Relationship of secondary metabolism to growth in oregano (Origanum vulgare L.) shoot cultures under nutritional stress

Environmental and Experimental Botany 65 (2009) 54–62

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Relationship of secondary metabolism to growth in oregano (Origanum vulgare L.) shoot cultures under nutritional stress Vincenzo Lattanzio a,∗ , Angela Cardinali b , Claudia Ruta c , Irene Morone Fortunato c , Veronica M.T. Lattanzio b , Vito Linsalata b , Nunzia Cicco d a

Dipartimento di Scienze Agro-Ambientali Chimica e Difesa Vegetale, Facoltà di Agraria, Università degli Studi di Foggia, Via Napoli, 25, 71100 Foggia, Italy Istituto di Scienze delle Produzioni Alimentari - CNR, Via Amendola 122/O, 70126 Bari, Italy Dipartimento di Scienze delle Produzioni Vegetali, Facoltà di Agraria, Università degli Studi di Bari, Via Amendola 165/A, 70126 Bari, Italy d Istituto di Metodologie per l’Analisi Ambientale - CNR, Contrada S. Loja - C.P. 27, 85050 Tito Scalo-Potenza, Italy b c

a r t i c l e

i n f o

Article history: Received 3 January 2008 Received in revised form 6 July 2008 Accepted 3 September 2008 Keywords: Proline Trade-off between growth and secondary metabolism Rosmarinic acid Lithospermic acid Lithospermic acid B Oregano tissue and callus cultures

a b s t r a c t Micropropagation of Origanum vulgare L. by shoot buds, as a potential model system for studying carbon skeleton diversion from growth to secondary metabolism as adaptive response to nutrient deficiency, has been performed. In addition, the antioxidant phenolic compounds, produced by shoots under nutritional stress or in response to exogenously added proline, have been studied. Caffeic acid, rosmarinic acid, and lithospermic acid B have been isolated in oregano shoot cultures by reversed-phase high-performance liquid chromatography, and their structures have been elucidated by tandem mass spectrometry. Both nutritional stress, which in turn causes a moderate increase of constitutive free proline, and exogenous proline affect growth and antioxidant phenolic content of oregano shoots, compared to control. The role of proline, and the associated redox cycle, as a form of metabolic signaling based on a transfer of redox potential amongst interacting cell pathways, which in turn elicit phenolic metabolism via stimulated carbon flux through oxidative pentose phosphate pathway, is discussed. Furthermore, the potential use of oregano tissue and callus cultures as a new strategy to enable the production of useful secondary metabolites on a commercial scale is also discussed. © 2008 Elsevier B.V. All rights reserved.

1. Introduction There is increasing evidence implicating the existence of a general stress response system in plants and this suggests some common adaptive responses of plants to adverse environmental conditions. In this connection, plant phenolics are ubiquitous secondary metabolites that function in plants to regulate the stress response. The accumulation of phenolics in plant tissues is a distinctive characteristic of plant stress: phenolic compound may be increased or de novo synthesized in plants as a response to various biotic and abiotic stresses, including nutrient deficiency. Both the oxidative pentose phosphate pathway (OPPP) and Calvin cycle can provide carbon skeletons in form of erythrose-4phosphate, which along with phosphoenolpyruvate, formed from glycolysis, serves as a precursor for phenylpropanoid metabolism via the shikimic acid pathway. Under stress conditions, reduced photosynthetic rates may necessitate higher rates of OPPP to provide carbon skeletons to phenolic metabolism. In addition,

∗ Corresponding author. Tel.: +39 320 4394738; fax: +39 0881 740211. E-mail address: [email protected] (V. Lattanzio). 0098-8472/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2008.09.002

several studies have suggested that OPPP is the source of reducing equivalents for phenolic compound biosynthesis, and that its activity is increased under conditions of increased flux into the phenylpropanoid pathway. Understanding the regulatory and biochemical mechanisms that control the types and amounts of phenolic compounds synthesized under different conditions, continues to be a high priority for research with an eye to crop plants that could be engineered to overproduce antioxidant phenolics (Robbins et al., 1985; Lattanzio et al., 1994; Dixon and Paiva, 1995; Fahrendorf et al., 1995; Hare et al., 1999; Winkel-Shirley, 2002). In many plants, free proline also accumulates in response to a wide range of biotic and abiotic stresses, including nutrient deficiency. Accumulation of proline could be due to de novo synthesis, decreased degradation, or both. Most attempts to account for the phenomenon have focused on the ability of proline to mediate osmotic adjustment, scavenge free radicals, and to act as a source of reducing power and a source of carbon and/or nitrogen. Accumulated proline has been proposed to protect enzymes, membranes, and polyribosomes during environmental perturbations (Kiyosue et al., 1996; Hare et al., 1998; Kavi Kishor et al., 2005).

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Furthermore, it has been suggested that since the primary capture of photon energy is insensitive to stress, plants under adverse conditions are frequently exposed to light intensities in excess than those that can be used for carbon assimilation. In these conditions the limited regeneration of NADP+ results in cellular redox imbalance. Therefore, it has been proposed that a stress-induced increase in the transfer of reducing equivalents into proline synthesis (cytosolic) and degradation (mitochondrial) cycle might enable sensitive regulation of cellular redox potential in cytosol. High levels of cytosolic proline synthesis, catalyzed by two enzymes, 1 -pyrroline-5-carboxylate-synthetase (P5CS) and 1 -pyrroline5-carboxylate-reductase (P5CR), both using NADPH as a cofactor, during stress may maintain NAD(P)+ /NAD(P)H ratios at values compatible with metabolism under normal conditions. In addition, the increased NADP+ /NADPH ratio, mediated by proline biosynthesis, is likely to enhance the activity of the OPPP: this would provide precursors to support the demand for increased secondary metabolite production, such as phenolic compounds, during stress (Merrill et al., 1989; Hare and Cress, 1997; Hare et al., 1998). A tight link between proline synthesis and OPPP activity has several precedents in both animal and plant systems (Smith et al., 1980; Yeh et al., 1984; Phang, 1985; Yeh and Phang, 1988; Kohl et al., 1990; Shetty, 1997; Shetty, 2004). The two dehydrogenases responsible for transforming glucose-6-phosphate into ribose-5-phosphate are primarily regulated by the NADP+ /NADPH ratio, with both enzymes being strongly inhibited by NADPH. Dehydrogenase reactions that consume NADPH and produce NADP+ would positively interfere with OPPP activity: the alternating oxidation of NADPH by proline synthesis and reduction of NADP+ by the two oxidative steps of the OPPP would link these two pathways (Hare and Cress, 1997; Kavi Kishor et al., 2005). The rationale for this research is based on the studies of Shetty and co-workers showing that phenolic metabolites in plants are efficiently produced through an alternative mode of metabolism, linking proline synthesis with the oxidative pentose phosphate pathway and that plant tissue culture techniques could be used for controlled production of useful antioxidant phenolics on demand (Shetty, 1997; Yang and Shetty, 1998; Zheng et al., 2001; Shetty and McCue, 2003; Shetty, 2004). Production of secondary metabolites by plants, in fact, is not always satisfactory. It is often restricted to a species or genus and might be activated only during a particular growth or developmental stage, or under specific seasonal, stress or nutrient availability conditions. For these reasons in the past years a lot of effort has been put into plant cell cultures as a possible production method for plant secondary metabolites of commercial interest (Grotewold et al., 1998; Verpoorte et al., 1999, 2002; Zhang et al., 2004). In the present paper, oregano (Origanum vulgare L.) shoots have been grown on Murashige and Skoog medium, with or without exogenous proline added, in order to study the effects of nutrient deficiency and/or exogenous proline on metabolic fluxes of carbon skeletons between primary and secondary metabolism, with reference to phenolic metabolism. The results presented in this study could be targeted for improving the production of compounds of interest or for the design of functional foods. More generally, this research has been carried out in order to understand the relationships between primary and secondary metabolism and their responses to environmental conditions. The research places emphasis on the fundamental understanding of metabolic pathways and their regulations, and on the interactions between competing and/or complementary pathways with the ultimate goal of a rational development of workable technologies that use the biosynthetic pathways of plant tissues for the production of useful secondary metabolites on demand.

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2. Materials and methods 2.1. Oregano shoot culture Oregano shoot cultures were obtained according to Yang and Shetty (1998). Origanum vulgare L. ssp. hirtum genotype from a 2-year-old oregano crop was used to establish micropropagation. Oregano axillary buds, 4–5 mm length, explants were induced to produce multiple shoots via adventitious shoot formation on basal medium (BM), containing the macronutrients according to Murashige and Skoog medium (MS) (Murashige and Skoog, 1962), the micronutrients of Nitsch and Nitsch medium (Nitsch and Nitsch, 1969), 25 mg/l Fe-EDTA, 0.4 mg/l thiamine HCl, 1 mg/l 6benzylaminopurine (BAP), 30 g/l sucrose, 7 g/l agar. The pH of the medium was adjusted to 5.6–5.8. Sterilization of culture media was performed in autoclave at 121 ◦ C for 20 min; the culture of the explants was instead carried out under a horizontal laminar flow hood to ensure the necessary sterile conditions. During the experiment, oregano explants were maintained in a growth chamber at 24 ± 1 ◦ C with a photoperiod of 16 h light under a light intensity of 30 ␮E m−2 s−1 . After a 45-day shoot multiplication, uninodal microcuttings (5–6 mm) were subcultured twice at intervals of about 3 weeks using the same medium conditions. To test the effects of nutrient deficiency and exogenous proline on antioxidant phenolic content, oregano shoots (three-nodal microcuttings) were transferred and cultured for 15 days on BAP (hormone) free MS medium, plus or without 0.5 mM proline, and BAP (hormone) free half-strength MS medium, plus or without 0.5 mM proline. MS medium without proline was used as control. On day 15, free proline content, total phenolic content, rosmarinic acid content, and fresh weight of oregano shoots were measured. Oregano leaves coming from in vitro plantlets were used as primary explants in the establishment of calli cultures on basal medium (BM) consisting of macronutrients (Murashige and Skoog, 1962) and micronutrients (Nitsch and Nitsch, 1969), FeEDTA (30 mg/l); thiamine HCl (0.4 mg/l); myo-inositol (100 mg/l); sucrose (30 g/l) and agar (7 g/l). For callus induction the BM was enriched with 2.2 mg/l of 2,4-dichlorophenoxyacetic acid (2,4 D). The plates were incubated in the dark at 24 ± 1 ◦ C. After 4 months, calli derived from individual leaves were separated from the explant tissue and transferred to medium, added with 2.2 mg/l of 2,4 D and 21.5 ␮g/l for the callus culture. Subculturing was then carried out at 2 months intervals with transfer of only the vigorously growing portions of calli on fresh medium.

2.2. Proline content Free proline content was determined according to the methods of Bates et al. (1973). 100 mg of plant material was homogenized in 2 ml of 3% aqueous sulfosalicylic acid. The homogenate was centrifuged at 13,000 g for 10 min and 1 ml of supernatant was placed in a reaction test tube. The sample was reacted with 1 ml of acid–ninhydrin (1.25 g of ninhydrin in 30 ml of glacial acetic acid and 20 ml of 6 M phosphoric acid) and 1 ml glacial acetic acid for 1 h at 100 ◦ C, and the reaction terminated in an ice bath. The reaction mixture was extracted with 2 ml of toluene and mixed vigorously with a vortex for 15–20 s in a fume hood. The test tube was allowed to stand at least 10 min at room temperature to allow the separation of toluene and aqueous phase, then the toluene phase was aspirated from the aqueous phase. The absorbance of chromophore containing toluene was measured at 520 nm, using toluene as the blank. Standard curves were prepared with each assay using standard proline in 3% sulfosalicylic acid solution. Proline content was expressed as micromoles per gram of FW of plant materials.

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2.3. Isolation and characterization of caffeic acid derivatives in oregano extracts For qualitative and quantitative determination of phenolic compounds, oregano shoots (1 g) were first homogenized for 3 min with 30 ml hot MeOH-EtOH (1:1) and then refluxed for 30 min (2×). After centrifugation and pooling of the extracts, the combined solutions were concentrated under vacuum and partitioned with petroleum ether (bp 40–70 ◦ C). The aqueous fraction was analyzed for total phenolic content, antioxidant activity, and HPLC determination of caffeic acid oligomers (rosmarinic acid, lithospermic acid, and lithospermic acid B). HPLC analyses were performed with a Hewlett Packard Series 1100 liquid chromatograph equipped with a binary gradient pump G1312A, a G1315A spectrophotometric photodiode array detector, and G1316A Column thermostat set at 45 ◦ C. The Hewlett Packard Chem Station (Rev. A. 06.03) software was used for spectra and data processing. An analytical Phenomenex (Torrance, CA, USA) Luna C18 (5 ␮) column (4.6 mm × 250 mm) was used throughout this work. The solvent system consisted of (A) MeOH and (B) acetic acid–water (5/95, v/v). The elution profile was as reported by Lattanzio and Van Sumere (1987). The flow rate was 1 ml/min. Samples of 25 ␮l were applied to the column by means of a 25 ␮l loop valve. HPLC–MS/MS analyses were performed on a QTrap MS/MS system, from Applied Biosystems (Foster City, CA, USA), equipped with an ESI interface and a 1100 series micro-LC system comprising a binary pump and a microautosampler from Agilent Technologies (Waldbronn, Germany). ESI interface was used in positive ion mode, with the following settings: temperature (TEM) 350 ◦ C; curtain gas (CUR), nitrogen, 30 psi; nebulizer gas (GS1), air, 10 psi; heater gas (GS2), air, 30 psi; ionspray voltage +4500 V. Full scan chromatograms were acquired in the mass range 100–800 amu, MS/MS chromatograms were acquired at collision energy of 20 V. LC conditions were as for HPLC–DAD analyses.

Fig. 1. Response of oregano shoot growth to treatments.

prepared by adding the solvent instead of the antioxidant solution (sample). Trolox, a water-soluble ␣-tocopherol analogue, was used as a positive control. The antioxidant activity was calculated as percent of inhibition of the control reaction rate and expressed as I50 , as interpolated by the dose–response curves. IC50 is the amount of phenolic antioxidants (micrograms) that caused 50% inhibition of control reaction, in the reaction volume, under the conditions described. 3. Results

2.4. Total phenolic content Total phenolic content of oregano shoot extracts was determined using a modified Folin-Ciocalteau spectrophotometric method (Marigo, 1973). 20 ␮l of oregano shoot extract was placed in a reaction test tube to which 1.58 ml of water and 100 ␮l of Folin-Ciocalteau reagent (Sigma) were added. The test tube was allowed to stand for between 30 s and 8 min, and then 300 ␮l 20% Na2 CO3 was added. After 20 min at 40 ◦ C, absorbance was measured at 750 nm. Total phenolic content was expressed as mg rosmarinic acid equivalents/g fresh weight. 2.5. Antioxidant activity Iron chelates, such as myoglobin, can form ferryl species in Fenton systems. In addition, incubation of haem protein with an excess of H2 O2 can cause haem breakdown to release iron ions. Both ferryl formation and the release of iron ions result in hydroxyl radical formation. The antioxidant activity of oregano shoot extracts against these radicals was measured as described by Miller et al. (1993). This spectrophotometric assay is based on the reduction of the blue–green ABTS•+ (2,2 -azinobis-(3-ethylbenzothiazoline-6sulphonic acid)) radical cation by hydrogen-donating antioxidants, which is measured by the suppression of its long wave absorption spectrum. ABTS•+ radical cation is generated by incubating the following reagents in a final volume of 1.5 ml for 6 min: KH2 PO4 –KOH buffer pH 7.4 (50 mM), NaCl (145 mM), sample to be tested, metmyoglobin (1.67 ␮M), ABTS (100 ␮M) and, finally, H2 O2 (50 ␮M) (final concentrations). Hydrogen peroxide is added to start the reaction. The absorbance was measured at 734 nm. Control reaction was

Oregano is a species of the family Lamiaceae traditionally used by the food industry because of its aromatic and antioxidant properties linked to its phenolic metabolites, such as rosmarinic acid (␣-O-caffeoyl-3,4-dihydroxyphenyl-lactic acid; RA). These properties have raised the interest in developing studies concerning the stimulation of antioxidant phenolics in shoot cultures of spices in order to develop biotechnological processes for the industrial production of such fine chemicals (Petersen and Simmonds, 2003). The results of this research show that both nutritional deficiency (MS1/2 ), which in turn causes a moderate increase of constitutive free proline, and exogenous proline affect negatively, compared to control (MS), fresh weight of oregano shoots grown 15 days on agar culture medium (Fig. 1). The addition of exogenous proline results in very high levels of intracellular free proline. At the same time the growth of oregano shoots on MS medium is inhibited 60% by 5 mM exogenous proline. This effect is probably related to the age and handling of plant tissues as well as to concentration of exogenously added proline. It is likely that the physiological status of tissues determined their response to exogenous proline, by perhaps differential efficiency of proline absorption1 (Handa et al., 1986; Rodriguez and Heyser, 1988; Hu et al., 1992). By contrast to this reduced growth of oregano shoots, the total phenolic content (carbon-based secondary metabolites) is greatly enhanced by exogenous proline (+120%) as well as by nutri-

1 In fact, in a different series of experiments (data not shown) the growth of oregano shoots, in absence of stress, has been enhanced (about 40% relative to untreated cell) by exogenously supplied proline 5 mM.

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Table 1 Oregano shoot fresh weight (FW) and free proline content, total phenolic content, rosmarinic acid content, and antioxidant activity of oregano shoot extract with different treatments Sample

Shoot weight (mg)

Proline (␮moles/g FW)

Rosmarinic acid (mg/g FW)

Control (MS) Exogenous proline5mm (MS)

110 44

0.32 8.90

0.95 3.18

3.40 7.50

5,40 3,46

66 51

0.42 8.40

2.45 5.06

7.50 10.90

3,70 2,77

Nutritional stress: MS1/2 MS1/2 + proline5mM

Total phenols (mg RA/g FW)

Antioxidant activity (IC50 )

Fig. 2. HPLC–MS/MS spectrum (positive ions) of: (a) rosmarinic acid (molecular ion [M+H]+ , m/z 361.1); (b) lithospermic acid (molecular ion [M+H]+ , m/z 539.1); and (c) lithospermic acid B (molecular ion [M+H]+ , m/z 719.0), identified in oregano shoots and calli.

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Fig. 2. (Continued ).

ent deficiency (+120%), and this increase is further stimulated by synergistic action of nutrient depletion of culture medium and exogenously added proline (+220%) (Table 1 ). The increased total phenolic content in stressed oregano shoots runs parallel with the increased content of intracellular free proline: accumulation of free proline is a typical stress response induced in plant tissues by different environmental (biotic and abiotic) stresses. The moderate increase of constitutive proline in stressed oregano tissues could be justified by utilization of the amino acid in increasing the net flux through the proline cycle (see Section 4, Fig. 3). The effect of both, exogenously added proline and stress elicited proline, on phenolic metabolism is, probably, linked to replenishment of the NADP+ supply to oxidative pentose phosphate pathway which, in turn, is a source of NADPH and carbon skeletons (erythrose4-phosphate that, along with phosphoenolpyruvate formed from glycolysis, serves as a precursor for phenylalanine biosynthesis via the shikimic acid pathway) for phenylpropanoid biosynthesis (Stewart et al., 1977; Hagedorn et al., 1982; Fahrendorf et al., 1995; Maggio et al., 1997). As a consequence of the increased total phenolic content, an increased antioxidant activity (measured as IC50 ) of oregano shoot extracts can be also observed (Table 1). From a qualitative viewpoint, the phenolic fraction of oregano shoot extracts, as determined by means of HPLC–MS, is characterized by the presence of caffeic acid, rosmarinic acid, and lithospermic acid B, a dimer of rosmarinic acid, besides other minor constituents (Fig. 2a and c). Rosmarinic acid and lithospermic acid B MS/MS spectra, as well as spectrum of lithospermic acid identified in callus (see Section 4), show a fragment peak of m/z 181.0 diagnostic of the presence of caffeic acid moiety. The most abundant phenolic compound present in oregano shoot extracts is rosmarinic acid, an ester of caffeic

acid with 3,4-dihydroxyphenyllactic acid, which is considered to be responsible for most of the observed antioxidant activity of the nonterpenoid component in spices of the family Lamiaceae (Dorman et al., 2003). Data presented in Table 1 show that there is a strong correlation between the induced rosmarinic acid level and antioxidant activity of extracts from treated oregano shoots. Both nutritional stress and exogenous proline induce a significant increase of the antioxidant activity of oregano shoot extracts, and this increase is essentially related to increased level of rosmarinic acid, whose level is enhanced by nutritional stress (+158%) and exogenous proline (+234%) more than total phenolic content (+120%). This means that rosmarinic acid is more likely to be responsible for most of the observed antioxidant activity of oregano extracts. Therefore, both nutritional stress and exogenous proline can represent two possible approaches in stimulating antioxidant phenolics, especially rosmarinic acid, in oregano tissue cultures. 4. Discussion Primary metabolism is an important source of precursors for the synthesis of secondary phenolic metabolites, which have a range of functions in metabolism, signaling, and defence against abiotic and biotic stress. Central metabolism requires high levels of limited plant resources and during intense growth the synthesis of phenolic metabolites may be substrate- and/or energy-limited. On the other hand, either abiotic or biotic stresses divert substantial amounts of substrates from primary metabolism into secondary defensive product formation and this could lead to constraints on growth. Plants, in fact, have limited resources to support their physiological processes, hence all requirements cannot be met simultaneously and trade-offs occur between growth and defence (Coley et al.,

Fig. 3. Scheme showing the relationships between primary and secondary metabolism and the role of endogenous and exogenous proline in stimulating phenylpropanoid pathway. The enzymes are: 1: Glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6PDH); 2: 6-Phosphogluconate dehydrogenase (EC 1.1.1.44, 6PGDH); 3: 1 -pyrroline-5carboxylate-synthetase (EC 2.7.2.11 + EC 1.2.1.41; P5CS); 4: 1 -pyrroline-5-carboxylate-reductase (EC 1.5.1.2; P5CR); 5: Proline dehydrogenase (EC 1.4.3; PDH); 6: 1 -pyrroline5-carboxylate-dehydrogenase (EC 1.5.1.12; P5CDH); 7: Phenylalanine ammonia-lyase (EC 4.3.1.5; PAL); 8: Glutamine synthetase (EC 6.1.1.3; GS); 9: Glutamate synthase (EC 1.4.1.14; GOGAT).

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1985; Herms and Mattson, 1992). Therefore, a principal feature of plant metabolism is the flexibility to accommodate developmental changes and to respond to the environment. On a short time-scale, metabolic regulation can be achieved through mechanisms such as altered enzyme kinetics in response to metabolite concentrations or via secondary modifications of protein structure, including phosphorylation and redox regulation. On a longer time-scale, gene transcription has also been shown to have a role in regulating metabolism (Logemann et al., 2000; Henkes et al., 2001; Nakane et al., 2003; Lloyd and Zakhleniuk, 2004; Leser and Treutter, 2005; Fritz et al., 2006). 4.1. Metabolic costs of adaptive responses to adverse environmental conditions Several hypotheses have been put forward to explain the effects of environmental factors on the trade-off between growth and resistance-related secondary compounds. Both the carbon/nutrient balance (Bryant et al., 1983) and the growth differentiation balance (Herms and Mattson, 1992) hypotheses suggest that there is a trade-off between growth and differentiation (the production of carbon-based secondary metabolites, such as phenolics). Plants respond to variation in resource availability by modifying allocation of carbon skeletons between primary and secondary metabolism: in conditions of high resource availability growth is dominant but, when shading and nutrient deficiency (or any other resource limitation) restrict growth more than photosynthesis, plants are predicted to use the excess of carbon skeletons to produce defensive phenolics (Lerdau and Coley, 2002). The protein competition model (Margna, 1977; Margna et al., 1989; Jones and Hartley, 1999) suggests that protein synthesis and phenylpropanoid synthesis compete for the use of the precursor phenylalanine and that, consequently, any environmental factor that affects plant growth and protein synthesis may also affect the availability of phenylalanine for the biosynthesis of phenolics. Finally, Close and McArthur (2002) have suggested that any factor that increases oxidative pressure may cause an increase in phenolic level and that the primary role of many phenolics is to protect plant tissues from photodamage not herbivores. This proposition raises the question concerning the costs of resistance in plants, relative to the demands for photosynthates by other metabolic functions, and the implications of such costs. Most plants produce a broad range of secondary metabolites that are toxic to pathogens and herbivores, either as part of their normal program of growth and development or in response to biotic stress. Both tolerance and resistance traits require the reallocation of host resources, therefore defensive chemicals are considered to be costly for plants, because resistance genes might impose metabolic costs (e.g., lower growth rates than their sensitive counterparts) due to the diversion of limited energy and resources away from primary metabolism. One way for a plant to reduce these costs is to synthesize defence compounds only after there has been some degree of initial damage by a pathogen or insect: this strategy is inherently risky because the initial attack may be too rapid or too severe for an effective defence response. Therefore, plants that are likely to suffer frequent and/or serious damages may be better off investing mainly in constitutive defences, whereas plants that are attacked rarely may rely predominantly on induced defences (Kombrink and Hahlbrock, 1990; Herms and Mattson, 1992; Purrington, 2000; Wittstock and Gershenzon, 2002; Brown, 2003). 4.2. Transduction pathway between nutrient depletion and enhanced polyphenol content Results from this study are consistent with the scheme proposed in Fig. 3, which involves a continuous cycling of proline and sug-

gests a link between free proline (stress-induced or exogenously added) and increased phenolic metabolism, via stimulated carbon flux through oxidative pentose phosphate pathway. This scheme is based on the fact that the plant cell is a highly integrated system, which ensures a tight regulation of interacting pathways by their coupling through common intermediates, including pyridine nucleotides (Hare and Cress, 1997). Following the imposition of a nutritional stress, the growth of oregano shoots is reduced (−40%), compared to control, and this reduction seems to be related to an energetic drain involved in building an increased level of phenolic compounds that diverts resources from the biomass production. Here must be again emphasized that photosynthesis and growth do not respond equally to gradient of nutrients. Growth processes are slowed considerably by even moderate shortage of nutrients, while net photosynthesis is not as sensitive to resource limitation. Thus, when nutrient deficiency imposes sink limitation upon growth, carbohydrates accumulate in excess of growth requirements, and, in turn, could be diverted into secondary metabolism (Hsiao, 1973; Chapin, 1980; Herms and Mattson, 1992). At the same time, as a consequence of the imposed nutritional stress, plant tissues exhibit a (moderate) increase of proline that enhances the tolerance of cellular components to reactive oxygen species (ROS), synthesized by plants experiencing stress conditions (Smirnoff, 1993). Here it must be stressed that OPPP is the source of reducing equivalents and carbon skeletons, via the shikimic acid pathway, for phenylpropanoid biosynthesis and that its activity must be increased under conditions of increased flux into the phenylpropanoid pathway (Fahrendorf et al., 1995). In addition, increased levels of proline synthesis under condition of nutritional stress may account, at least in part, for reduced growth rates associated with exposure to adverse conditions. In these conditions the increased synthesis of proline maintains NAD(P)+ /NAD(P)H ratios at values compatible with metabolism under normal conditions, being proline synthesis accompanied by the oxidation of NADPH, and this may constitute a form of metabolic response, triggered in the signal transduction pathway between perception of nutritional stress and physiological response, within the plant cell. The increased NADP+ /NADPH ratio, mediated by proline biosynthesis, is likely to enhance the activity of the oxidative pentose phosphate pathway (Chandler and Thorpe, 1987; Chen and Kao, 1995; Hare and Cress, 1997). The increase in total phenolics and RA content in response to exogenous proline suggests that mitochondrial proline oxidation could drive the oxidative pentose phosphate pathway by recycling glutamic acid into the cytosol to generate a proline redox cycle (Zheng et al., 2001). In addition, Fig. 3 shows that cytosolic glutamic acid may be also utilized for recycling ammonium ions, produced in the first step of the phenylpropanoid biosynthesis, by means of the glutamine synthetase and glutamate synthase (GS/GOGAT) cycle. It has been suggested that the ammonium ion released during active phenylpropanoid metabolism is not made available for general amino acid/protein synthesis. Rather it is rapidly recycled back to regenerate phenylalanine, thereby providing an effective means of maintaining active phenylpropanoid metabolism with no additional nitrogen requirement. The ammonium ion released during lysis is metabolized via GS/GOGAT cycle to generate glutamate thereby permitting arogenate synthesis, via prephenate transamination, which, in turn, regenerates phenylalanine (van Heerden et al., 1996). As far as the development of a new strategy to enable the production of useful secondary metabolites on a commercial scale is concerned, any progress made in the basic understanding of metabolic pathways and regulatory mechanisms may be addressed to exploit the plant cell and tissue culture potentials to produce food additives, such as antioxidant phenolics. The results of this study shows that the potential of tissue cultures in the use of

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plant tissue technology for the production of oregano shoot cultures with an increased content of antioxidant phenolics is not satisfactory because of their decreased biomass production, compared to control, in both stressed and proline treated cultures. In addition, the results of this research also suggest that, as an alternative to tissue cultures, oregano callus cultures can be used to form cell suspension cultures that can be grown in bioreactors to produce antioxidant phenolics. Indeed, high-performance liquid chromatography/tandem mass spectrometry of leaf callus extracts have shown that oregano calli, besides caffeic acid, rosmarinic acid, and lithospermic acid B identified in oregano shoots extracts, also contain lithospermic acid (Fig. 2b), a caffeic acid–rosmarinic acid conjugate, with antioxidative, antibacterial and antiviral activities (Jiang et al., 2005).

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