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Abstract. Given the essential role played by phenol metabolism in many resistance responses to different types of stress, the aim of the present work was to ...

Plant Growth Regulation 41: 173–177, 2003.  2003 Kluwer Academic Publishers. Printed in the Netherlands.

173

Role of Ca 21 in the metabolism of phenolic compounds in tobacco leaves (Nicotiana tabacum L.) ´ Juan M. Ruiz, Rosa M. Rivero, Inmaculada Lopez-Cantarero and Luis Romero* Department of Plant Physiology, Faculty of Science, University of Granada, Av. Fuentenueva s /n E-18071, Granada, Spain; * Author for correspondence (e-mail: [email protected] ugr.es; fax: 134 958 248 995) Received 28 January 2003; accepted in revised form 2 June 2003

Keywords: Calcium, Nicotiana tabacum, Oxidation, Phenols, Synthesis

Abstract Given the essential role played by phenol metabolism in many resistance responses to different types of stress, the aim of the present work was to determine how different application rates of calcium may influence this metabolic process. Increased calcium in the nutrient solution in which tobacco plants were grown considerably reduced the foliar concentration of phenolic compounds. Calcium clearly exerted a positive influence on the activities of enzymes (phenylalanine ammonia-lyase, polyphenol oxidase and peroxidase) involved in the metabolism of the phenolics. High dosages of calcium (5 mM) promoted more oxidation than synthesis of these compounds, thus explaining the lower concentration of the phenolics. Abbreviations: BSA – bovine serum albumine; PAL – phenylalanine ammonia-lyase; POD – peroxidase; PPO – polyphenol oxidase

Introduction Phenols, among the most widely distributed natural products in the plant kingdom, carry strong physiological and ecological implications (Ruiz and Romero 2001). The metabolism of phenolics involves a series of enzymes that participate in the synthesis and the oxidation of these compounds. The reaction catalysed by phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the deamination of L-phenylalanine to produce transcinnamate, is commonly regarded as a key step in the biosynthesis of phenolics and is affected by a number of factors (Hao et al. 1996). On the other hand, phenolics are degraded to diquinones by peroxidases (POD, EC 1.11.1.7) and principally polyphenol oxidases (PPO, EC 1.14.18.1) ¨ ¨ 1995; Thypyapong et al. 1995). Many (Soderhall studies have demonstrated that both enzymes increase in response to biotic and abiotic stress (Kwak et al. 1996; Ruiz et al. 1998, 1999a).

The processes which have been most thoroughly studied and which most directly involve phenolics are ¨ related to pest and disease resistance (Dubeler et al. 1997; Ruiz et al. 1999a). In addition, the metabolism of phenolics has been associated with injuries (Smith 1982), with resistance to thermal stress (Christie et al. 1994), with tolerance against exposure to UV rays and ozone (Rasmussen et al. 1991) and finally to changes in the nutritional state of nutrients such as boron ¨ (Cakmak and Romheld 1997; Ruiz et al. 1998, 1999a), nitrogen (Wojtaszek et al. 1993) and calcium ˜ ´ (Castaneda and Perez 1996; Penel et al. 1999). Calcium (Ca 21 ), one of the essential nutrients for plants, plays a major role in the initiation of many signal transduction processes in higher plant cells, including bud formation, polar growth, gas-exchange regulation, secretion, movements and light- and hormone-regulated growth and development (Hepler and Wayne 1985). In addition, this nutrient actively influences one of the processes most vital to plant

174 growth, nitrogen metabolism (Ruiz et al. 1999b; ´ Lopez-Lefebre et al. 2000). The role of Ca 21 in phenolic metabolism has been described by various authors. For example, the first work that demonstrated a direct role of Ca 21 in the synthesis of phenols was ˜ ´ performed by Castaneda and Perez (1996). In this work, the authors observe that the foliar application of 10 m M of CaCl 2 increases PAL activity, and therefore the accumulation of phenols, prompting resistance to infection by the fungus Alternaria alternata in citrus. However, the literature remains controversial on the effect of Ca 21 on the enzymes responsible for ¨ ¨ (1995) and Wan and phenol oxidation. Soderhall Heinrich (1997) demonstrated that increased concentrations of CaCl 2 in the nutritive solution augment POD activity. Conversely, other researchers have reported that higher levels of Ca 21 diminished PPO and POD activities (Kawai et al. 1995; Tomasbarberan et al. 1997). Recently, Penel et al. (1999) proposed that Ca 21 indirectly activates POD, as this cation induces the cross-linking of polygalacturonan chains into a structure which can be recognized by isoperoxidase. Given the essential role played by phenol metabolism in many resistance responses to different types of stress, the rapid and effective manipulation of this metabolic process could be of great help in developing plants with greater resistance against adverse conditions. Therefore, the objective of this work was to analyse how the application of different amounts of Ca 21 in the nutrient solution influences synthesis and oxidation of phenols.

Materials and methods Plant material and culture conditions Seeds of Nicotiana tabacum L. cv Tennessee 86 were sown in March 1998. The seedlings were grown in an experimental greenhouse in southern Spain (Granada) for 45 days and then transferred to a cultivation chamber under controlled environmental conditions with relative humidity of 60–80%, temperature 30 / 20 8C (day / night), and 16 / 8 h photoperiod at a PPFD of 350 m mol m 22 s 21 (measured at the top of the plants with a 190 SB quantum sensor, LI-COR Inc., Lincoln, NE, USA). The plants were grown in individual pots (25 cm upper diameter, 17 cm lower

diameter, 25 cm in height) of 8 l volume, filled with vermiculite. For 1 month (from day 45 until day 75 after sowing), before the experimental treatments, the ´ plant received a modified Hoagland medium (LopezLefebre et al. 2000). The nutrient solution (pH 5.5– 6.0) was renewed every 3 days and the vermiculite rinsed with Millipore-filtered water. At 75 days after sowing, we applied different levels of Ca 21 , the initial level being 1.25 mM for the Ca1 treatment, 2.5 mM for the Ca2 treatment, and 5.0 mM for the Ca3 treatment. The application rates of Ca 21 in the present work were similar to those used in other works performed by our research group (Ruiz and ´ Romero 1998; Ruiz et al. 1999b; Lopez-Lefebre et al. 2000), and these rates, in addition to influencing the physiology of the plant, do not provoke symptoms of Ca 21 deficiency or toxicity and therefore are ideal for these types of studies. The experimental design was a randomized complete block with three treatments, arranged in individual pots with six plants per treatment, each one replicated three times. Plant sampling The plants were sampled twice, beginning at the 14leaf stage just before the onset of flowering, using three plants per sampling. At the first sampling, day 105 after sowing, leaves were picked from nodes 10 and 11. At the second sampling, 2 weeks later, leaves from nodes 12 and 13 were picked. The leaves were rinsed three times with distilled water after disinfecting with non-ionic detergent at 1%, then blotted on filter paper. The leaves from nodes 10 (first sampling), and 12 (second sampling) were used fresh for the analysis of enzymatic activities and phenolic compounds. The leaves from node 11 (first sampling), and 13 (second sampling) were dried in a forced air oven at 70 8C for 24 h and were used for the analysis of Ca 21 content. Plant analysis Enzymatic analysis The extraction of PAL was carried out following the method proposed by Lister et al. (1996) and its activity was assayed by an adaptation of the methods of Zucker (1965) and McCallum and Walker (1990), determined from the yield of cinnamic acid, estimated from absorbance at A 290 in the presence and absence of phenylalanine.

175 Table 1 Effect of CaCl 2 ?2H 2 O treatments on the foliar concentration of Ca 21 and the metabolism of the phenolics compounds in the leaves of tobacco plants. Data are means 6 S.E. (n 5 6). The least significant difference (LSD) is given for each plant. Treatments (mM)

Total Ca 21

Total phenolics

PAL

PPO

POD

1.25 2.50 5.00 Significance LSD at 5%

502.7 6 54.4 675.2 6 62.9 833.7 6 67.1 *** 157

2467 6 176 2028 6 158 1715 6 166 *** 239

1.10 6 0.21 1.54 6 0.27 1.73 6 0.36 ** 0.33

3.52 6 0.65 5.06 6 0.80 6.55 6 0.79 *** 1.15

2.57 6 0.43 3.35 6 0.63 3.92 6 0.74 * 0.74

Total Ca 21 : m mol g 21 D.W.; Total phenolics: m g caffeic acid g 21 F.W.; PAL: m mol cinnamic acid produced mg 21 protein min 21 ; PPO: m mol caffeic acid oxidised mg 21 protein min 21 ; POD: m mol guaiacol oxidised mg 21 protein min 21 .

The extraction method used for the determination of PPO was that proposed by Thypyapong et al. (1995) and its activity assayed as described by Nicoli et al. (1991), measured by the change in A 370 of the assay mixture (30 8C) based on the enzymatic oxidation of caffeic acid. The method used for the determination of POD was a modified version of that proposed by Kalir et al. (1984) and Badini et al. (1990) and its activity was determined by following the change of A 485 due to guaiacol oxidation (Kalir et al. 1984; Ruiz et al. 1998). To test whether the reaction was due to peroxidase, control assays contained catalase from bovine liver (EC 1.11.1.6) (Fluka). In all cases, the samples extracts were boiled and assayed to determine whether the reactions were enzymatic. Protein in the samples extracts were estimated by the method of Bradford (1976) using BSA as a standard. Extraction and quantification of total phenols Leaves were ground to a fine powder with a pestle and extracted at a ratio of 100 mg fresh weight to 1 ml methanol. Total phenolic content was assayed quantitatively with Folin-Ciocalteau reagent (Singlenton and Rossi 1965; Singlenton et al. 1985). The results obtained were expressed as m g of caffeic acid g 21 fresh weight (FW). Extraction and quantification of total Ca 21 Total Ca 21 was analysed by atomic-absorption spectrophotometry (Hocking and Pate 1977), after digestion of dry and milled material with 12 N H 2 SO 4 and H 2 O 2 . The content of Ca 21 was expressed as m mol g 21 dry weight (DW). Statistical analysis Standard analysis of variance techniques were used to

assess the significance of treatment means. The data shown are mean values 6 S.E. Differences between treatment means were compared using the LSD at the 0.05 probability level. Levels of significance are represented by * at P , 0.05, ** at P , 0.01, *** at P , 0.001, and ns: not significant.

Results and discussion As confirmed in previous work, the highest foliar accumulation of Ca 21 , in treatment Ca3 (Table 1), was due to the fact that, after Ca 21 absorption by the root cells, this cation is transported through the xylem towards the shoot, accumulating in zones where transpiration is greatest (Bharti et al. 1996; Ruiz and Romero 1998; Ruiz et al. 1999b). Foliar PAL activity (Table 1), was affected significantly by the different Ca 21 treatments applied, the highest activities being registered in the Ca3 treatment, which also showed the highest foliar levels of total Ca 21 , thus presenting a positive and significant relationship (r 5 0.941***) between these two parameters. With reference to the response of PAL ˜ ´ activity to Ca 21 application, Castaneda and Perez (1996), using lemon seedlings injured or treated with fungal elicitors, suggested that the increase in this activity was not directly caused by the Ca 21 , but that this cation participates in the cell response, triggering a series of transduction signals. Our results show that even in uninfected tobacco leaves, the application of different dosages of Ca 21 stimulated PAL activity. On the contrary, the results showed a fall in the 21 concentration of the phenolics with increasing Ca level (Table 1), the highest concentration appearing at Ca1 and the lowest at Ca3. Although the highest PAL activity occurred in the Ca3 treatment, this treatment contained the lowest concentration of total phenolics.

176 This behaviour appears to explain the negative relationship between these parameters (r 5 20.989***). The activity of the enzymes POD and PPO was affected positively by the application of Ca 21 . Like PAL activity, the PPO and POD activities increased in the Ca3 treatment, by 85 and 52%, respectively as compared to the lowest activities occurring in the Ca1 treatment (Table 1). In agreement with other reports, in our experiment the enzymes responsible for phenolic oxidation, PPO and POD, were very positively affected by the presence of Ca 21 (Ca 21 concentration–PPO, r 5 0.987***; Ca 21 concentration–POD, r 5 0.981***). This activation of PPO by Ca 21 has been described elsewhere, and it has been reported that Ca 21 acts on PPO, which normally is found in its latent form, modifying the conformational state of this enzyme ¨ ¨ 1995). The and thus boosting its activity (Soderhall enzyme POD also reportedly reacts positively to Ca 21 , and Penel et al. (1999), recently, demonstrated that this cation is necessary for the action of this enzyme to induce the cross-linking of polygalacturonan chains into a structure which could be recognized by the peroxidase. In our experiment, we found an inversely proportional relationship between the activity of these enzymes and phenolic accumulation (total phenolics– PPO, r 5 20.992***; total phenolics–POD, r 5 20.999***). Thus, we conclude that in response to Ca 21 application, the metabolism of phenolic compounds is affected by a stimulation in the rate of oxidation as compared to that of synthesis.

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