284 Plant, fungal and habitat diversity investigation and conservation • Proceedings of IV BBC – Sofia ' 2006

Secondary metabolites: tools for stress protection in plants

Aglika M. Edreva1, Violeta B. Velikova2, Tsonko D. Tsonev2, Emilia Z. Gesheva1, Soleia Z. Dagnon3, Francesco Loreto4, Aynur Gürel5, Hüseyin Hakerlerler6, Bülent Yağmur6 & Hüseyin Akdemir7

1 Acad. D. Kostoff Institute of Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria, e-mail: edreva5@yahoo.com (corresponding author);

2 Acad. M. Popov Institute of Plant Physiology, Bulgarian Academy of Sciences, Acad. G. Bonchev St., Bl. 21, 1113 Sofia, Bulgaria;

3 Tobacco and Tobacco Products Institute, 4108 Plovdiv, Bulgaria;4 Istituto di Biologia Agroambientale e Forestale, CNR, Via Salaria Km 29,300 – 00016

Monterotondo Scalo (Roma), Italy;5 Department of Bioengineering, Faculty of Engineering, Aegean University,

35100 Bornova – Izmir, Turkey;6 Department of Soil Science, Faculty of Agriculture, Aegean University,

35100 Bornova – Izmir, Turkey;7 Department of Crop Science, Faculty of Agriculture, Aegean University,

35100 Bornova – Izmir, Turkey

Abstract. The protective role of secondary metabolites against environmental constraints is substantiated by studying various plant – abiotic stress stimuli systems. It was shown that phenylamides, conjugates of hydroxycinnamic acids and polyamines, are induced in tobacco and bean leaves, subjected to water excess and heat shock, respectively. ROS-scavenging ability of phenylamides was evidenced. Anthocyanins were found to dramatically accumulate in cotton leaves due to Na/K unbalance; shifts from mono- to orthodihydroxy substitution in the B-ring of anthocyanin aglycone was established, with this conferring a higher ROS-scavenging capacity of the molecule. The protective effect of a gaseous secondary metabolite, isoprene, against ozone fumigation and heat shock was shown. The ROS-scavenging ability of isoprene was demonstrated. Altogether, the data provide evidence that secondary metabolites can be involved in the non-enzymatic plant defence strategy by their antioxidant and antiradical properties.

Key words: anthocyanins, antioxidants, isoprene, phenylamides, reactive oxygen species (ROS)

Introduction

In addition to primary metabolites, such as amino acids, fatty acids, nucleotides, carbohydrates, cyto-chromes, chlorophylls, and metabolic products of the anabolic and catabolic pathways which occur in all plants and have the same metabolic functions, plants also contain a large variety of compounds, named sec-ondary metabolites (Heldt 1999). The plant physiol-ogist Albrecht Kossel was the first to coin the term "secondary" for these seemingly non-functional com-pounds, stating that "while primary metabolites are present in every living cell capable of dividing, the sec-ondary metabolites are present only incidentally, and

are not of paramount significance for plant life" (Kos-sel 1891). Although occurring in all living organisms – from bacteria and fungi to mammals, secondary me-tabolites are particularly largely distributed in plants. As sedentary organisms incapable of fleeing from un-favourable cues and lacking an immune system, plants have evolved the strategy of exploiting an extremely diversified network of constitutive and inducible sec-ondary metabolites, allowing them to deter, avoid or tolerate stress constraints, to complete their life cycle and survive. Thus, secondary metabolites play a ma-jor role in the adaptation of plants to their constantly changing environment and the generation of diversity at the organism level (Bourgaud & al. 2001; Osbourn

285Scientific Area C • Plant ecology. Vegetation and habitat diversity

& al. 2003; Grotewold 2005). More than 100 000 sec-ondary compounds have been described displaying a large complexity of chemical types, structures and bi-ological implications. Functions of secondary metab-olites comprise attractants, such as colour pigments and scents, repellents such as antifeedants against in-sects and herbivores, and toxins that affect microbi-al agents. They can also act as signals in plant–plant, plant–herbivore and plant–microbe interactions, and are components of the signal-transducing cascades in plants (Yang & al. 1997; Baldwin & al. 2002; Hadacek 2002; Foley & Moore 2005). Some secondary metab-olites absorbing light and UV radiation act as photo-protectants, while high molecular compounds such as lignin stabilize cell walls thus constituting a phys-ical anti-stress barrier (Dixon & Paiva 1995; Steyn & al. 2002; Edreva 2005). Recently, increasing amount of evidences substantiates the functioning of second-ary metabolites as antioxidants assisting the plant to cope with oxidative stress arising in stress situations (Grace & Logan 2000; Gould & al. 2002; Grassmann & al. 2002).

The aim of the present paper was to summarize our data on the involvement of secondary metabo-lites in several plant – abiotic stress systems, by as-suming that these molecular species could counteract the state of oxidative stress arising in a harmful, hos-tile environment. Phenylamides in tobacco and bean, anthocyanins in cotton and isoprene in reed were the subject of our research.

Phenylamides are typical bifunctional compounds produced by the conjugation of polyamines with hy-droxycinnamic acids (Fig. 1), combining the proper-ties of both parent compounds. The cationic character conveyed by the polyamines can determine an inter-fering in pH, ion and osmotic cell balance, as well as interactions with negatively charged loci in nucleic ac-ids, proteins, cell walls and membranes, this yielding a structure stabilizing effect. The hydroxycinnamic acid moiety of phenylamides can confer an ability to form dimers catalysed by peroxidase – H2O2 system, and free radical scavenging ability. Thus, when bound to

cell walls, phenylamides could bridge and cross-link polysaccharide chains contributing to cell wall consol-idation; as free radical scavengers they may counteract oxidative stress (Bors & al. 1989; Facchini & al. 2002). The involvement of phenylamides in the responses of plants to pathogenic agents has long been reported and later confirmed (Martin-Tanguy 1985; Bouchereau & al. 1999), while paucity of data is available on the phe-nylamides in relation to abiotic challenges.

Red pigments of various chemically unrelated types (red carotenoids, betacyanins, anthocyanins) are largely reported to accumulate in the vegetative parts of plants in stress conditions, such as low tem-perature, excess light, UV radiation, mineral unbal-ance, etc., with anthocyanins being recognized as the most important group of stress-responsive red col-oured compounds (Ibdah & al. 2002; Steyn & al. 2002; Han & al. 2003; Hormaetxe & al. 2004). Light attenu-ating, UV-screening, osmoregulatory and ROS-scav-enging properties of these compounds were proposed as a rationale of putative protective functions (Chalk-er-Scott 1999; Steyn & al. 2002; Hughes & al. 2005). An abnormal red colouration of cotton growing in the Aegean region of Turkey and other countries has long been observed but the type and putative function of the red pigments remained unknown.

An emphasis was laid on the isoprene (C5H8, 2-me-thyl-1,3-butadiene), a gaseous metabolite emitted in the atmosphere by a range of plant species, including reed (Kesselmeier & Staudt 1999). In the last decade intensive research has been devoted to this highly re-active volatile organic compound. Besides being im-plicated in the tropospheric chemistry by a series of complex interactions (Fehsenfeld & al. 1992), isoprene was assigned an important physiological role relevant to defence performance in plants (Sharkey & Singsaas 1995; Loreto & al. 2001; Sharkey & al. 2001).

Materials and methods

Experimental design

In our research several model systems were used em-bracing plant species of different genera and stress fac-tors of various origin.

Tobacco (Nicotiana tabacum L.) – water excess. Heavy rainfalls during the late vegetation periods when tobacco leaves become "over-riped" caused the formation of dot-like necrosis on leaf lamina named

HO

HO

(CH2)4NH NH2

O

C CHCH

Fig. 1. A phenylamide conjugate of caffeic acid and putrescine (caffeoylputrescine).

38 • Proceedings of IV BBC – Sofia ' 2006

286 Plant, fungal and habitat diversity investigation and conservation • Proceedings of IV BBC – Sofia ' 2006

"sharilka", referred to as a physiological disorder typi-cal for Oriental tobacco (Edreva & al. 1972a, b). Pres-ence of phenylamides was tested in "sharilka"-affected leaves, healthy leaves serving as controls.

Bean (Phaseolus vulgaris L.) – heat shock. Plants were exposed to 50oC for 5 h, this resulting in leaf wilting (Edreva & al. 1998). Phenylamides were de-termined in leaves of high temperature-treated bean plants, using non-treated controls.

Cotton (Gossypium hirsutum L.) – Na+ overaccu-mulation. Leakage of K and accumulation of Na+ in the soil brought about K depletion and Na+ overac-cumulation in the leaves of plants that was accompa-nied by an abnormal red colouration of leaves (Edreva & al. 2002; Yağmur & al. 2003). Anthocyanins content and pattern were analysed in reddening cotton leaves, green leaves serving as controls. Antioxidant and anti-radical capacity was also determined.

Reed (Phragmites australis L.) – ozone fumigation and heat shock. Leaves of reed plants emitting iso-prene and those in which isoprene was specifically in-hibited by fosmidomycin were used. Ozone fumiga-tion caused damage symptoms (flecking) on the leaves of isoprene-deficient plants, while not affecting the isoprene-emitting ones (Loreto & al. 2001). Similar-ly, in isoprene-deficient leaves heat shock produces se-verer damage than in isoprene-evolving leaves (Velik-ova & Loreto 2005; Velikova & al. 2005). Contents of hydrogen peroxide (H2O2) and lipid peroxides in reed leaves were analysed as markers of oxidative stress.

The ability of isoprene to quench 1O2 was evalu-ated in isoprene-emitting reed leaves as compared to leaves in which isoprene synthesis was previously in-hibited (Velikova & al. 2004), while the 1O2 quench-ing ability of phenylamides was determined using syn-thetic compounds (Velikova & al. 2007).

Details on the plant material and stress treatments are given in the corresponding above-cited papers.

MethodsPhenylamides were analysed by thin layer chroma-tography (Delétang 1974). 1O2 quenching ability of phenylamides was tested in vitro by using Rose Ben-gal photosensitizer as a 1O2 generator, and a Hansate-ch oxygen electrode. The 1O2 quenching ability of iso-prene emitting- and inhibited reed leaves was similarly determined (Velikova & al. 2004). Anthocyanins were analysed by HPLC (High Performance Liquid Chro-matography) according to Woodall & Stewart (1998).

Antioxidant capacity was estimated by the FRAP (fer-ric reducing antioxidant power) assay (Benzie & Strain 1999), and antiradical capacity – by the DPPH• (2,2-diphenyl-1-picrylhydrazyl radical) test (Brand-Wil-liams & al. 1995). Lipid peroxide content was deter-mined by the malondialdehyde assay (Heath & Packer 1968), and hydrogen peroxide content – as described by Velikova & al. (2004).

Results and discussion

Phenylamides

In tobacco leaves exposed to water excess ("sharil-ka") we detected phenylamides as conjugates of the polyamines spermidine and putrescine with feru-lic and caffeic acids as hydroxycinnamic acid com-ponents. Impressively, phenylamides lacked in the healthy leaves being induced by the stress imposed (Edreva & al. 1995). Similarly, induction of pheny-lamides following abiotic stress was reported for to-bacco leaves suffering K+, Ca2+, Mg2+ and P deficiency (Delétang 1974) or fumigated with ozone (Langebar-tels & al. 1991), as well as for S-starved or heat-treat-ed tobacco cell suspension cultures (Klapheck 1983; Königshofer & Lechner 2002). In our experiments we identified the same polyamine and hydroxycinnamic acid components of phenylamides as those reported in the above tobacco – stress systems.

In a different plant – abiotic stress couple, name-ly bean – heat shock we have also established forma-tion of phenylamides. High temperature treatment of the plants induced the appearance of conjugates of pu-trescine, spermidine and cadaverine with ferulic, p-coumaric and caffeic acids in the leaves (Edreva & al. 1995), with this being the first report on the occur-rence of phenylamides in bean. Cadaverine is an un-common polyamine characteristic for Leguminosae plants (Flores 1990), and was found to highly accu-mulate in a free form in the leaves of heat-treated bean plants (Edreva & al. 1998). The appearance of cadaver-ine as a conjugate in stress conditions may be relevant to its versatile employment in the protective mecha-nisms of bean plants.

Our data and those cited above point that the in-duction of phenylamides is not confined to a certain plant or a specific abiotic challenge, as also shown by the formation of phenylamides following herbicide application in spinach (Suzuki & al. 1981) and heat

287Scientific Area C • Plant ecology. Vegetation and habitat diversity

treatment of alfalfa (Königshofer & Lechner 2002), but can be commonly involved in the responses of plants to abiotic stresses. Given the largely report-ed involvement of phenylamides in biotic stress situ-ations (Flores & Martin-Tanguy 1991; Facchini & al. 2002), these secondary metabolites could be regard-ed as tools in a general mechanism triggered by plants to withstand adverse stress constraints of various or-igin. It can be assumed that the multiplicity of prop-erties conferred to the molecule of conjugates by their parent compounds may underlie a protective role. Since oxidative stress is recognized as a primary event in plant exposed to harmful environment (Foyer & Noctor 2005), the ROS-scavenging ability of pheny-lamides may be of considerable protective importance by contributing to the regulation of ROS level and pre-vention of ROS-induced damage. In vitro scavenging of free radicals by phenylamides was reliably demon-strated (Bors & al. 1989; Son & Lewis 2002), and sup-posed to be due to the hydroxicinnamic acid moiety (Bors & al. 1989). By in vitro experiments for the first time we provided evidence that phenylamides scav-enge singlet oxygen, a ROS particularly damaging to the photosynthetic apparatus. Potentiation of the 1O2 scavenging ability of the conjugates relative to both components of their molecule was established (Velik-ova & al. 2007). A view is developed that conjugation is a means by which plants moderate the high cytotox-icity proper to free hydroxycinnamic acids, keeping or even potentiating their beneficial characters (Martin-Tanguy 1997). Detailed in vivo experiments are need-ed to ascertain the exact mechanisms of phenylamide involvement in stress protection of plants.

AnthocyaninsWe reported that the abnormal red colouration of leaves of cotton was provoked by Na/K imbalance and Na+ overaccumulation in the soil and plants (Yağmur & al. 2003). It was demonstrated that the red colour of cotton leaves is due to the tremen-dous accumulation of anthocyanins and not to other red-coloured chemical types, whereas in the green leaves very low levels of anthocyanins were presented (Edreva & al. 2006). Of particular inter-est is the different character of the main aglycones in red leaves (cyanidin) and green ones (malvidin) which are distinguished by distinct substitution pattern of the B-ring, namely presence of o-dihy-droxy grouping in cyanidin and one hydroxy/two

methoxy groups in malvidin (Fig. 2). The increase of Na+ level in plants was shown to result in over-production of toxic oxygen species, particularly the strongly damaging hydroxyl free radical (OH•) (Alia & al. 1993). Since anthocyanins are recognized as free radical scavengers by in vitro tests (Kähkönen & Heinonen 2003; Kim & al. 2003), their protec-tive role in cotton reddening could be reasonably supposed. In support to this assumption we have shown that extracts of red cotton leaves performed higher antioxidant and antiradical capacity than ex-tracts of green leaves (Table 1). The data are in line

OH

OH

OH

OH

HO H O +

Cyanidin

B

A

OCH3

OH

OH

OH

HO OCH3O+

Malvidin

B

A

Fig. 2. Structural formulae of the main anthocyanin aglycones in the green (malvidin) and red (cyanidin) cotton leaves.

Table 1. Antioxidant and antiradical capacities (μmol g-1 fm) in red cotton leaves. Green leaves served as controls.

Variants Antioxidant capacity

Antiradical capacity

Green leaves 12.07 1.70Red leaves 16.00 2.29

288 Plant, fungal and habitat diversity investigation and conservation • Proceedings of IV BBC – Sofia ' 2006

with findings that cyanidin glycosides possess high-er antioxidant and antiradical activity than malvi-din glycosides (Aaby & al. 2004). This character is common for o-diphenols (Rice-Evans & al. 1997), and can be accounted for by the facilitated electron exchange in the o-dihydroxy – o-quinoid transi-tions. Hence, the shift from malvidin to cyanidin aglycones, i.e. to o-dihydroxy substitution in the B-ring of anthocyanins during cotton leaf reddening can contribute to an increased protective capacity against oxidative stress in the red cotton leaves. A shift from mono- to o-dihydroxy substitution in the B-ring of flavonoids has been observed in UV-toler-ant rice, mustard, Arabidopsis and Petunia plants as a response to UV-radiation (Markham & al. 1998; Olsson & al. 1998; Ryan & al. 2001, 2002); this has been proposed as a mechanism used by plants to overcome the UV-induced oxidative stress. Our re-sults (Edreva & al. 2006) are in line with the above findings, suggesting that the hydroxylation pattern of the B-ring in anthocyanins can also be important in stress protection.

IsopreneBy using an efficient model system of reed leaves normally emitting isoprene, and leaves in which isoprene biosynthesis was previously inhibited, we were able to demonstrate a differential effect of stress factors in both situations. Ozone fumiga-tion and high temperature exposure produced se-vere damage to the photosynthetic performance of isoprene-deficient leaves while stress-induced inju-ry was attenuated in isoprene-evolving leaves (Lo-reto & Velikova 2001; Velikova & Loreto 2005). We hypothesized that the stress tolerance in isoprene-emitting leaves can be mediated by antioxidant in-teractions of isoprene, interfering with events lead-ing to functional and structural cell damage. The contents of H2O2 and lipid peroxides were assessed by assuming that both metabolites are recognized as markers of oxidative stress in plants, with the formation of malondialdehyde (MDA) being indic-ative of membrane fragmentation (Heath & Pack-er 1968). When ozone fumigation and heat shock were imposed, enhancement of both metabolites was registered in isoprene-inhibited leaves whereas the isoprene-emitting ones sustained lower leaves of H2O2 and MDA. Moreover, in control (stress-free) leaves isoprene inhibition per se brought about

a certain degree of oxidative stress (Loreto & Ve-likova 2001; Velikova & Loreto 2005). Altogeth-er the data point to the ability of isoprene to coun-teract oxidative stress possibly by ROS-scavenging mechanisms. In fact, higher 1O2 scavenging capac-ity of isoprene-evolving reed leaves as compared to isoprene-deficient leaves was clearly demonstrated (Velikova & al. 2004). Similarly to carotenoids, con-jugated double bond structure of isoprene (Fig. 3) may quench 1O2 by facilitating energy transfer and heat dissipation (Cantrell & al. 2003); thus isoprene can act as a mean to elevate the antioxidant power of cell. Moreover, as a small lipophilic molecule iso-prene may assist in hydrophobic lipid–lipid, lipid–protein and protein–protein interactions. If allocat-ed to membranes, it may contribute to membrane stabilization with this providing an additional pro-tective mechanism (Sharkey & Yeh 2001).

��2 � �� ��2

��3

Fig. 3. Chemical structure of isoprene.

In conclusion it can be stated that secondary metab-olites constitute a remarkably flexible biochemical system integrated into a complex network of func-tions that have evolved both divergently or conver-gently over millions of years of plant evolution. Their involvement in the non-enzymatic defence strategy can play a major role in plant adaptation to the envi-ronmental challenges and the generation of diversi-ty at the organism level (Shirley 1996; Bourgaud & al. 2001). The plant protection that secondary metabo-lites can confer and the benefits of their use as phar-maceuticals and health-promoting nutraceuticals in animals and humans substantiate, according to Dix-on & Steele (1999), their being "a gold mine for met-abolic engineering".

Acknowledgments. The financial support of the National Sci-ence Fund (project SS–1503/05), NATO Reintegration Grant (No 981279/2004–2007), and Bilateral projects within the framework agreement between Italian National Research Council and Bul-garian Academy of Sciences (2004–2006), as well as between TÜ-BITAK, Turkey, and Bulgarian Academy of Sciences (2000–2002) is gratefully acknowledged.

289Scientific Area C • Plant ecology. Vegetation and habitat diversity

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