazynowa

cyjanidyna

Fig. 9. Structures of pelargonidine, cyanidine and delphinidine Rys. 9. Struktura pelargonidyny, cyanidyny i delfinidyny

The example of flavanons is hesperidin, found as glycoside of hesperitine in the orange fruit and also in the leaves of pepper mint. Pinobanksin as a flavanonole is found in Pinus banksiana and in been lute [KOHLMÜNZER 2003].

Catechin and epicatechin, the most commonly found flavanols are natural optic isomers. A specially high concentration of these compounds was found in tea, chocolate and wine. Their importance is connected with the antioxidant role.

Chalcons are relatively instable compounds, being in equilibrium with flavons and preceding the cyclisation of the pirone ring. They are primary metabolites on the path of the anthocyanins synthesis (Fig. 10). They are yellow, lypophilic compounds, that can be found in the inflorescence of Helichrysum bracteatum. Aurons are synthesized from chalcons. According to DIXON and PAIVA [1995] they may play the role of antibiotic in plants.

OH

O O

H

OH

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Fig. 10. Chalcone structure Rys. 10. Struktura chalkonu

Stilbenes are synthesized as a result of connection of 3 molecules of malonyl- CoA and p-coumaroyl-CoA (after decarboxylation) via threeketonic intermediate. The latter undergoes condensation, hydrolysis of thioester and decarboxylation, which leads to stilbene synthesis (Fig. 11). The most known stilbene is resveratrol accumulated in the skin of red grapes, in peanuts and in Rheum (it is frequently added to food as an antioxidant and colouring ingredient). In plants some stilbenes act as phytoalexins [GEHM et al. 1997].

Fig. 11. Stilbene structure Rys. 11. Struktura stilbenu

Lignans are composed of two phenylpropanoid units, mainly conipheryl or sinapic alcohols. They are used in lignin biosynthesis. The basic structure of lignans forms are lignane and cyclolignane (Fig. 12). The example of cyclignans is podophylotoxin, which is found in the resin of Podophyllum peltatum and P. emodi.

This compound blocks the function of the cell karyokinetic spindle bounding with tubuline molecules [KOHLMÜNZER 2003].

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1

2

Fig. 12. Structure of 1) lignan and 2) cyclolignan Rys. 12. Struktura 1) lignanu i 2) cyklolignanu Isoflavonoids

They are synthesized from flavonoids after the shift of B ring from the carbon atom of the 2^nd to the 3^rd position. That way isoflavons are synthesized from flavons, while isoflavans from flavans. This group includes rotenone, the poison isolated from the tropical plant Derris eliptica. Rotenone blocks the electron transport between NADH and ubiquinone and was used for many years in fishing not only by South American Indians. It was also applied by Norwegian which resulted in the pollution of rivers and lakes posing some time ago a serious ecological problem.

Isflavonoids also include specific compounds, structurally similar to human sex hormones which may bound to the receptors of estrogenes or androgenes located on the cell surface. This group of compounds includes daidzeine and genisteine, found in relatively high concentrations in soy-bean. On the other hand gliceolin, also present in soy-bean plants is an isoflavonoid of phytoalexin properties [KOHLMÜNZER 2003].

Lignin and suberin

As it was mentioned above, lignin is formed via the polymerization of phenolic alcohols (the reaction is demonstrated bellow), while suberin is a hydrophobic mixture composed of polyaromatic and polyaliphatic domain. The monomers of polyaromatic domain are derivatives of hydroxycinnamic and ferulic acids.

Typical reactions of plant phenolics and their physiological properties

O

H OH

OH OH

O

H OH

O O

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Oxidation

Phenolics are oxidized by polyphenoloxidases, which belong to two groups: the first-those of monophenoloxidase properties that oxidizes monophenols to diphenols (Fig. 13) and the second-those of diphenoloxidase properties which oxidizes diphenols to quinones (Fig. 14).

monophenol diphenol monofenol difenol

Fig. 13. Scheme of reaction catalyzed by monophenoloxidase Rys. 13. Schemat reakcji katalizowanej przez oksydazę monofenolową

2

catechol o-benzoquinone katechol o-benzochinon

Rys. 14. Scheme of reaction catalyzed by diphenoloxidase Rys. 14. Schemat reakcji katalizowanej przez oksydazę difenolową

The best known diphenoloxidases are catecholase and laccase, where catecholase oxidizes o-diphenols, while laccase oxidizes p-diphenols. Polyphenoloxidase may also oxidize aromatic amines or o-amine phenols, that are structurally similar to mono- and diphenols. The oxidation is a two-step-reaction realized via hydroxylation and

R OH

+ O

R

OH OH

OH

OH O

₂

O O

2 + 2 H

2

O

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oxygenation in the presence of atmospheric oxygen, and copper ions as the cofactors and at neutral pH (5 < pH < 8) of medium [POURCEL et al. 2006].

Polymerization

Lignin is synthesized in the free radical polymerization of phenolic alcohols [LEWIS, YAMAMOTO 1990]. The reaction is catalyzed by cell wall peroxidases. These enzymes remove hydrogen atom from alcohol molecule, leaving an unpaired electron in oxygen atom (Fig. 15). As a result free radical is produced. This electron is transferred to other atoms of the molecule, forming five radicals, being in a dynamic resonance equilibrium. These radical molecules combine, coupling their unpaired electrons into stable carbon or oxygen bridges. As a consequence free radicals are scavenged. The structure of lignin is additionally strengthen by numerous hydrogen bridges [OLSON, VARNER 1993].

OH OH

O OH

•

OH

O

•

OH

O

•

OH

O

•

OH

O

•

Fig. 15. The production of free radicals of coumaric acid in presence of peroxidase (PX) Rys. 15. Powstawanie wolnych rodników kwasu kumarowego przy udziale peroksydazy (PX)

Condensation

In this process melanins or tannins are synthesized. In neutral medium and in the presence of atmospheric oxygen diphenol oxidase oxidizes tyrosine to quinones that forms brown melanins. Non-hydrolyzing tannins are produced from catechin or epicatechin via C-C bound between the 8^th C atom of the last molecule of polymer and the 4^th C atom of a new monomer molecule (Fig. 16).

O H

OH

OH OH

O

O H

OH

OH OH

O

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Fig. 16. Procianidine structure - polymer of epicatechin Rys. 16. Struktura procyjanidyny - polimer epikatechiny

The molecules of gallic or epigallic acids can be joined to the ring B of catechin or epicatechin. Their structure is also strengthen with hydrogen bridges. The term of condensed tannins is often used, however, its proper name is proanthocyanindins [HASLAM 1989].

Reactions with proteins and sugars

Carboxyl and hydroxyl groups of phenolics react with the carboxyl, hydroxyl or amine groups of proteins or with the hydroxyl of sugars forming ester bonds or oxygen bridges (Fig. 17).

HOOC

HO

H₂N OH

COOH

–OH + –OH –O– + H₂O –COOH + – OH –COO– + H₂O

Protein Białko

Fig. 17. Creation of ester and oxygen bounds between phenolic and protein molecules Rys. 17. Powstawanie wiązań estrowych i mostków tlenowych pomiędzy cząsteczką fenolu i

białkiem

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Phenolics as antioxidants

Phenolics are effective scavengers of free radicals. They fulfill principal demands imposed on these type compounds. Firstly, they are electron donors, secondly they possesses at least one hydroxyl group [RICE-EVANS et al. 1997]. Phenolics are also the substrates for peroxidases in decomposition of hydrogen peroxide (Fig. 18). Peroxidases transfer hydrogen atoms from hydroxyl group of reduced phenolic on hydrogen peroxide molecule, leaving an unpaired electron in oxygen atom. Products of this reaction are water and phenol radical. Unpaired electrons couple (as an effect of inner molecular shifting) creating neutral quinones. Molecular mechanism of this reaction is presented in Figure 19.

H₂O₂+ PhenH₂ 2 H₂O + Phen peroxidase

peroksydaza

PhenH₂- reduced form of phenolic

zredukowana forma związku fenolowego Phen- oxidated form of phenolic

utleniona forma związku fenolowego

Fig. 18. Scheme of reaction catalyzed by peroxidase scavenging hydrogen peroxide using phenolic as a substrate

Rys. 18. Schemat reakcji katalizowanej przez peroksydazę „zmiatającą” nadtlenek wodoru przy uŜyciu związku fenolowego jako substratu

OH

OH + R ^•

peroxidase peroksydaza

O^•

OH + R ^•

peroxidase peroksydaza

O^•

O ^•

O

Fig. 19. Molecular scheme of free radical scavenging catalyzed by peroxidase using phenolic as a substrate

Rys. 19. Molekularny schemat „zmiatania” wolnych rodników katalizowanego przez peroksydazę przy uŜyciu związku fenolowego jako substratu

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+ HO

^•

coumarin hydroxycoumarin

kumaryna hydroksykumaryna

Fig. 20. Coumarin as an Rys. 20. Kumaryna jako antyoksydant rodnika hydroksylowego

O O

OH

O O

OH

+

H – O – O

^•

hydroperoxyl radical rodnik wodoronadtlenkowy

+

H

2

O

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Coumarin neutralizes strongly reactive hydroxyl radical in two ways. As an effect of dismutation of coumarin, hydroxycoumarin and water are created, while after oxidation and elimination hydroxycoumarin and hydroperoxide radical (being in equilibrium with superoxide radical), are produced (Fig. 20). So, instead of very dangerous for the cell hydroxyl radical (which is not decomposed by any specific antioxidant), hydroperoxide radical is synthesized and it may be inactivated by SOD (superoxide dismutase) [LOUIT et al. 2005].

Free radical production

Phenolic compounds, apart from their antioxidant properties, can produce free radicals. The coumarin when is excited by the UV radiation initiates the creation of reactive oxygen forms. In the excited coumarin C-O bond is broken (Fig. 21). After oxygen removing an unpaired electron is left at carbon atom creating acyl radical. Two oxygen atoms couple to singlet oxygen molecule. The latter can take an unpaired electron creating the superoxide radical, which after accepting the next electron and two protons transforms into hydroxyl radical [LYNCH et al. 2001]. Because of this property plants containing coumarin in strong sun radiation are dangerous for human skin.

coumarin kumaryna

•

• 2 OH

^•

hydroxyl radical rodnik hydroksylowy

O

2

•

singlet Oxygen tlen singletowy

O O O O

O

acyl free radical rodnik acylowy

+ 2 x atomic Oxygen tlen atomowy

e

O

2

• −

superoxide radical

e + 2H

⁺

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Fig. 21. Coumarin as a „producer” of free radicals Rys. 21. Kumaryna jako „producent” wolnych rodników UV absorbtion

Phenolics demonstrate protective properties towards the radiation of high energy.

In some coloured compounds like chlorophyll, electron which has absorbed quantum of visible light „jumps” from basal orbital on the next one of higher energy.

fluorescence fluorescencja

orbital ππππ

orbital anty ππππ UV

e e e

ππππ

ππππ benzen ring

pierścień benzenowy

Fig. 22. Scheme of UV dispersion by phenolic compounds Rys. 22. Schemat rozpraszania energii UV przez związki fenolowe

If absorbed energy is not high enough for ionization, the electron comes back to the basal orbital emitting an excess of energy as fluorescence (red one in the case of chlorophyll). In phenolic molecules below and above benzene ring two clouds of π electrons are formed. In chlorophyll as well as in other coloured compounds electrons also form two π clouds, however, they absorb lower energy as compared to phenolics.

When electrons reach anti-π orbital (which is highly unstable), they come back emitting blue fluorescence (Fig. 22).

Physiological role of phenolics

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Regulation of growth and flowering

Salicylic acid (SA) is included among plant growth regulators, sometimes it is called plant hormone. Being a signal molecule it may join the specific membrane receptors, forming an active complex initiating signal transduction in plants. SA receptor is homologic to catalase. It is composed of four subunits, and can decompose hydroperoxide to water and oxygen. Plant hormones initiate de novo protein synthesis, activate formerly synthesized proteins or initiate their degradation. In some plants SA stimulates seed germination, root growth, biosynthesis of chlorophyll, flowering and metabolite transport through cell membranes. In Araceae the temperature of flowers may increase a dozen of Celsius degrees above the level of the neighborhood temperature. This phenomenon is observed in giant lilly Amorphophallus titanum.

Flowers of this plant family produce specific compounds luring pollinating insects.

However, these compounds consist of hardly volatile amines and indoles, which evaporate at higher temperature. This temperature increase is caused by SA, so it is called calorygene [RASKIN 1992].

Growth inhibitors

The majority of the plant growth inhibitors belong to phenolics. They inhibit seed germination and also play a role in allelopathy. More information about allelopathy is given in the chapter Antibiotic activity. These inhibitory compounds, accumulated in the seed cover are responsible for seed dormancy. Germination inhibitors involve, for example, scopoletin (found in caraway seeds), quercetin, chlorogenic acid or coumarin [JANKIEWICZ 1997].

Signaling role in systemic acquired resistance (SAR)

Salicylic acid controls some mechanisms of plant resistance to stresses. Its role is important because it increases concentration of hydrogen peroxide - other signaling molecule responsible for plant resistance to abiotic (UV, ozone, heavy metals, drought, cold) and biotic stresses [GANESAN, THOMAS 2001]. SA activates SOD responsible for synthesis of H₂O₂ and breaks activities of catalase and peroxidase - enzymes decomposing H₂O₂. SA stimulates the synthesis of such PR-proteins (pathogenesis- related) as chitinase or glucanase. SA is recognized as a signal compound controlling resistance mechanism called SAR. It was described for the first time in tobacco plants infected with tobacco mosaic virus [seeRASKIN 1992]. In infected tobacco leaves fast necrotisation was observed. In these leaves the presence of PR proteins as well as lignin, reactive oxygen forms and also SA were detected. However, also in healthy leaves of this plant the same compounds were found. It was proved that SA is responsible for the transmission of information about infection from the infected to healthy parts of plant.

SA may be transported systemically as a free acid or as volatile methyl salicylate, which evaporates from healthy plant parts and penetrates cells initiating the synthesis of defence compounds [NEILL et al. 2002].

Antibiotic activity

Chlorogenic acid is a strongly antifungal, antibacterial and antiviral agent. Plants showing higher concentration of the acid are more resistant to pathogens [NICHOLSON, HAMMERSCHMIDT 1992]. Lignin and melanins from cell walls seperate the infection center from healthy tissue, which prevents pathogen spread. The lignin synthesis is one of processes induced during HR (hypersensitive reaction) [HAMMOND-KOSACK,JONES 1996].

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PHYSIOLOGICAL ROLE OF PHENOLICS 43 Phytoalexins play an important role in the resistance to stress. They are low molecular derivatives of phenolics, which accumulate in plant under the influence of such stresses as heavy metals, drought, frost, UV, pathogens, elicitors or fungicides.

Term phytoalexins originates from Greek alexein - to protect and phyton - plant. In the case of pathogen attack phytoalexins are produced ca. two hours after plant inoculation.

The compounds belong to various groups of chemicals, but most of them are isoflavonoids. The activity of phytoalexins depends on their chemical structure, particularly on the number and type of substituents joined to aromatic rings. In Papilionaceae this activity increases in the following order: isoflavons < isoflavans <

pterocarpans < isoflavans. Antifungal activity of phytoalexins in Papilionaceae can result from their structural similarity to steroid molecule. They disturb the metabolism of fungal steroids or directly change the fungus cell membranes. The first identified phytoalexin was pisatin, produced in pods of pea infected with fungus Monilinia fructicola. Other main phytoalexins are: medicarpin, the derivative of pterocarpan synthesized in alfalfa, resveratrol belonging to stilbens found in grape fruit skin, and gliceolin produced in soybean plants [GRAHAM,GRAHAM 1991; JANKIEWICZ,SOBICZEWSKI

1997].

Antibiotic action of phenolics is also important in respect to interactions between plants or between plants and microorganisms under field conditions, to eliminate „rival”

plants. This phenomenon is known as allelopathy. The term was proposed by Molish in 1937, as combination of Greek: allelon - mutually and patheia - pain. The allelopathic compounds were divided into four groups in relation to interacting organisms. The compounds playing the role in the interaction between two plants are called colins, secreted by higher plant but acting on microorganisms are phytoncides, produced by microorganisms but acting on plant are marasmines, and finally antibiotics controlling interactions between microorganisms. The group of allelopathic phenylpropanoids includes such phenolic acids as chlorogenic, p-coumaric, ferrulic, sinapic, syringic and vanillic and other compounds as quercetin, kempherol, myricetin, 5- hydroxynaphtochinon (juglone) and tannins.

The allelopathic compounds secreted by plants inhibit seed germination as well as growth and development of neighbour plants, decreasing permeability of vascular elements by deposition of callose. They also decrease the rate of protein biosynthesis, yield of both photosynthetic and oxidative phosphorylation, and finally they decline chlorophyll accumulation and permeability of cell membranes [OLESZEK 1997].

Phenolics in pharmacology

Phenolics are found in numerous pharmaceutical plants, where their action is multidirectional. Because of their antioxidative properties they are used as medicines.

For example, melanins contained in human skin are important protectants against sun radiation. Melanins react with free radicals because their molecules contain numerous o- quinone (oxidative) as well as o-hydrochinone (reducing) groups. Brown and black eumelanins are more effective scavengers of free radicals than yellow or red feomelanins. Procyanidines demonstrate strong antioxidative activity ca. 50-times stronger than C and E vitamins. Procyanidines are contained in considerable amount in apple pulp but during juice production 90% of them are decomposed [KOSMALA, KOŁODZIEJCZYK 2006].

Table 1 demonstrates the content of main phenolics in red and white wine. Red wine contains a few times more antioxidative phenolics as compared to white one [RICE- EVANS et al. 1996]. The fact explains the „French paradox”. Against common opinion relating to dietary French cuisine, in many regions of France diet is reach in fat.

However, people drink a lot of red wine, which prevents deposition of cholesterol. As a

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consequence, inhabitants of these regions do not suffer of blood system illness.

Table 1; Tabela 1 Phenolic content in red and white wine

Zawartość związków fenolowych w czerwonym i białym winie

Phenolic compounds Związki fenolowe

Red wine Wino czerwone

(mg⋅dm^-3)

White wine Wino białe (mg⋅dm^-3)

Catechin; Katechina 191 35

Epicatechin; Epikatechina 82 21

Gallic acid; Kwas galusowy 95 7

Cyanidines; Cyanidyny 3 0

Rutin; Rutyna 9 0

Quercetin; Kwercetyna 8 0

Caffeic acid; Kwas kawowy 7.1 2.8

Resveratrol; Rezweratrol 1.5 0

Anthocyanins are applied in ophthalmology. Their uptake improves eye vision contrast due to they increase elasticity and decrease permeability of blood vessels, specially effectively in the iris veins. They demonstrate anti-inflammatory and antioxidant properties and also accelerate the regeneration of rodopsine necessary for registration of light.

Chlorogenic acid decreases glucose content secreted to the blood, hence it can help in slim down procedure and may be also used in the case of diabetes. Kempherol possesses weak spasmolytic action. Rutoside, known also as rutin, is one of the commonly occurring glycosides of quercetin, for example, it can be found in Viola tricolor or Sambucus nigra. It prevents decomposition of C vitamin and seals the blood vessels (in medicine it is known as Rutinoscorbin). Hesperidin has an antiviral property, while myricetin produced in grape fruits, berries and vegetables acts as anti- inflammatory, anti-cancerogen and antiviral drug. It blocks karyokinetic spindle by bounding with tubuline. Genistein, used in specific hormonal therapy also shows antitumor properties. It also acts as the cytostatic and cytotoxic agent to tumor cells via inhibition of tyrosine kinases, and directs tumor cells to programmed cell death.

Echinacea purpurea is abounding in phenolics. The extract from this plant contains:

chlorogenic acid and its isomer - iso-chlorogenic acid, 2,3-dicaffeic-ilochinic acid, phenolic glycoside - echinacoside (the derivative of 3,4-dioxyphenyl-ethylalkohol), verbascoside, flavonoids (quercetin, kempherol, rutoside, flavons - luteolin and apigenin, flavonol - isoramnetin). The extract accelerates the metabolism, acts as immunostimulator, antibacterial, antiviral, antifungal, analgesical, anticontractor, cholagogic, antifebrile, anti-inflammator agent, stimulating also regeneration processes.

It is no wonder that Echinacea made a career among homeopathic medicines [KOHLMÜNZER 2003].

Reassuming

As it follows from information presented above phenolics are the secondary metabolites of various properties and applications. With respect to the oxidative stress, phenolic characteristic is their dual action: they may both produce and scavenge free

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PHYSIOLOGICAL ROLE OF PHENOLICS 45 radicals. Hence, we can assume that their main function is to maintain the stable concentration of free radicals. Phenylpropanoids often exhibit their reducing properties, hence their physiological function may be shown by regulation of cell redox potential.

These compounds are produced in plants, where they „fight” against other organisms via their allelopathic and antibiotic traits. They are synthesized not only in stress, but also in physiological conditions. They regulate numerous physiological processes, both in plant, animal and human organisms, so they are used in medicine. In the studies of particular abiotic or biotic stresses in plants usually total phenolic pool is analyzed.

However, we suggest to pay more attention to the physiological function of individual phenolics.

References

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1085-1097

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GEHM B.D.,MCANDREWS J.M,CHIEN P.,JAMESON J.L.1997. Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for the estrogen receptor. Proc. Natl.

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GRAHAM M.Y., GRAHAM T.L. 1991. Rapid accumulation of anionic peroxidases and phenolic polymers in soybean cotyledon tissues following treatment with Phytophthora megasperma f. sp. glycinea wall glucan. Plant Physiol. 97: 1445-1455.

HAHLBROCK K.,SCHEEL D.1989. Physiology and molecular biology of phenylpropanoid metabolism. Annu. Rev. Plant Phisiol. Plant Mol. Biol. 40: 347-369.

HAMMOND-KOSACK K.E., JONES J. 1996. Resistance gene-dependent plant defense re- sponse. Plant Cell 8: 1773-1791.

HASLAM E.1989. Plant polyphenols. Vegetable tannins revisited. Cambridge University Press: Cambridge, U.K.).

JANKIEWICZ L.S. 1997. Inne inhibitory i mniej znane naturalne regulatory roślinne, w:

Regulatory wzrostu i rozwoju roślin. L.S. Jankiewicz (Ed.), Wydawn. Nauk. PWN Warszawa: 103-107.

JANKIEWICZ L.S.,SOBICZEWSKI P.1997. Fitoaleksyny i inne substancje związane z od- pornością roślin przeciwko patogenom, w: Regulatory wzrostu i rozwoju roślin. L.S.

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124-134.

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LOUIT G.,FOLEY S.,CABILIC J.,COFFIGNY H.,TARAN F.,VALLEIX A.,RENAULT J.P.,PIN S.

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MÉNDEZ-ALBORES A.,DEL RIO-GARCIA J.C.,MORENO-MARTINEZ E. 2007. Decontamination of aflatoxin duckling feed with aqueous citric acid treatment. Animal Feed Science and Technology 135: 249-262.

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NICHOLSON R.L.,HAMMERSCHMIDT R.1992. Phenolic compounds and their role in disease resistance. Annu. Rev. Phytopathol. 30: 369-389.

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Key words: antioxidants, condensation, deterring, lignin, melanins, polyme-rization, polyphenoloxidases, UV absorbtion

Summary

Phenylpropanoids are a numerous group of the secondary metabolites. The pathway of phenolic biosynthesis is induced in plants under the treatment of various unfavorable factors. Phenylpropanoid compounds act twofold: they can be toxic for plant, inhibiting their growth and development, and, on the other side, they protect plants from stress effect. In the paper the most important phenolics, their properties and influence on plant metabolism, the typical reactions and application in pharmacy were discussed. The molecular explanation of oxidation reactions, lignin polymerization, tannin condensation, UV absorbtion and decomposition and the production of reactive oxygen species were demonstrated.

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PHYSIOLOGICAL ROLE OF PHENOLICS 47 In plant physiology phenylpropanoid compounds are grouped into simple and composed phenylpropanoids. Simple pheylpropanoid compounds involve mainly phenolic acids and alcohols, vanilin and coumarins. Chlorogenic acid demonstrates antibiotic properties, while salicylic acid (SA) is a plant growth and development regulator, playing also a signal role in plant defence response to numerous stresses. SA initiates synthesis of PR (pathogenesis-related) proteins, hydrogen peroxide production and controls systemic acquired resistance (SAR). Phenolic alcohols polymerize to lignin, which strengths cell wall and builds natural barrier against pathogen attack.

Compounds like vanilin, strong aromatic, attract insects and are used in cosmetic and food industry. Coumarins show phototoxic effect and also demonstrate a growth inhibitor action.

Composed phenylpropanoids involve tannins, flavonoids and isoflavonoids.

Tannins protect plants from pathogens and deter preying insects. Flavonoids are pigments of flowers and leaves, and can protect cell structures and organic compounds from cold, UV radiation and free radicals. Isoflavonoids are characterized mainly by insecticide feature. Many isoflavonoids belong to phytoalexins, specific compounds synthesized within defence mechanism against stresses. They inhibit fungal spore germination and act osmotically to penetrating hyphae. Moreover, these compounds may imitate steroid molecules joining to specific steroid receptors disturbing numerous metabolic processes. Among the best known phytoalexins pisatin, phaseolin and medicarpin are mentioned. Phenylpropanoids also play an allelopathic role secreted by roots into soil, and inhibiting germinating and growth of other plants.

FIZJOLOGICZNA ROLA FENYLOPROPANOIDÓW

Agnieszka PłaŜek¹, Franciszek Dubert²

1 Katedra Fizjologii Roślin, Akademia Rolnicza im. H. Kołłątaja w Krakowie

2 Instytut Fizjologii Roślin im. F. Górskiego PAN w Krakowie

Słowa kluczowe: absorbcja UV, antyoksydanty, kondensacja, lignina, melaniny, odstraszanie, oksydaza polifenolowa, polimeryzacja

Streszczenie

Fenylopropanoidy są bardzo liczną grupą metabolitów wtórnych. Synteza tych związków jest inicjowana pod wpływem wielu niekorzystnych czynników. Działają one w podwójny sposób: są toksyczne dla roślin hamując ich wzrost i rozwój, a z drugiej strony chronią je przed efektami licznych stresów. W artykule przedstawiono wpływ fenylopropanoidów na metabolizm roślin, podstawowe ich właściwości i reakcje, a takŜe omówiono krótko ich wykorzystanie w farmakologii. Przedstawiono molekularne wyjaśnienie reakcji utlenienia, polimeryzacji ligniny, kondensacji tanin, absorbcji promieniowania UV oraz rozkładania i tworzenia reaktywnych form tlenu.

Fenylopropanoidy zostały pogrupowane na fenole proste i złoŜone. Do prostych zalicza się kwasy i alkohole fenolowe i ich pochodne, wanilinę i kumaryny. Kwas chlorogenowy wykazuje właściwości antybiotyczne, podczas gdy kwas salicylowy (SA) jest roślinnym regulatorem wzrostu, oraz odgrywa rolę sygnalną w reakcjach odpornościowych na stresy abiotyczne i biotyczne. SA indukuje syntezę białek typu PR (ang. pathogenesis-related), zwiększa stęŜenie nadtlenku wodoru, innej cząsteczki sygnalnej, oraz kontroluje systemiczną odporność nabytą (SAR - systemic aquired resistance). Alkohole fenolowe polimeryzują do ligniny, która wzmacnia ścianę

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komórkową oraz tworzy naturalną barierę ograniczającą rozprzestrzenianie się patogenów w tkance. Wanilina jako związek o silnych właściwościach aromatycznych przywabia owady, a jednocześnie jest wykorzystywana w przemyśle kosmetycznym i spoŜywczym. Kumaryny wykazują właściwości fototoksyczne i są inhibitorami wzrostu.

Do fenylopropanoidów złoŜonych naleŜą taniny, flawonoidy i izoflawonoidy.

Taniny chronią rośliny przed owadami i chorobotwórczymi mikroorganizmami.

Flawonoidy są bardzo liczną grupą metabolitów. Nadają zabarwienie kwiatom przywabiając owady, wykazują właściwości antyoksydacyjne, rozkładając reaktywne formy tlenu, a takŜe mogą być ich producentami, ponadto absorbują promieniowanie UV. Izoflawonoidy charakteryzują się głównie właściwościami antybiotycznymi i owadobójczymi. Do tej grupy zaliczane są związki podobne strukturalnie do ludzkich hormonów płciowych, stąd teŜ znalazły one zastosowanie w leczeniu róŜnych schorzeń ludzi. Do fenylopropanoidów zaliczane są teŜ tzw. fitoaleksyny, które syntetyzowane są w ramach mechanizmu odpornościowego na róŜne stresy, zwłaszcza na atak patogenów.

Ich działanie polega głównie na zwiększeniu przepuszczalności membran cytoplazmatycznych strzępek grzybów, hamowaniu kiełkowania zarodników oraz, poprzez strukturalne podobieństwo do steroidów, na zaburzaniu róŜnych szlaków metabolicznych. Do najbardziej popularnych fitoaleksyn zalicza się medikarpinę, pizatynę i fazeolinę. Fenylopropanoidy wykazują ponadto właściwości allelopatyczne, ograniczając wzrost konkurencyjnych roślin.

Dr hab. inŜ. Agnieszka PłaŜek Katedra Fizjologii Roślin

Akademia Rolnicza im. H. Kołłątaja ul. PodłuŜna 3

30-239 KRAKÓW

e-mail: rrplazek@cyf-kr.edu.pl

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ZESZYTY PROBLEMOWE POSTĘPÓW NAUK ROLNICZYCH 2008 z. 524: 45-54

CZY TEMPERATURA POMIARU

AKTYWNOŚCI PEROKSYDAZY ASKORBINIANOWEJ I KATALAZY MOśE ZABURZYĆ

OCENĘ CHŁODOODPORNOŚCI SIEWEK KUKURYDZY?

Renata Bączek-Kwinta

Katedra Fizjologii Roślin, Akademia Rolnicza im. H. Kołłątaja w Krakowie

Wstęp

Stres oksydacyjny, czyli nadprodukcja aktywnych form tlenu do których za- liczany jest nadtlenek wodoru, zawsze towarzyszy stresowi chłodowemu. Chłodo- odporność jest cechą zaleŜną od wielu przystosowań, zarówno anatomicznych, jak i fizjologiczno-biochemicznych. Rośliny chłodoodpornych genotypów gatunków chłodowraŜliwych wykazują zatem (między innymi) zdolność do uaktywniania systemu antyoksydacyjnego, czyli do syntezy i (lub) aktywacji określonych przeciwutleniaczy.

WaŜnymi enzymatycznymi przeciwutleniaczami - zmiataczami H₂O₂ są katalaza i peroksydaza askorbinianowa [HODGES i in. 1997; BĄCZEK-KWINTA i in. 2005].

Katalaza (CAT) jest enzymem obecnym w mitochondriach, peroksysomach i apoplaście [FEIERABEND 2005]. Charakteryzuje się wysoką aktywnością molekularną, sięgającą 5 mln cząsteczek substratu przekształconych w ciągu 1 minuty [KĄCZKOWSKI

1987]. CAT przejawia takŜe aktywność peroksydazową, dzięki której katalizuje utlenianie alkoholi, chinonów, mrówczanu i azotynów. Peroksydaza askorbinianowa (APX) występuje w postaci izoform plastydowych, mitochondrialnych, cytozolowych i mikrosomalnych [MITTLER,POULOS 2005]. Powinowactwo APX do jej substratu jest duŜo większe niŜ powinowactwo katalazy. MoŜliwe jest zatem przejmowanie roli izoform katalaz przez róŜne izoformy APX [DE GARA i in. 2000; FATH i in. 2001].

Według standardowych procedur pomiarowych, ze względu na konieczność zapewnienia optymalnego przebiegu reakcji, analizy aktywności APX i CAT naleŜy przeprowadzić w temperaturze 25°C [NAKANO, ASADA 1981; AEBI 1984]. W praktyce stosuje się temperaturę 20-25°C, często po prostu temperaturę pokojową. Rodzi się zatem pytanie: czy aktywność enzymów badana w takich warunkach (in vitro) pozwala na prawidłową ocenę aktywności ujawniającej się in vivo, temperaturze chłodowej?

MoŜna wyrazić obawę, iŜ aktywności enzymów wyznaczone w temperaturze wyŜszej od aktualnie panującej w czasie wegetacji (np. 5°C) nie odzwierciedlają

„rzeczywistych” aktywności. W związku z tym wnioskowanie dotyczące relacji pomiędzy genotypami podczas chłodzenia moŜe zostać zaburzone. Większość białek enzymatycznych charakteryzuje się bowiem zróŜnicowaną wraŜliwością na temperaturę, która wpływając na oddziaływania wewnątrzcząsteczkowe, zmienia konformację tych protein, a co się z tym wiąŜe - ich aktywność. Podczas działania chłodu lub mrozu na rośliny, aktywność poszczególnych enzymów in vivo znacznie odbiega od wartości uzyskanych in vitro - w wyŜszej temperaturze i przy znacznie

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R. Bączek-Kwinta 46

większym, niŜ ma to miejsce w tkankach, pomiarowym stęŜeniu substratu. Dlatego niektórzy badacze postulują stosowanie temperatury pomiarowej równej temperaturze wegetacji lub doń zbliŜonej [HUNER i in. 1993; LEIPNER,STAMP 1999] albo wykonywanie oznaczeń aktywności enzymu w duŜym zakresie temperatur [HAKAM, SIMON 1996].

Natomiast badanie w temperaturze chłodowej parametrów kinetyki reakcji katalizowanej przez określone białko moŜe pozwolić na uzyskanie odpowiedzi na pytanie, czy przyczyną spadku aktywności enzymu jest zmniejszone powinowactwo enzymu do substratu (w tym przypadku nadtlenku wodoru), wywołane na przykład zmianami konformacyjnymi części białkowej.

W związku z powyŜszym celem doświadczeń było ustalenie bezpośredniego wpływu temperatury pomiaru (5 lub 20°C) na aktywność enzymów rozkładających nadtlenek wodoru w liściach form mieszańcowych. Uwzględniono aktywność zarówno konstytutywną, jak i ujawniającą się podczas chłodu. Wybrano genotypy o ustalonej, zróŜnicowanej chłodoodporności [BĄCZEK-KWINTA,KOŚCIELNIAK 2003]. Zbadano ponadto niektóre parametry kinetyki reakcji enzymatycznej katalizowanej przez CAT w nadziei na poznanie przyczyny niŜszej aktywności tego enzymu u mieszańca wraŜliwego na temperaturę chłodową w porównaniu z odpornym. Relacje takie wykazano w pracach prowadzonych przez BĄCZEK-KWINTĘ [2002] oraz BĄCZEK-KWINTĘ iKOŚCIELNIAKA

[2003].

Materiał i metody

Materiał roślinny

Nasiona badanych genotypów kukurydzy (KOC 9431 i K103 X K85) pochodziły z firmy „Nasiona Kobierzyc”. Ziarniaki zaprawione zaprawą nasienną T (50- procentowy Thiuram) wysiano po 10 sztuk do plastikowych wazonów o pojemności 5 dm³, z podłoŜem o składzie torf + gleba brunatna + piasek (proporcja objętościowa 3 : 2 : 1). Wegetację wstępną prowadzono w klimatyzowanej szklarni, przy fotoperiodzie 15/9 godz. (dzień/noc). Do momentu skiełkowania ustalono temperaturę 20°C, następnie utrzymywano termoperiod 20°C/17°C (dzień/noc). Wilgotność względna powietrza (RH) wynosiła około 60%, oświetlenie około 500 µmol(kwantów)⋅m^-2⋅s^-1. W dni pochmurne rośliny doświetlano lampami matalohalogenkowymi. Niezbędne do wegetacji makro- i mikroskładniki dostarczano roślinom od momentu wykształcenia przez nie trzeciego liścia, w postaci poŜywki Hoaglanda stosowanej w ilości zapewniającej osiągnięcie polowej pojemności wodnej gleby. W momencie osiągnięcia przez rośliny fazy trzeciego liścia (z pojawiającym się liściem czwartym), materiał przeniesiono do komór wegetacyjnych o temperaturze 5°C. Utrzymywano poprzedni fotoperiod, RH i poziom oświetlenia.

Oznaczenia aktywności enzymów antyoksydacyjnych wykonywano na próbkach pochodzących z trzeciego liścia, utrwalonych w ciekłym azocie. W kaŜdym przypadku jeden liść stanowił jedno powtórzenie biologiczne. Oznaczenia dla kaŜdej grupy prowadzono w pięciu powtórzeniach biologicznych, a dla kaŜdej próbki w dwóch powtórzeniach instrumentalnych. Stałość temperatury podczas pomiarów (5 lub 20°C) zapewniono wykorzystując termostatowany pokój laboratoryjny.

Izolacja enzymów

Po rozdrobnieniu w ciekłym azocie i rozmroŜeniu, tkanki homogenizowano przy pomocy homogenizatora elektrycznego (prod. Ultraturrax, Ika Labortechnik, Niemcy), w 0,05 mol⋅dm^-3 buforze fosforanowym o pH 7,0 z dodatkiem 0,5% albuminy (BSA;