INFLUENCE OF ULTRAVIOLET RADIATION ON PLANT SECONDARY METABOLITE PRODUCTION

Katerova Z. * , D. Todorova, K. Tasheva, I. Sergiev

Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bldg. 21, 1113 Sofia, Bulgaria

Received: 22 January 2013 Accepted: 13 June 2013 Summary: Classification of major secondary metabolite groups is described. A short account is

also given to ultraviolet (UV) climatology and UV response in plants. Investigations regarding secondary metabolite production in plants, in vitro cultivated plant cell and tissue cultures under UV radiation, particularly UV-B (280-315 nm) and UV-C (200-280 nm) are reviewed. The capacity of plants, callus and tissue cultures to accumulate secondary metabolite compounds after exposure to UV is discussed. The cell and tissue cultures possess high potential for production of valuable secondary metabolites under controlled conditions, and it seems perspective to enlarge the investigations in this direction by using low doses of UV as elicitors of such compounds.

Citation: Katerova Z., D. Todorova, K. Tasheva, I. Sergiev. Influence of ultraviolet radiation on plant secondary metabolite production. Genetics and Plant Physiology, 2012, 2(3–4), 113–144.

Keywords: Calli; plant secondary metabolites; UV-B; UV-C. Abbreviations: PAR – photosyntheticaly active radiation; ROS – reactive oxygen species; UV

– ultraviolet radiation.

Contents

1. UV radiation – classification and impact on plants

2. Secondary metabolites – an introduction

3. UV radiation and secondary metabolites

3.1. Impact of UV-C on the synthesis of secondary metabolites

3.1.1. In vitro cultured plant cells, tissues and calli

3.1.2. Plant organs

3.2. Impact of UV-B on the synthesis of secondary metabolites

3.2.1. In vitro cultured plant cells, tissues and calli

3.2.2. Plant organs

4. Concluding remarks

114 Katerova et al.

1 . UV radiation – classification and impact on plants

Ultraviolet (UV) wavelength (400 – 200 nm) is a small part of the solar radiation reaching the Earth’s surface but with significant biological impact on the living organisms, including plants. According to the International Commission on Illumination this wavelength region is divided into UV-A (315 – 400nm), UV-B (280 – 315nm) and UV-C (200 – 280nm). The negative effect of UV radiation increases towards the shorter wavelengths. Therefore, due to its highest energy, UV-C quickly provokes high levels of injuries and it is most detrimental for the living organisms (Stapleton, 1992; Hollósy, 2002; Häder et al., 2007). Both UV-C and UV-B possess enough energy to damage different chemical bonds causing photochemical reactions, which is the main reason for the negative biological effects (Kovács and Keresztez, 2002). The composition of the UV radiation is modified due to its absorption by the atmosphere . Usually the short-wave UV-C radiation is fully absorbed with exception of the high mountain locations (Häder et al., 2007), while UV-B radiation is only absorbed by the stratospheric ozone and small part of it reaches the Earth’s surface. During the last decades the surface solar UV-B radiation was found to increase which corresponds with depletion of the stratospheric ozone caused by the increased release of anthropogenic pollutants such as chlorofluorocarbons and other gaseous emissions. Along with the atmospheric ozone amount, the spectral irradiance of the environmental UV depends also on the angle at which the solar radiation reaches the Earth’s atmosphere, i.e. the “solar zenith angle” (including time of day, season and

latitude), altitude, clouds, surface reflection, aerosols and even air pollution (Diffey, 1991; Paul, 2001; McKenzie et al., 2007). Since there is not selective absorber for the long-wave UV-A radiation, it is affected mainly by the light scattering, its intensity is much higher than UV-B but it is not so biologically relevant (Stapleton, 1992; Vass et al. 2005). Additionally, it was found that UV-A irradiation partially protected PSII reaction center from damages caused by UV-B by activating xanthophyll cycle by preserving the level of β-carotene in cluster bean chloroplasts during the steady phase of leaf development (Joshi et al., 2007). The report supported previous observations showing that environmentally relevant UV-A doses possess ameliorating effect on UV-B triggered damage (Newsham et al., 1998; Bischof et al., 2003). In fact, the presence of realistic doses of both UV-A and PAR are highly important in order to obtain environmentally adequate results since they both moderate the negative UV-B effects (Caldwell et al., 1994; Flint et al. 2003, Kakani et al., 2003; Dolzhenko et al., 2010).

High doses of UV-B and UV-C radiation affect negatively growth, development, photosynthesis, and other important processes in plants, leading to overproduction of reactive oxygen species (ROS) and development of oxidative stress, acting negatively on macromolecules, may decrease cell viability and cause cell death (Alexieva et al., 2001; Jansen, 2002; Frohnmeyer and Staiger, 2003; Zacchini and de Agazio, 2004; Procházková and Wilhelmová, 2007; Takeuchi et al., 2007; Danon and Gallois, 1998; Toncheva- Panova et al., 2010; Schreiner et al., 2012). However, low ROS concentrations were found to play a key role in the signaling

UV radiation and secondary metabolites

processes during plant acclimation (Dat et al., 2000). Along with the nucleic acids (Kucera et al., 2003; Takeuchi et al., 2007), proteins and lipids , the main target sites of UV radiation are known to be amino acids, membranes, quininones, pigments, photosynthetic machinery, mainly because of the UV absorbing aromatic chemical groups (Jansen et al., 1998; Hollósy, 2002; Jansen, 2002; Vass et al., 2005; Edreva, 2005). UV-induced effects depend also on the plant sensitivity (Lavola et al., 2003; Zu et al., 2011). Low UV-B or UV-C doses may trigger acclimation responses in plants, including activation of enzymatic and non- enzymatic defense systems (Loyall et al., 2000; Jansen, 2002; Lavola et al., 2003; Katerova and Todorova, 2009; Katerova et al., 2009; Katerova and Todorova, 2011; Rai et al., 2011), but high UV doses

could activate repair mechanisms in order to cope with the stress (Frohnmeyer and

Staiger, 2003). It was documented that the defense or tolerance to UV-B can

be related to the induction of different signal transduction pathways, secondary metabolite production, and DNA repair mechanisms (A-H Mackerness, 2000; Brown et al., 2005; Ishibashi et al., 2006). Application of low UV-C doses (0.5– 9.0kJm -2 ) has been considered to be of commercial prospect by causing hormetic (beneficial) effects to prevent pathogen diseases and delay senescence during fruit storage (Shama and Alderson, 2005).

Although the role of some secondary metabolites such as anthocyanins is still under question (Sarma and Sharma, 1999; Hada et al., 2003), most authors claim that the production of these compounds (mainly flavonoids and UV-B absorbing metabolites) in plants subjected to low UV-B doses is a major part of the complex

plant defense system (Solovchenko and Schmitz-Eiberger, 2003; Kucera et al., 2003; Schmitz-Hoerner and Weissenböck, 2003; Frohnmeyer and Staiger, 2003; Jansen et al., 2008). Bashandy et al. (2009) also assume that the accumulation of non-pigmented flavonoids in leaves of the double ntra ntrb (lacking NADPH-

dependent thioredoxin reductases) Arabidopsis mutant might lead to the observed UV-C tolerance. The authors strongly support the proposed theory by the fact that UV-C tolerance was lost after crossing the double mutant with the tt4 (mutation in the gene encoding the first enzyme of the flavonoid biosynthesis) showing that production of flavonoids in

the ntra ntrb mutant could protect plants against UV-C. In addition, the mRNA

level of Chs gene (chalcone synthase gene, involved in flavonoid production) was also induced in UV-C treated plants. The authors suggest that NADPH- dependent thioredoxin reductases could

be a new negative regulator of flavonoid biosynthesis.

In opposite to the high UV-fluency rate, relatively low UV-B or UV-C doses led to an increased production of secondary metabolites (Kreft et al., 2002; Antognoni et al., 2007; Nadeau et al., 2012; Schreiner et al., 2012). As the pathways for secondary metabolite production are interrelated, the fact that some of these compounds increase and other decrease is not unexpected, and the biosynthesis prevails mostly to compounds possessing higher ROS scavenging activity or UV-shielding properties (Jansen et al., 2008). In the current review, we focus on secondary metabolites, which have been reported to alter predominantly after UV-B and/or UV-C treatment.

Katerova et al.

2. Secondary metabolites – an

reported (Bell, 1980; Gershenzon,

introduction

2002). They are comparable to common The primary metabolites are vital

amino acids and occur in a free form or for every living cell. On the other hand,

as ingredients of low molecular weight the secondary metabolites are present

compounds. The non-protein amino acids only incidentally and are not essential could be divided into several subgroups –

for plant life (Edreva et al., 2008). neutral aliphatic amino acids; acidic amino They are organic compounds derived

acids; basic amino acids; heterocyclic through methylation, hydroxylation or

amino acids; aromatic amino acids; imino glycosylation from primary metabolites

acids; sulphur-containing amino acids; (carbohydrates, proteins, amino acids,

and selenium-containing amino acids lipids) (Korkina, 2007). Secondary

(Bell, 1980).

metabolites could be classified into Plant amines often derive from amino several categories according to various acids by decarboxylation (Smith, 1980). features like their chemical structure,

Several subcategories of plant amines are solubility in different solvents, or the

identified in relation to number of amino pathway of their biosynthesis. Another

groups in their structure – simple aliphatic important classification is related to

monoamines; aliphatic diamines; aliphatic the presence or absence of nitrogen in

polyamines, amines containing various their chemical structure (Gershenzon

heterocyclic groups. Several plant amines 2002). Thus, secondary metabolites

serve as precursors in biosynthesis of form two major groups: 1) nitrogen

polyamine alkaloids.

containing – alkaloids, non-protein Cyanogenic glycosides are natural amino acids, amines, cyanogenic

compounds containing cyanide group in glycosides, and glucosinolates; and 2)

their structure and can release HCN by without nitrogen – terpenes (mono-,

hydrolysis (Conn, 1980; Gershenzon, sesqui-, di-, tri-, tetraterpenes, steroids,

2002). They also derive from common saponins), phenolics (phenolic acids

amino acids and could be classified on the and phenylpropanoids), polyketides and

basis of the glycosylated group. polyacetylenes.

Glucosinolates are sulfur- and More than 12000 alkaloids are

nitrogen-containing compounds, dis- synthesized in plants (Gershenzon, 2002;

tributed predominantly in dicotyledonous Zhang and Bjorn, 2009), and derived from

plant families and like other nitrogen- amino acids such as ornithine, lysine,

secondary metabolites phenylalanine, tyrosine, tryptophan,

containing

are synthesized from common amino histidine, and aspartic acid. Five major

acids (Underhill, 1980; Gershenzon alkaloid subgroups are identified and

2002). Glucosinolates are precursors of representative alkaloids are shown in Fig.

mustard oils and similarly to cyanogenic

1. glycosides, they can also release toxic Beside the well known 20 essential

volatiles like isothiocyanate. amino acids involved into protein

Up to date, approximately 29000 structures more than 500 other uncommon

terpenoids/terpenes are discovered. non-proteinogenic amino acids are

Terpenes derive from their precursor

UV radiation and secondary metabolites

Figure 1. Major alkaloid subgroups and chemical structures of some representatives. isopentenyl diphosphate and are for generation of the ubiquinone prenyl

classified by the number of isoprene group in mitochondria (Croteau et al., units. There are two major pathways for

2000). The key biosynthetic enzymes biosynthesis of isopentenyl diphosphate:

are prenyltransferases and monoterpene

1) acetate/mevalonate pathway in cytosol synthase/cyclases. Some major repre- and endoplasmatic reticulum and 2) sentatives of the terpene group are

glyceraldehyde

presented in Fig. 2. Steroids and saponins pathway in plastids (Croteau et al., 2000;

phosphate/pyruvate

are secondary metabolites closely related Gershenzon, 2002). Additionaly, the

to terpenoids (Grunwald, 1980, Croteau, acetate/mevalonate pathway is implicated

et al. 2000).

Katerova et al.

Figure 2. Terpenes classification based on isoprene unit numbers. More than 11000 plant phenolics

derivatives (C 6 -C 1 skeleton), so called are synthesized mainly by two major

phenolic acids and phenylpropanoids (C 6 - pathways: shikimic acid pathway and

C 3 skeleton) (Fig. 3).

malonic acid pathway (Gershenzon, 2002). Phenolic acids are common substances The plant phenols could be classified

widespread in plant species. Several well- into two major categories – benzoic acid

known plant acids belong to this category:

UV radiation and secondary metabolites 119

Figure 3. Classification of plant phenolics and some important representatives.

Katerova et al.

salicylic acid, vanillic acid, gallic acid, 4-phenylcoumarins (neoflavanoids), and etc. (Harborne, 1980).

isocoumarins are recognized. Phenylpropanoids are classified into

Stilbenes (C 6 -C 2 -C 6 ) consist of a several subgroups: flavonoids, hydroxy-

trans (or cis ) ethene bond substituted cynnamic acids, cynnamic aldehydes,

with a phenyl group on both carbon coumarins, lignins, lignans, stilbenes and

atoms of the double bond. The most suberins.

abundant natural stilbenes are resveratrol

Hydroxycynnamic acids (C 6 -

and lunularic acid (Harborne, 1980).

C 3 ) are derivatives of cinnamic acid Suberins are lipid-derived bio- (Harborne, 1980). They are formed from

polymers consisting of long-chain (C 16 trans -cinnamic acid by a series of

to C 22 ) dicarboxylic acids, long-chain hydroxylations and O -methylations to

(C 20 to C 26 ) components like acids and yield compounds like p -coumaric, caffeic,

alcohols and substantial amount of ferulic and sinapic acid. Hydroxycynnamic

phenolic compounds (Thompson, 1980). acids rarely occur in free forms and

Among the phenylpropanoids in usually exist as conjugates, mostly as

plants flavonoids are the most plentiful esters of glucose or various organic acids

group including more than 9000 or amides, and less often as glycosides.

representatives. They are synthesized Lignins (C 6 -C 3 ) n are complex in plants via the flavonoid branch of the phenolic heteropolymers based on

phenylpropanoid and acetate-malonate phenylpropanoid units resulting pathway. Flavonoids comprise 15 carbon

from the oxidative polymerization of atoms - two aromatic rings (A and B) hydroxycinnamoyl alcohol derivatives connected with a 3-carbon bridge (C

(Ibrahim, 2001b; Gershenzon, 2002;Vogt, ring). The basic flavonoid skeleton can 2010).

tolerate a large number of substitutions,

for example hydroxyl groups, methyl group of phenylpropanoids which are

Lignans (C 6 -C 3 ) 2 constitute a different

groups, sugars (e.g. glucose, galactose, biochemically related to lignins. Lignans

rhamnose), etc. Introducing a second are synthesized from phenylalanine via

hydroxyl group at o -position in the B dimerization of substituted cinnamic

ring of flavonoids is responsible for alcohols to yield monolignol-derived

the enhancement of the antioxidant dimers and some oligomers (Harborne,

capacity of the resulting compounds 1980; Ibrahim, 2001b).

(Edreva et al., 2006, 2008). Sugars and Coumarins (C 6 -C 3 ) are structurally hydroxyl groups increase the water considered as the lactone derivatives of

solubility of flavonoids, while methyl 2-hydroxy-(cis)-cinnamic acids which

and isopentyl groups make flavonoids result in an apyrone nucleus. More than

lipophilc. Flavonoids are divided into 500 naturally occurring coumarins are

several subclasses: flavones, flavanones, known and they are mostly spread only

flavonols, flavanols, anthocyanidins, in few plant families like Umbelliferae

isoflavonoids (Ibrahim 2001a; Buer et and Rutaceae (Harborne, 1980, Ibrahim

al., 2010).

2001b). Beside the typical coumarins, Flavones are a class of flavonoids some other classes as furanocoumarins,

based on the basic flavone structure

UV radiation and secondary metabolites

with substituents mainly on 4’, 5, and pelargonidin, peonidin, and petunidin.

7 carbon atoms and lack of –OH group Isoflavonoids (isoflavones) are in position 3. Apigenin and luteolin and

similar to flavones, but the B ring is their respective glycosides are commonly

attached to C 3 of the C ring. The major found in many herbaceous plant species

representatives are the isoflavones (Harborne, 1980).

genistein and daidzein and their Flavanones are presented in high

respective glycosides daidzin and concentrations in citrus fruits. Flavanones

genistin. They are found almost

exclusively in leguminous plants with the flavone structure. The most common

have no double bond between C 2 and C 3 of

highest concentrations in soybean. flavonones are hesperetin, naringenin,

Tannins are secondary metabolites and their glycosides hesperidin and which can be divided in two categories naringin.

– hydrolyzable (gallotannins) and The molecule of flavonols has a

condensed (Harborne, 1980, Gershenzon double-bonded oxygen atom attached

2002). The hydrolyzable tannins usually to position 4 and double bond between

contain glucose and phenolic acids

2 C 3 and C of the flavone structure, (mainly gallic acid). Condensed tannins and –OH group at C 3 . Flavonols are (or polyflavonoid tannins, catechol-type

present in a wide variety of fruits and tannins, pyrocatecollic type tannins, vegetables mainly as O-glycosides. The

non-hydrolyzable tannins or flavolans) most abundant flavonols are quercetin

are polymers formed by the condensation (main glycosides – rutin and quercitrin),

of flavans and they do not contain myricetin (glycoside myricitrin), and

sugar residues. Tannins have molecular kaempferol. More than 200 different

weights ranging from 500 to over 3000 sugar conjugates of kaempferol are

(gallic acid esters) and up to 20000 discovered.

(proanthocyanidins).

Flavanols (catechins and epi- Quinones are aromatic dicarbonyl catechins) are flavonoids without double

compounds. The two carbonyl groups bonds in the C ring, and have –

usually are in p -position and form colored OH group at C 3 . They occur in a number

pigments. Most of the naturally occurring of plant species, but predominantly in

quinones contain a long isoprenoid side cocoa, green tea and some woody species

chain, and are divided in two major as birch, pine and apple (Harborne, 1980;

structural groups – naphtoquinones Kostina et al. 2001; Lavola et al. 2003;

and benzoquinones. Usually naturally Solovchenko and Schmitz-Eiberger,

occurring quinone pigments contain 2003). Another two classes of flavanols

phenolic or metoxyl constituents and are flavan-4-ol and flavan-3,4-diol.

have important role in vital physiological Anthocyanidins have a positive

processes in plants (Harborne, 1980). charge in the C ring and two double

Polyketides are secondary meta- bonds in the C ring. Anthocyanins are

bolites from plants that are usually anthocyanidin glycosides, and most

synthesized in a similar to fatty common in plants are the glycosides acids biosynthetic process through of cyanidin, delphinidin, malvidin,

decarboxylative

condensation of

122 Katerova et al.

malonyl-CoA. The polyketide chains produced by a polyketide synthase are often further modified into bioactive metabolites (Thompson, 1980). Poly-

acetylenes are natural products containing carbon-carbon triple bond functionality. There are discovered approximately 2000 polyacetylenes, and near 1200 of them are found in plants of Asteraceae (Compositae) family. Polyacetylenes derive from fatty acids and polyketide precursors (Minto and Blacklock, 2008).

Secondary metabolites play an important role in many plant physiological

and developmental processes such as root nodule formation; determination of pollen germination and pollen functionality; leaf and petal pigmentation; gravity responses; regulation of auxin binding and transport; inhibition of certain enzymatic activities; influence on cellular protein phosphorylation. They also contribute to stress responses as signaling molecules, potent scavengers of ROS, and to the

protection against pathogens and UV irradiation

(Winkel-Shirley,

Ibrahim, 2001a; Gershenzon, 2002; Velikova et al., 2004; Edreva, 2005; Velikova et al., 2007; Korkina, 2007, Edreva et al., 2007; Edreva et al., 2008; Buer et al., 2010; Samanta et al., 2011). Furthermore, plant secondary metabolites possess biological activities which are important for human life and health. A number of articles have documented the benefits of plant secondary metabolites and their use in traditional and modern medicine, food industry, perfumery and cosmetics (Havsteen, 2002; Korkina, 2007; Dinkova-Kostova, 2008; Jansen et al., 2008; Zhang and Bjorn, 2009; Caputi and Aprea, 2011; Wijesinghe and Jeon, 2011). Most of the known functions

of alkaloids are related to protection against insects, but they also contribute to animal metabolism as important neurotransmitters. Many alkaloids are used in medicine as antiarrhythmics, anticholinergics, antitumors, vasodilata- tors, antihypertensives, anesthetics, analgesics, as well as muscle relaxants, inhibitors

of acetylcholinesterase, antipyretics, and antiprotozoal agents. Terpenes and terpenoids are important components of plant essential oils that are also extensively used as

natural flavor additives in food, as fragrances in perfumery, and in traditional and alternative medicine

such as aromatherapy. Plant-derived phenylpropanoids (especially flavonoids) and their derivatives are among the most

common biologically active components in food, wines, beer, spices, aromas, fragrances, and essential oils. Taking in account their defensive roles, these compounds are of great medicinal interest, especially as free radical scavengers, antioxidants, UV screens, anticancer, antivirus, anti-inflammatory, wound healing, antibacterial, and metal chelating agents.

3. UV radiation and secondary metabolites

3.1. Impact of UV-C on the synthesis of secondary metabolites

3.1.1. In vitro cultured plant cells, tissues and calli

UV-C light exposure of sterile cultures of Scenedesmus quadricauda (Chlorophyceae) over 1h did not

influence total soluble phenols and flavonoids (Kováčik et al., 2010).

UV radiation and secondary metabolites

Phenolic acids were altered differently derivatives of boswellic acid - active by UV-C - vanillic acid increased;

metabolite produced in Boswellia serrata gallic,

Roxb. (endangered medicinal plant). p -coumaric acids were decreased,

The authors found that 5 min of UV-C while protocatechuic and salicylic acids

irradiation was effective for production did not change significantly. Selected

and accumulation of acetyl-11-keto-β- flavonols (quercetin and kaempferol)

boswellic acid (10-fold) and β-boswellic were not detected after UV-C treatment.

acid (7-fold) in the callus culture. The The authors concluded that the exposure

synthesis of stilbenes was extensively time to UV light was not sufficient

investigated in grape calli systems to

(Liu et al., 2010). The authors reported changes of the phenolic metabolites in

stimulate more

considerable

induced by UV-C in vitro production of Scenedesmus quadricauda. Exposure resveratrols and their glucoside (piceids)

of Chlamydomonas nivalis (so-called in four grape genotypes and three tissue snow alga) cells to UV-C light resulted

types of each genotype. UV-C irradiated in a three-fold increase in free proline

calli accumulated stilbenes which have occurred within two days after exposure

been already reported to have a number to UV-C, accompanied with a 12–24%

of health-beneficial properties, such as increase in phenolics after 7 days of

antioxidant capacity, cardioprotective exposure (Duval et al., 2000). The

effects, and anti-mutagenic, estrogenic authors report that UV-C light exposure

and anti-cancer activity (Hung et al., can stimulate phenolic-antioxidant

2000; Sgambato et al., 2001). Further by production in aplanospores of C. nivalis

methylation of resveratrol via O-methyl- which supports the idea that there is

transferases can be generated its methyl

a considerable biotechnological and ether pterostilbene (trans-3,5-dimethoxy- pharmaceutical potential incorporated 4′-hydroxystilbene) which is known to

within the genome of this UV-tolerant possess fungicidal, antioxidant, anti- snow alga. Moreover, UV-induced

cancer , and antiinfective properties. secondary

Xu et al. (2012) isolated VpROMT similar to that in C. nivalis , may provide

pseudoreticulata resveratrol

a valuable source of pharmacological O -methyltransferase) gene from the products targeted for anticancer,

Chinese wild plant V. pseudoreticulata, anticoagulant, antimicrobial, or anti-

which had 98.9% and 98.3% identity inflammatory treatments (Duval et

to the resveratrol O -methyltransferase al., 2000). Additionally, by using blue

gene of V. vinifera at the nucleotide and autofluorescence method Lesniewska

amino acid levels and found that gene et al. (2004) also found that UV-C light

expression level was rapidly induced by forced Vitis vinifera cells to produce UV-C irradiation in suspension culture phytoalexins - secondary metabolites

cells of Vitis romanetii. Ku et al. (2005) with antimicrobial properties.

also found that synthesis of resveratrol Ghorpade et al. (2011) used tissue

and piceatannol (stilbenoids) were culture techniques and examined the

promoted by UV-C radiation in callus effect of UV-C on the synthesis of four

cultures of peanut.

Katerova et al.

3.1.2. Plant organs Nadeau et al. (2012) studied the Total polyphenols and phenol-

effect of hormetic UV-C dose on carboxylic acids in potato and buckwheat

glucosinolates - secondary metabolites were examined by Orsák et al. (2001)

derived from amino acids which are who found that the content of secondary

the precursors of bioactive compounds metabolites studied was enhanced by

with anti-cancer properties such as UV-C irradiation. Schmidlin et al. (2008) sulforaphane and indole-3-carbinol.

reported that a whole range of stilbene The authors showed that UV-C tended derivatives (including trans -resveratrol,

to enhance 4-methoxyglucobrassicin, trans - and cis-piceid, trans-ε and trans-δ

4-hydroxyglucobrassicin and gluco- viniferins, and trans-pterostilbene),

raphanin in broccoli florets. So they are induced in leaves of grapevine

suggested that hormetic dose of UV-C (Vitis vinifera , Cabernet Sauvignon

had biochemical significance to enlarge variety) by UV-C (6 min, 90 mW cm -2 ).

potential health effect of broccoli in Balouchi et al. (2009) investigated the

cancer prevention by increasing bioactive changes in photosynthetic pigments and

compounds.

other physiological and biochemical traits of durum wheat leaves exposed

3.2. Impact of UV-B on the synthesis of

to UV-C radiation. Their results

secondary metabolites

showed that carotenoids, anthocyanins, flavonoids and proline content increased

3.2.1. In vitro cultured plant cells, tissues significantly by UV-C as compared

and calli

to the control. Other authors (Boveris Accumulation and tissue localization et al., 2001) found that the pigment

of phenolic compounds in response to apigeninidin -2 (3-deoxyanthocyanidin) UV-B radiation (up to 40 d, 0.74 W m )

accumulated in the epidermal areas of was studied in two strains of Camellia soybean cotyledons irradiated for 60

sinensis L. (tea plant) callus cultures, min with UV-C light. Interestingly, the

which varied in biosynthetic capacity authors stated that this pigment was not

(Zagoskina et al., 2003). UV-B treatment verified in soybean species principally

affected negatively culture growth and and only UV-C (not UV-B) led to its

size of the callus-forming cells. However, induced accumulation. The in vitro

UV-B radiation induced a considerable test showed that apigeninidin had the

increase in soluble phenolics and flavans ability to quench some semiquinone

but the rise in polymeric forms as lignin radicals as ascorbyl and lipid radicals in

was negligible. Phenolic deposition in

a dose-dependent manner and possessed cell walls and intercellular space as well antioxidant capacity. Twenty four hour-

as the deposition of lignin-resembling UV-C irradiation was also effective in the

substance on the callus cultures surface reddening of yellow saffron thistle florets

also rose. Further, the strain possessing to yield carthamin which is applied as a

a higher rate of phenolic compounds colour additive for processed foods, in

accumulation revealed greater tolerance cosmetic and medicinal industry (Saito,

to the UV-B radiation, demonstrating 2001).

the key role of these metabolites in cell

UV radiation and secondary metabolites

protection to UV-B light. -2 37.9 kJ m ) the optimal was found to be

UV-B irradiation induced a rise of -2 25.3 kJ m . It was reported that exposure nitric oxide (NO) production, activities of

of P. quadrangularis calli to the optimal nitric oxide synthase and phenylalanine dose increased the production of all ammonia lyase (leading to flavonoid

studied flavonoids, which was 6 to 40- synthesis), as well as flavonoid level in

fold higher than elicitation with methyl Ginkgo biloba callus (Hao et al., 2009).

jasmonate. In addition, UV-B treatment The authors reported that both inhibitors

led to a higher antioxidant activity of nitric oxide synthase and nitric oxide

compared to non-treated calli. Further, reduced phenylalanine ammonia lyase UV-B exposure of callus cultures for 7 activity and the production of flavonoids.

days caused production of isoorientin It was noted that both phenylalanine

similar to the quantities found in fresh ammonia lyase activation and flavonoids

leaves from glasshouse-grown plants. synthesis in UV-B treated G. biloba

The induction of monoterpenoid callus were induced mainly by NO

indole alkaloids camptothecin and signaling molecule. Further, Loyall et

10-hydroxycamptothecin by phyto- al. (2000) demonstrated a contribution

hormones, heavy metals, hydrogen of the non-enzymatic and enzymatic

peroxide and UV-B radiation were antioxidants glutathione and glutathione evaluated in the Chinese medicinal tree

S-transferase in the initial events of UV- Camptotheca acuminate cell culture (Pi dependent signaling to the gene encoding

et al., 2010). UV-B and salicylic acid the key enzyme chalcone synthase (Chs)

showed the most prominent effects as in parsley (Petroselinum crispum) cell elicitors of the studied alkaloids, which

cultures. Thus, using several short pulses are valuable due to their considerable of UV-B radiations, the authors proved

anti-tumor actions. UV-B irradiation that the oxidative cell status played a role

of Catharanthus roseus multiple shoot of a central regulating element.

cultures and cell suspension cultures was The effect of elicitation by methyl

shown to induce a considerable rise in the jasmonate and/or UV-B radiation on the

production of terpenoid indole alkaloids, production of four C-glycosyl flavonoids

along with precursors of the dimeric (isoorientin, orientin, isovitexin, vitexin)

alkaloids vinblastine and vincristine was examined in callus cultures from leaf

(composed of both vindoline and explants of Passiflora quadrangularis

catharanthine), known to be effective in (Antognoni et al., 2007). Flavonoids

the treatment of leukemia and lymphoma shield UV-B radiation, play a defense

(Binder et al., 2009). Other authors role against pathogen attacks, function

exposed stationary phase cell suspension as attractants to pollinators, and because

cultures of C. roseus to low UV-B dose of their high antioxidant activity, they are

and succeeded to enhance substantially considered to posses health-promoting

the amounts of catharanthine and assets for humans and to provide

vindoline without affecting cell growth protection against cardiovascular disease,

and viability (Ramani and Chelliah, cancer, and age-related disorders. Among

2008). The concentrations of these all UV-B doses tested (12.6, 25.3 and

secondary metabolites were found to be

Katerova et al.

highest 48-72h after UV-B treatment. authors assumed that the octadecanoid In general, cell cultures of C. roseus

pathway did not actively control the produce terpenoid indole alkaloids, but

generation of terpenoid indole alkaloids fail in vindoline production, noted to

under normal or UV-B stress conditions

be an important component of the anti- in C. roseus . Leaf concentration of tumor dimeric alkaloids. Further, Ramani

another monoterpene indole alkaloid and Chelliah (2007) showed that cell

brachycerine, possessing antioxidant and surface receptor(s), calcium, medium

antimutagenic activities was also noted alkalinization, ROS, Ca +2 -dependent to increase significantly in UV-B-treated

protein kinase and mitogen-activated cuttings of Psychotria brachyceras protein kinase have an important role Müll. Arg. (do Nascimento et al., 2013).

in UV-B signaling, in transcriptional The authors supposed that brachycerine activation of triptophan decarboxylase

probably participated in acute UV-B (Tdc) and strictosidine synthase responses and at least partially its

(Str ) genes, which encode enzymes accumulation might be regulated at participating in biosynthesis of terpenoid

transcriptional level. The expression of indole alkaloids, and subsequent

the majority genes involved in peppermint accumulation of catharanthine.

(Mentha x piperita L.) terpenoid biosynthesis were also modulated by

3.2.2. Plant organs exposure to UV-B (7.1 kJm -2 day -1 UV BE ) Ouwerkerk et al. (1999) reported

radiation of plants grown in field and in a that UV-B specifically induced a Tdc-

growth chamber, but it did not correlate gusA construct in tobacco. In addition,

with the amount of most essential oil UV-B induced expression of Tdc gene compounds (Dolzhenko et al., 2010). and accumulation of terpenoid indole

The authors documented enhanced alkaloids, but the percentage induction

phenolic compounds like flavonoids of catharanthine and vindoline was

eriocitrin, hesperidin and kaempferol not markedly enhanced (14 and 11%,

7-O-rutinoside in UV-B treated plants. respectively) in C. roseus leaves. Binder

The interaction between terpenoid and et al. (2009) revealed that up to 168h

flavonoid production in response to after UV-B exposure an augmented

UV-B was proven by the higher essential lochnericine and reduced hörhammericine

oil amount in the growth chamber plants amounts were found in hairy roots of

associated with lower total phenolic

C. roseus . When UV-B exposure time contents; and the decreased terpenoid was increased up to 20 min a rise in

concentrations in field grown peppermint lochnericine, serpentine, and ajmalicine

related with increased content of phenolic and decline in hörhammericine was

compounds. Expectedly, it was concluded noted. Peebles et al. (2009) examined

that field grown plants were better the role of the endogenous production of

adapted to increasing UV-B irradiation jasmonic acid via octadecanoid pathway

than peppermints in the growth chamber in the production of terpenoid indole

due to enhanced flavonoid concentration alkaloids in C. roseus hairy roots using (Dolzhenko et al., 2010). Similarly, octadecanoid pathway inhibitors. The

Johnson et al., (1999) found that the

UV radiation and secondary metabolites

broad-leaf variety of sweet basilicum plants to different spectral quality of (Ocimum basilicum) containing light (red, blue, white) and UV-B showed phenylpropanoids in its essential oils,

the highest melatonin concentration after after UV-B exposure showed a strong

exposure to high intensity UV-B radiation increase of the phenylpropanoids

for 3 days, which decreased after longer (eugenol and methyl-eugenol) and the

exposure period. The authors assumed terpenoids (1,8-cineole, linalool, trans-

that melatonin protected G. uralensis β-ocimene, α- and β-pinene, sabinene,

plant against UV-B-triggered oxidative β-myrcene, limonene, α-terpinolene,

damage. In addition, Solhaug et al. borneol, α-terpineol, α-trans-bergamo-

(2003) showed that induction of melanin tene, γ-cadinene, germacrene D). UV-B

and parietin (anthraquinone, possessing induction of these secondary metabolites

antifungal activity) synthesis in Lobaria was found to be strongest in the five-

pulmonaria and Xanthoria parietina leaf than in two-leaf plants. Further,

lichens required presence of UV-B. UV-B-induced accumulation of the

Secondary metabolites, including monoterpene trans -ocimene was also

flavonoids and terpenoids, are important observed in leaves (predominantly in

for UV-B induced lessening of plant mature than in developing leaves) of

tissue quality required for microbial and linalool-rich commercial variety of sweet

herbivory pathogenes (Bassman, 2004; basilicum lacking phenylpropanoids in

Roberts and Paul, 2006; Izaguirre et al., its essential oil (Ioannidis et al., 2002). In

2007). Izaguirre et al. (2007) reported addition, it was reported that UV-B was

that both UV-B and simulated herbivory necessary for the normal development

induced the accumulation of several leaf of oil glands, in particular for the filling

phenolic compounds (chlorogenic acid of glandular trichomes of sweet basil.

and dicaffeoylspermidine isomers) in Other authors revealed that short term

Nicotiana attenuata and N. longiflora high intensity (3d, 1.13Wm -2 ) and long plants. The flavonoid rutin was specifically

term low intensity (15d, 0.43Wm −2 ) increased by UV-B irradiation. In another UV-B irradiation induced nearly 1.5-fold

study (Kreft et al., 2002), rutin and higher glycyrrhizin (an oleanane-type

tannin concentrations were reported to triterpenoid saponin, natural sweetener

be reduced in the following order: under possessing anti-tumor and anti-viral

ambient > UV-B enhanced (simulating activities) production in roots of only

17% O 3 depletion) > UV-B depleted

3 month-old Glycyrrhiza uralensis (using Mylar foil) conditions in field than in control plants (Afreen et al.,

grown Fagopyrum esculentum Moench 2005). Using a similar model system,

(buckwheat). The highest amounts of Afreen et al. (2006) compared the

these compounds were determined in concentration of melatonin (N-acetyl-5-

flowers, followed by leaves and stems. methoxytryptamine, an indole amine) in

Tattini et al. (2004) identified and different tissues (seed, root, leaf and stem)

quantified the polyphenol spectrum of G. uralensis and noted the highest of Ligustrum vulgare leaves grown amount in root tissues, which increased

outdoors and exposed to increasing with plant development. Exposure of

sunlight (receiving PAR, UV-A and UV-

Katerova et al.

B). A prominent rise in the accumulation availability in pine seedlings. A similar of singlet oxygen ( 1 O

investigation was done with silver birch flavonoids (quercetin 3-O-rutinoside and

2 )-scavengers as

(Betula pendula ) showing that UV-B luteolin 7-O-glucoside occurring in both

exposure did not increase phenolic acids adaxial epidermis and palisade tissue) or condensed tannins, but significantly and hydrohycinnamates (echinacoside,

enhanced flavonoids (quercitrin, hyperin, occurring mainly in abaxial tissues) was

kaempferol-3-rhamnoside and myricetin- reported in response to solar radiation. 3-galactoside), which are important

The authors assume that a coordinated UV-B shields (de la Rosa et al., 2001). control system exists between flavonoids

The concentration of some flavonoids and hydroxycinnamate pathways and

was also found to depend on UV-B dose. flavonoids may serve antioxidant

In another study with silver birch it was functions in L. vulgare exposed to excess

shown that the amount of condensed light. Different physiological parameters

tannins and anthocyanins in leaves was were measured in Scots pine (Pinus

not altered by UV-B light (Tegelberg et sylvestris ) subjected to different UV-B

al., 2004). The authors showed that UV-B levels for one growing season (Lavola

treatment increased the concentration of et al. 2003). The authors suggested that

quercetins, kaempferols, and chlorogenic pine plants was adequately protected

acids but along all measured plant growth against supplemental irradiation. It parameters only leaf area was negatively was noted that UV-B affected mainly

affected. In another study, Kostina et al. secondary metabolites. Under moderate

(2001) demonstrated a rise in (+)-catechin, nutrient availability, the accumulation

quercetin, cinnamic acid derivatives, of flavonols in P. sylvestris needles was

apigenin and pentagalloylglucose in highest at the ambient (4.3 kJ m -2 day -1 )

leaves of birch seedlings exposed or near to ambient UV-radiation doses.

to enhanced UV-B radiation. The At elevated nutrient level the UV-B

significant negative correlations between doses higher than ambient (up to 13.1

apigenin, and mainly quercetin amounts kJ m -2 day -1 ) specifically enhanced the

and levels of lipid peroxidation revealed accumulation of diacylated flavonols

the antioxidant role of these secondary (dicoumaroyl-trifolin,

dicoumaroyl- metabolites (Kostina et al. 2001). Wulff isorhamnetin,

dicoumaroyl-astragalin et al. (1999) also documented UV-B- and dicoumaroyl-isoquercitin) in a

dependent accumulation of quercetin dose-dependant manner. Non-acylated

3-glycoside in European silver birch flavonols were increased to a lesser extent

(Betula pendula Roth.) seedlings by UV-B treatment but condensed tannins

exposed to high UV-B radiation

were not enhanced. The major effects -1 levels (14.4 or 22.5 kJ m d UV-B BE ) of UV-B radiation were on the pathway

accompanied with a transient increase division converting dihydroflavonols to

of Chs mRNA assuming induction of flavonols. The authors assume that the

flavonoid biosynthesis. The reported production of secondary metabolites

weaker induction of Chs mRNA levels through flavonoid pathway is multi-step

in the higher UV-B dose exposure led regulated by UV-B exposure and nutrient

to the suggestion that a substantial DNA

UV radiation and secondary metabolites

damage took place, which could partially deciduous as V. myrtillus , but possessed inhibit the transcription of Chs as well.

leaves with structural similarity to V. The concentrations of flavonol conjugates

vitis-idaea . Kumari and Agrawal (2010) and betacyanins in the halophyte

also applied supplemental to the ambient Mesembryanthemum crystallinum

was -2 d UV-B radiation (1.8 and 3.6 kJ m -1 ) on also reported to increase after exposure

the aromatic perennial herb Cymbopogon to very low wavelength UV-B (like

citratus (D.C.) Staph in field conditions 280 or 295 nm) and the amount of

and reported that only the high dose feruloylglucose (the precursor of flavonol

inhibited biomass production. A reduction conjugates and acylated betacyanins)

of chlorophyll content without significant was much higher in leaves than in leaf

alteration in photosynthesis, and increase tips (Ibdah et al., 2002). The authors

of carotenoids and phenolic compounds reported that accumulation of flavonols

was noted in UV-B treated plants. and betacyanins could be illustrated by a

The authors demonstrated the positive weakly sigmoid dose function along with

outcome of the supplemental low dose of an exponential reduction of the response

UV-B radiation on volatile oil production function of the plant with increasing

acompanied by dense waxy deposition wavelength.

on the adaxial surface of the leaves. Other authors observed that three

Further, using 45 species from different closely related species of the sub-Arctic

genera, Holmes and Keiller (2002) dwarf shrubs (Vaccinium myrtillus L.,

demonstrated that one of the functions Vaccinium vitis-idaea L., and Vaccinium

of leaf waxes is shielding of UV-B. In uliginosum L.) grown outdoors showed

another study, a reduction of epicuticular different strategies in UV-B response

wax, photosynthetic pigments and concerning the content and distribution

flavonoid content in needles of Korean of UV-absorbing phenolic compounds

pine (Pinus koraiensis Sieb. et Zucc) was in leaves (Semerdjieva et al., 2003). • associated with a rise in ROS ( OH, H

2 O 2 ) Methanol-extractable UV-B absorbing

and malondialdehyde amounts as well as compounds were highest in V. myrtillus.

catalase activity after UV-B exposure (Zu They increased with UV-B irradiation,

et al., 2011). The authors reported that and were distributed all over the leaf

Korean pine had considerable sensitivity but concentrated in cells containing

to supplementary UV-B exposure and chlorophyll. The majority of phenolic

concluded that the induced antioxidant compounds in V. vitis-idaea were cell-

defense system was not efficient against wall bound, concentrated in the walls of

UV-B triggered injuries. Supplemental epidermis and their pool was enhanced

UV-B radiation applied for 3 months with UV-B dose. The authors assumed

to three-year-old Taxus chinensis var. that the difference in strategies for UV

mairei led to a significant augmentation screening found in those two plants could

of taxol (a diterpenoid) and flavonoid

be connected with leaf longevity. The content in fully expanded leaves, which response of V. uliginosum to UV-B was

play an important role in the observed found to be flexible. This plasticity was

plant tolerance (Zu et al., 2010). The taxol explained with the fact that the plant is

production of this shrub is well known

130 Katerova et al.

and it is used as an anti-tumor agent for treating breast and ovarian cancer, however up to now the major source of this molecule is obtained via extraction from T. chinensis var. mairei.