Response of phenolic metabolism to the application of carbendazim plus boron in tobacco

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Response of phenolic metabolism to the application of carbendazim plus boron in tobacco
  Copyright  ©  Physiologia Plantarum  1999  PHYSIOLOGIA PLANTARUM 106: 151–157. 1999 Printed in Ireland   —  all rights reser  ed ISSN   0031-9317  Response of phenolic metabolism to the application of carbendazim plusboron in tobacco Juan M. Ruiz*, Pablo C. Garcia, Rosa M. Rivero and Luis Romero Department of Plant Biology ,  Faculty of Sciences ,  Uni   ersity of Granada ,  18071 - Granada ,  Spain*Corresponding author ,  e - mail  :   lromero@goliat . ugr . es Received 16 November 1998; revised 22 March 1999 In view of the essential role of phenolic compounds in the with only carb, and six combinations of carb with eachdevelopment of pathogen resistance in plants, and given the concentration of B. The results indicated that the foliarinfluence that fungicides and boron (B) exert over phenolic application with carb alone led to increases in phenylalaninemetabolism, the aim of the present study was to determine the ammonia-lyase (PAL; EC activity and a foliar accu-mulation of phenols. This effect of the carb alone could signifyindividual effect of the application of a fungicide, as well as toan additional tolerance mechanism to pathogenic infection,determine the joint effect of the fungicide and B on thegiven the participation of phenolic compounds in the lignifica-metabolism of phenolic compounds in tobacco plants ( Nico -tion of plant cell walls. The joint application of carb and B tiana tabacum  L. cv. Tennessee 86). The fungicide applied wasincreased both the biosynthesis and the oxidation of thecarbendazim (carb), a preventative fungicide, with a purity of phenolic compounds, especially in carb plus B 3 , while the100% at a concentration of 2.6 m M  . Boron was applied in theform of H 3 BO 3  at: 1.6 m M   (B 1 ), 4 m M   (B 2 ), 8 m M   (B 3 ), 16 application of carb plus B 5  or carb plus B 6  reduced thesem M   (B 4 ), 32 m M   (B 5 ), or 64 m M   (B 6 ). In all, there were eight processes as well as the foliar biomass.treatments: one without carb and without B (control), one Introduction Plants react to pathogen attack through a variety of activeand passive defense mechanisms. At the site of infection, ahypersensitive response is often initiated in resistant plants,which is frequently manifested as necrotic lesions resultingfrom host cell death. In addition, the distal uninfected partsof the plant can develop systemic-acquired resistance, whichresults in enhanced long-lasting defense against the same oreven unrelated pathogens (Fodor et al. 1997, Wendehenne etal. 1998). Both the hypersensitive response and systemic-ac-quired resistance are associated with increased expression of a large number of defense or defense-related genes (Klessigand Malamy 1994). Examples of the defense reactions in-clude the lignification and suberization of the plant cell wall(Stein et al. 1993), deposition of callose (Benhamou 1992), de novo synthesis of pathogenesis-related proteins (Siegristet al. 1994), production of active oxygen species (Low andMerida 1996), and biosynthesis of secondary metabolites(Daayf et al. 1997). Much evidence suggests that the in-creases in salicylic acid levels are essential for the inductionof systemic-acquired resistance (Tenhaken and Ru¨bel 1997).Phenolic compounds are among the most influential andwidely distributed secondary products in the plant kingdom.Many of these play important physiological and ecologicalroles, being involved in such diverse processes as: rhizogene-sis (Curir et al. 1990), vitrification (Kevers et al. 1984), resistance to different types of stress (Delalonde et al. 1996),and participation in redox reactions (Takahama and Oniki1992). Nevertheless, the processes that have been mostthoroughly studied and that most directly involve phenoliccompounds are related to pest and disease resistance (Na-garathna et al. 1993, Du¨beler et al. 1997). In relation to this,increased activity of polyphenol oxidase (PPO), peroxidaseand phenylalanine ammonia-lyase (PAL) has been reportedin plants treated with various abiotic and biotic inducers of  Abbre  iations  – carb, carbendazim; DTT, 1,4-dithio- DL -threitol; EDTA, ethylenediamine tetraacetic acid; PAL, phenylalanine ammonia-lyase; PMSF, phenylmethanesulfonyl fluoride; PPO, polyphenol oxidase; PVP, polyvinylpyrrolidone; SDS, sodium dodecyl sulfate; TFA,trifluoroacetic acid. Physiol. Plant. 106, 1999  151  resistance (Smith-Becker et al. 1998). Given the scope of theprocesses in which phenolic compounds are involved,knowledge of the factors that regulate the metabolism of these compounds allows for the manipulation of their syn-thesis or degradation, depending on the conditions chosenor the results desired.Prominent among the factors that directly influence phe-nolic metabolism are pesticides and herbicides (Molina et al.1998), with the effects of herbicides having been most exten-sively studied (Lydon and Duke 1989). Some herbicideswere found to increase the activities of enzymes involved inthe accumulation of hydroxyphenolic compounds in severalplant species (Scarponi et al. 1992), whereas others de-creased enzyme activity (Hoagland 1990). On the otherhand, in relation to the action of fungicides, Molina et al.(1998) have demonstrated that the systemic-acquired resis-tance signal transduction pathway, a salicylic acid-depen-dent plant-defense mechanism, mediates fungicide action inthe plant.Finally, boron (B) has been related to changes in thecontent of phenolic compounds and metabolism (Ruiz et al.1998). The accumulation of phenolic compounds is charac-teristic of B-deficient tissues due to increased synthesis andinhibited utilization of phenolic compounds in cell-wall syn-thesis. In response to high accumulation of phenolic com-pounds, PPO and peroxidase activities rise in B-deficienttissues (Cakmak and Ro¨mheld 1997). This oxidation of phenolic compounds has also been found under appropriatefoliar levels of B in tobacco plants (Ruiz et al. 1998).In the present study, we analyze how the fungicide car-bendazim (carb) alone, as well as in combination with B,influences the phenol metabolism in tobacco plants, giventhe recognized role of these compounds in this metabolicprocess. In addition, taking into account the function of phenols in plant-pathogen relationships, we examine thefeasibility of reducing the application of fungicides withoutdiminishing the resistance of the plants to pathogen attack,through the application of lower fungicide dosages accom-panied by B. Materials and methods Crop design and plant sampling The plants chosen for this experiment were tobacco, becauseprior research in our laboratory has revealed in these plantsa substantial influence of B in the metabolism of phenoliccompounds (Ruiz et al. 1998). Seeds of   Nicotiana tabacum L. cv. Tennessee 86 were sown in May 1997 in southernSpain (Granada). The seedlings were grown in individualpots of peat in an experimental greenhouse for 45 days, andthen were transferred to individual pots (25 cm upper di-ameter, 17 cm lower diameter, 25 cm in height) filled withvermiculite. The plants were grown in a cultivation chamberunder controlled environmental conditions with relative hu-midity of 60–80%, temperature of 30 / 20°C (day / night), anda 16-h photoperiod at a photosynthetic photon flux density(PPFD) of 350   mol m − 2 s − 1 (measured at the top of theplants with a 190 SB quantum sensor; Li-Cor Inc., Lincoln,NE, USA). For 1 month (from day 45 until day 75 aftersowing), before the experimental treatments, the plants re-ceived a nutrient solution of: 6 m M   KNO 3 , 2 m M  NaH 2 PO 4 , 1.5 m M   CaCl 2 , 1.5 m M   MgSO 4 , 5   M   Fe-ethylenediamine tetraacetic acid (EDTA), 2   M   MnSO 4 ,1   M   ZnSO 4 , 0.25   M   CuSO 4 , 0.1   M   (NH 4 ) 6 Mo 7 O 24 , and2.5   M   H 3 BO 3 . The nutrient solution (pH 5.5–6.0) wasrenewed every 3 days.At 75 days after sowing, we performed a foliar applica-tion of the different treatments. The treatments were appliedto plants to runoff as aqueous foliar sprays containing thesurfactant Tween 20 (0.5% v / v), using a stainless steelsprayer. The fungicide applied was carb (bencimidazol 2-ilmethyl carbamate; C 9 H 9 N 3 O 2 ) with a purity of 100% at therecommended concentration of 2.6 m M  , this being therecommended concentration. This fungicide was used in ourexperiment because (1) it is one of the most widely usedfungicides in southeastern Spain, a zone of intensive agricul-ture, and because (2) it has a broad preventive spectrum,and therefore can be applied to a large proportion of thecrop that is not yet infected by pathogens. The fungicidecarb is recommended for the control of diseases caused by Botrytis  spp.,  Cercospora  spp.,  Coryneum  spp.,  Erysiphe graminis ,  Fusarium  spp.,  Fusicoccum  spp.,  Gaeumannomyces graminis ,  Monilia  spp.,  Piricularia oryzae ,  Rhizoctonia  spp., Sclerotium  spp.,  Septoria  spp.,  Taphrina  spp.,  Thiela  ia  spp., Venturia  spp., etc. This fungicide is applied normally tocereals, fruit (pome, stone, citrus, currants, strawberries,bananas, pineapples, mangoes, avocados, pawpaws, etc.),vines, hops, ornamentals, coffee, cotton, rice, flax, beet,sugar cane, peanuts, oilseed rape, cucurbits, rubber, to-bacco, turf, mushrooms, and other crops (Tomlin 1994).Boron was applied in the form of H 3 BO 3 : 1.6 m M   (B 1 ), 4m M   (B 2 ), 8 m M   (B 3 ), 16 m M   (B 4 ), 32 m M   (B 5 ), or 64 m M  (B 6 ). In all, there were eight treatments: without carb andwithout B (control), with only carb, and six combinations of carb with each concentration of B (carb-B x ). The experimen-tal design was a randomized complete block with eighttreatments, arranged in individual pots with four plants pertreatment, each replicated three times.Each treatment was applied three times fortnightly. Theplants were sampled beginning approximately at the 14-leaf stage, just before the onset of flowering. From the sameplants, leaves were sampled once, on day 120 after sowing,from nodes 10–13. All the sampled leaves were in themature state, with lengths of more than 10 cm. The materialwas rinsed three times in distilled H 2 O after disinfecting withnon-ionic detergent at 1% (v / v) (Decon 90, Merk) (Wolf 1982), then blotted on filter paper. A subsample of leaveswas used fresh for the analysis of enzymatic activities of PAL, PPO, peroxidase, and total phenols, performing tripli-cate assays for each extraction, while the other subsamplewas dried in a forced air oven at 70°C for 24 h. Dry weightwas recorded and expressed as g dry weight (leaf) − 1 . Plant analysis Extraction and assay of PAL  (  EC   4  . 3  . 1 . 5)  The extraction was carried out following the method pro-posed by Lister et al. (1996). Fresh plant material was Physiol. Plant. 106, 1999 152  ground at 4°C in buffer (50 m M   Na 2 HPO 4 / KH 2 PO 4 , pH7.0, 5% polyvinylpyrrolidone (PVP; M r = 44000), 50 m M  Na ascorbate, 18 m M   mercaptoethanol, and 0.1% (v / v)Triton X-100). The homogenate was filtered through fourlayers of cheesecloth and centrifuged at 20000  g   for 10 min.(NH 4 ) 2 SO 4  was added to the supernatant (to 35% satura-tion), which was then centrifuged for 20 min at 20000  g   toremove the PVP. More (NH 4 ) 2 SO 4  was added to this super-natant to reach a final saturation of 80%. This fraction wascentrifuged at 20000  g   for 20 min and the pellet resuspendedin extraction buffer (without PVP and Triton). This solutionwas used for PAL assays. Protein was estimated by themethod of Bradford (1976), using bovine serum albumin(BSA) as a standard.PAL activity was assayed by an adaptation of the meth-ods of  Zucker (1965) and McCallum and Walker (1990).The assay mixture consisted of 0.06  M   Na borate buffer, pH8.8, and crude enzyme. The reaction was started by theaddition of 11 m M   L -phenylalanine. Tubes were incubatedat 30°C for 60 min and the reaction stopped by the additionof 35% (w / v) trifluoroacetic acid (TFA). Tubes were thencentrifuged for 5 min at 5000  g   to pellet the denaturedprotein. PAL activity was determined from the yield of cinnamic acid, estimated from absorbance at A 290  in thepresence and absence of phenylalanine. To determinewhether the reaction was enzymatic, a sample extract wasboiled and assayed. Extraction and assay of PPO  (  EC   1 . 14  . 18  . 1)  The extraction method used was that proposed by Thipya-pong et al. (1995) with some modifications. Leaves wereground to a fine powder with a pestle and extracted at aratio of 150 mg fresh weight to 1 ml extraction buffer (100m M   Tris-HCl, pH 7.0, 100 m M   KCl, 1 m M   phenyl-methanesulfonyl fluoride (PMSF), and 3% [w / v] PVP) con-taining sodium dodecyl sulfate (SDS) at 0, 0.5, 1, 2, or 4%(w / v) each. The homogenates were centrifuged at 12000  g  for 15 min, and the supernatant was used to measure theprotein concentration by the method of  Bradford (1976),using BSA as standard. PPO was also assayed. All theseprocedures were carried out at 0–4°C.The PPO activity was assayed as described by Nicoli et al.(1991) with some modifications. Optimum activity wasreached using SDS at 2% (data not shown). The assaymixture consisted of 30   M   caffeic acid in 100 m M   buffer(Na 2 HPO 4 / KH 2 PO 4 ), pH 7.0, through which air was bub-bled for 5 min. Catalase (420 units) from bovine liver (EC1.11.1.6) (Fluka) was added in 0.1 ml H 2 O to preventperoxidation of the substrate (Vaughn and Duke 1981). Theassay was initiated by the addition of enzyme extract. PPOactivity was measured by the change in A 370  of the assaymixture (30°C) based on the measurement of the disappear-ance of caffeic acid by enzymatic oxidation. To determinewhether the reaction was enzymatic, the sample extract wasboiled and assayed. Extraction and assay of peroxidase  (  EC   1 . 11 . 1 . 7)  The method used was a modified version of that proposedby Kalir et al. (1984) and Badani et al. (1990). Fresh plantmaterial was ground with 50 m M   Tris-acetate buffer, pH7.5, 5 m M   2-mercaptoethanol, 2 m M   1,4-dithio- DL -threitol(DTT), 2 m M   EDTA, 0.5 m M   PMSF, and 1% (w / v) PVP.The homogenate was filtered through two layers of Mira-cloth and centrifuged for 30 min at 37000  g  . The pellet wasdiscarded and the supernatant used for peroxidase assaysand protein concentration by the method of Bradford(1976), using BSA as standard.Peroxidase activity was determined by following thechange of A 485  due to guaiacol oxidation (Kalir et al. 1984,Ruiz et al. 1998). The reaction mixture contained 100   M  Tris-acetate buffer, pH 5.0, 1   M   guaiacol, and 0.003   M  H 2 O 2 . To test if the reaction was due to peroxidase, controlassays contained catalase from bovine liver (EC (420 units in 0.1 ml H 2 O). To determine if thereaction was enzymatic, the sample extract was boiled andassayed. Extraction and quantification of total phenols The phenolic compounds of the plant material were extractedwith methanol. Total phenolic content was assayed quantita-tively by A 765  with Folin-Ciocalteau reagent (Singleton andRossi 1965, Singleton et al. 1985). The results obtained wereexpressed as   g of caffeic acid (g fresh weight) − 1 . Estimation of total boron Total B was analyzed after digestion of dry and milled leaf material with 6  M   H 2 SO 4  and H 2 O 2 . To measure B concen-tration in leaf tissues, the azomethine-H + method wasfollowed, and absorbance was read at A 410  (Wolf 1974). Theconcentration of B was expressed as   g (g dry weight) − 1 . Statistical analysis The data shown are mean values  SE . Differences betweentreatment means were compared using the  LSD  at the 0.05probability level. Levels of significance are represented by *at  P  0.05, ** at  P  0.01, *** at  P  0.001, and NS: notsignificant. Results and discussion Effect of carbendazim The metabolism of phenolic compounds is regulated by theactivity of various enzymes. The first necessary step forbiosynthesis of the phenylpropanoid skeleton in higherplants is the deamination of   L -phenylalanine to yield trans-cinnamic acid and ammonia. This reaction is catalyzed byPAL and is commonly regarded as a key step in thebiosynthesis of phenolic compounds (Ro¨sler et al. 1997).PAL activity is affected by a number of factors includinglight, temperature, growth regulators, inhibitors of RNAand protein synthesis, wounding, and mineral nutrition(Jones 1984, Ruiz et al. 1998). Another factor that affects the metabolism of phenoliccompounds, and more specifically PAL activity, is the appli-cation of herbicides. Some herbicides, by increasing or di-minishing PAL activity, reportedly cause the accumulation Physiol. Plant. 106, 1999  153  Fig. 1. PAL activity in tobacco leaves in response to the applicationof carb or carb plus B. Data are means  SE , n = 3. Fig. 3. PPO activity in tobacco leaves in response to the applicationof carb or carb plus B in tobacco plants. Data are means  SE ,n = 3. or loss of foliar phenolic compounds (Scarponi et al. 1992,Nemat Alla and Younis 1995). In our experiment, the foliarapplication of the fungicide carb increased PAL activitytwo-fold over the enzymatic activity of control plants ( P  0.001; Fig. 1). In the plants treated with carb, the foliarlevels of total phenols (Fig. 2) were 35% higher than incontrols ( P  0.001) due to the close relationship betweenthe PAL activity on the one hand, and the synthesis andaccumulation of phenolic compounds on the other. Finally,the relationship between the PAL activity and content of total phenols was positive and significant (PAL activity-totalphenols, r 2 = 0.95***).On the other hand, the metabolism of the phenolic com-pounds also involves oxidative enzymes, such as PPO, whichcatalyze the oxidation of phenols to quinones (Thipyaponget al. 1995). A large number of studies have demonstratedthat this enzyme increases in response to biotic and abioticstress (Thipyapong et al. 1995). In addition, PPO has beenidentified as a pathogenesis-related protein and a proteinaseinhibitor, and has been suggested to have a defensive roleagainst herbivores or pathogens (Lamb et al. 1989). In ourexperiment, the plants treated with carb showed no signifi-cant effect on PPO ( P  0.05; Fig. 3) and peroxidase ( P  0.05; Fig. 4) activities.Nemat Alla and Younis (1995) related the changes and,more specifically, the decline in secondary metabolic pro-cesses, to the growth reduction of plants treated with certainherbicides. In our experiment, the application of carb re-sulted in a significant increase in foliar dry weight withrespect to controls ( P  0.001; Fig. 5). This fact could beexplained by the positive effect of the fungicide on the PALactivity and the foliar accumulation of phenols, as there isevidence that in secondary metabolism these have a regula-tory role over plant growth (Nemat Alla and Younis 1995).In short, according to our results, the application of carbin tobacco plants not afflicted by biotic or abiotic agentsboosts phenol biosynthesis and accumulation. This mayimply an increased resistance of plants to infection by Fig. 2. Leaf concentration of total phenols in response to theapplication of carb or carb plus B in tobacco plants. Data aremeans  SE , n = 3.Fig. 4. Peroxidase activity in tobacco leaves in response to theapplication of carb or carb plus B in tobacco plants. Data aremeans  SE , n = 3. Physiol. Plant. 106, 1999 154  Fig. 5. Leaf dry weight accumulation in response to the applicationof carb or carb plus B in tobacco plants. Data are means  SE ,n = 3. (B 2 , B 3 , and B 4 ) contained foliar concentrations of thismicronutrient lower than in the control, the carb and thecarb plus B 1  treatments (Fig. 6), in which the B concentra-tion applied was lower.Recent research on B mobility has shown evidence forretranslocation of this micronutrient, principally after foliarapplication. Translocation is primarily by the phloem to-wards the meristem tissues (Brown and Shelp 1997). Thismobility of B could account for the decreased foliar concen-trations of this element in the carb plus B 2 , carb plus B 3 ,and carb plus B 4  treatments.The different B treatments also induced significant differ-ences in the enzymatic activity of PAL ( P  0.001; Fig. 1).The highest enzymatic activities were in the plants treatedwith carb plus B 2 , carb plus B 3 , and carb plus B 4 , while thelowest was recorded for the high concentrations of B (carbplus B 5  and carb plus B 6 ). The different B treatments alsocaused significant effects on total phenols ( P  0.001; Fig.2). In this case, the trend was completely opposite to that of the enzyme PAL (Fig. 1), since the intermediate treatments(carb plus B 2 , carb plus B 3 , and carb plus B 4 ) gave thelowest foliar concentrations of these compounds (Fig. 1).The relationship between these parameters was negative andsignificant (PAL activity-total phenols, r 2 =− 0.86***).Again, the B treatments induced significant differences inthe PPO ( P  0.001; Fig. 3) and peroxidase ( P  0.001; Fig.4) activities. The highest activities of the PPO and peroxi-dase enzymes appeared in the carb plus B 3  treatment, some179 and 190% greater, respectively, than the lowest PPO andperoxidase activities recorded with carb alone and carb plusB 1 . In general, the application of B boosted PPO andperoxidase activities, and the highest values were registeredfor B 2 , B 3 , and B 4  (Figs. 3 and 4, respectively). We suggestthat high PPO and peroxidase activities induced by B 2 , B 3 ,and B 4  account for the low total phenols observed in thesetreatments.The results of all the parameters of phenol metabolismstudied reflect an effect or direct involvement of B in thismetabolic process. The accumulation of phenolic com-pounds is characteristic of B-deficient tissues due to in-creased synthesis by induction of the enzyme PAL. Inresponse to high phenolic accumulation, PPO activity risesin B-deficient tissues (Cakmak and Ro¨mheld 1997). In ourexperiment, although without deficient conditions (Fig. 6), itis evident that the treatments with the lower B concentra-tions registered the highest PAL (synthesis), PPO, and per-oxidase (oxidation) activities, the relationships betweenthese parameters being negative and significant (B-PALactivity, r 2 =− 0.89***; B-PPO activity, r 2 =− 0.70**; B-peroxidase activity, r 2 =− 0.79**). The phenol-oxidationprocesses in the plants treated with carb plus B 2 , carb plusB 4  and, especially, with carb plus B 3 , could imply an in-crease in the resistance of these plants to infection bypathogens. Oxidation of phenolic compounds by PPO andperoxidase in leaves leads to the production of quinones(Thipyapong et al. 1995). Quinones are known to be highlytoxic and responsible for the production of active oxygenspecies (O 2 − , OH − , and H 2 O 2 ) (Pillinger et al. 1994). One of the principal events in the early phase of plant-pathogeninteractions is the rapid and transient production of activepathogens, given the essential role of phenolic compounds inthe lignification and suberization of the plant cell wall(Nagarathna et al. 1993, Daayf et al. 1997, Du¨beler et al. 1997). Finally, it is noteworthy that the application of carbdid not increase PPO or peroxidase activities, possibly be-cause these enzymes are activated only when the plant reactsto a pathogen (Siegrist et al. 1994). Combined effect of carbendazim-boron There were significant differences in B concentrations inleaves between treatments ( P  0.001; Fig. 6). The maximumB concentration appeared in the carb plus B 5 , and carb plusB 6  treatments (Fig. 6). In general, all the plants had B levelswithin the range normally observed for plants, except forthose that received B 5  and B 6  concentrations that can beconsidered toxic (Shelp 1993). In fact, these plants werecharacterized by toxicity symptoms such as chlorotic burnand necrotic patches on the margins and tips of older leaves(Nable et al. 1997). Plants receiving the intermediate B rates Fig. 6. B concentration in tobacco leaves in response to the applica-tion of carb plus B. Data are means  SE , n = 3. Physiol. Plant. 106, 1999  155
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