Effect of Different Elicitors and Preharvest Day Application on the

Article pubs.acs.org/JAFC

Effect of Different Elicitors and Preharvest Day Application on the Content of Phytochemicals and Antioxidant Activity of Butterhead Lettuce (Lactuca sativa var. capitata) Produced under Hydroponic Conditions Jesús Omar Moreno-Escamilla,† Emilio Alvarez-Parrilla,† Laura A. de la Rosa,† José Alberto Núñez-Gastélum,† Gustavo A. González-Aguilar,‡ and Joaquín Rodrigo-García*,§ †

Departamento de Ciencias Químico-Biológicas, Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez, Anillo envolvente del PRONAF y Estocolmo s/n, Ciudad Juárez, Chihuahua 32310, México ‡ Coordinación de Tecnología de Alimentos de Origen Vegetal, Centro de Investigación en Alimentación y Desarrollo, Carretera a la Victoria Km 0.6, Hermosillo, Sonora CP 8300, México § Departamento de Ciencias de la Salud, Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez, Anillo envolvente del PRONAF y Estocolmo s/n, Ciudad Juárez, Chihuahua 32310, México ABSTRACT: The effect of four elicitors on phytochemical content in two varieties of lettuce was evaluated. The best preharvest day for application of each elicitor was chosen. Solutions of arachidonic acid (AA), salicylic acid (SA), methyl jasmonate (MJ), and Harpin protein (HP) were applied by foliar aspersion on lettuce leaves while cultivating under hydroponic conditions. Application of elicitors was done at 15, 7, 5, 3, or 1 day before harvest. Green lettuce showed the highest increase in phytochemical content when elicitors (AA, SA, and HP) were applied on day 7 before harvest. Similarly, antioxidant activity rose in all treatments on day 7. In red lettuce, the highest content of bioactive molecules occurred in samples treated on day 15. AA, SA, and HP were the elicitors with the highest effect on phytochemical content for both varieties, mainly on polyphenol content. Antioxidant activity also increased in response to elicitation. HPLC-MS showed an increase in the content of phenolic acids in green and red lettuce, especially after elicitation with SA, suggesting activation of the caffeic acid pathway due to elicitation. KEYWORDS: Lactuca sativa L., preharvest day, phytohormones, antioxidant activity, polyphenols

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INTRODUCTION The intake of functional foods has increased due to the presence of molecules other than macronutrients that benefit the consumer’s health. These molecules, known as phytochemicals, can be found mainly in fruits and vegetables. Their concentration depends on factors such as agriculture practices, climate, and postharvest conditions. Butterhead lettuce (Lactuca sativa var. capitata) is a highly popular vegetable among consumers because of its flavor and texture. However, when compared with other lettuce varieties, butterhead lettuce presents lower concentrations of phytochemical content.1 Genetic modification,2 agronomic techniques,3 and elicitor application4 are the instruments employed to increase phytochemical contents in fruits and vegetables. Among these, elicitors have become popular because of their low cost and simplicity of usage. Elicitors can be either biotic or abiotic and, when applied at low doses or concentrations, trigger defense responses that enhance the production of secondary metabolites in plants. The most popular abiotic elicitors used in lettuce are UV light5 and water stress.6 On the other hand, phytohormones such as methyl jasmonate (MJ), arachidonic acid (AA), and salicylic acid (SA) have also been used as elicitors to trigger the production of phytochemicals by activating defense mechanisms in lettuce, broccoli, and moringa.7−9 Harpin protein (HP) has been used as elicitor in tomato to control its decay while increasing its phenolic © 2017 American Chemical Society

contents. HP affects lettuce as well, increasing its antioxidant activity.10,11 Furthermore, the type of elicitor used may activate different pathways that increase the phytochemical contents.8 Also, the application time of each elicitor may change the strength of the response and, therefore, influence the final phytochemical content in the plant at harvest time.12 Nevertheless, as far as we know, there are few systematic studies that report how the time of elicitor application influences the phytochemical contents of lettuce or any other plants.13 It is for this reason that this study aims to test how AA, SA, MJ, and HP affect phytochemical contents and the antioxidant activity of both green and red butterhead lettuce, as well as the impact of the moment of application day of elicitor during harvest days.

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MATERIALS AND METHODS

Chemicals and Reagents. 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,4,6-tripyridyl-s-triazine (TPTZ), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), Folin−Ciocalteu phenol reagent, sodium carbonate, methyl jasmonate, arachidonic acid, salicylic acid, monobasic potassium phosphate, dibasic potassium Received: Revised: Accepted: Published: 5244

April 12, 2017 June 7, 2017 June 13, 2017 June 14, 2017 DOI: 10.1021/acs.jafc.7b01702 J. Agric. Food Chem. 2017, 65, 5244−5254

Article

Journal of Agricultural and Food Chemistry phosphate, L-ascorbic acid, ferric chloride, sodium nitrate, aluminum chloride, sodium hydroxide, metaphosphoric acid, trichloroacetic acid, thiourea, β-carotene, α-tocopherol, δ-tocopherol, and γ-tocopherol were purchased from Sigma-Aldrich Chemical Co. (Oakville, ON, Canada). All solvents, except for the ones used for HPLC analysis, were purchased from Fisher Scientific (Nepean, ON, Canada) and were of ACS grade or better. HPLC solvents were purchased from Tedia (Farfield, OH, USA) and were HPLC grade. Plant Materials, Growing Conditions, and Elicitor Administration. Samples used in this research were planted, transplanted and harvested at InnoBio Hidroponia Inc. facilities by its employees. Butterhead lettuces plants (Lactuca sativa L. var. capitata) of green (FVM02 seed) and red (FRM02 seed) varieties were hydroponically grown (April−May 2016) in a recirculating system in a greenhouse with an average photoperiod of 10 h/day, a temperature of 25−28 °C, relative humidity of 40−60%, and 35% of sunlight blocking. Mineral nutrients consisted of N (16%), P (4%), K (17%), and a stage II micronutrient solution mix (prepared by InnoBio Hidroponia Inc.; pH 6.8, EC = 1800 mS). To evaluate the effect of application day of different elicitor treatments on green and red lettuce, the highest concentration for each elicitor, where no toxic effect has been reported, was chosen from previously published results. For AA,8 SA,14 and MJ,15 90 μM was chosen, while 120 mg/L was used for HP.10 All elicitors were dissolved in deionized water. Deionized water was used as control (C). The elicitors and control were administered at InnoBio facilities through three foliar aspersions (1.70 mL approx.) to each lettuce plant. Each elicitor was programmed for a single preharvest aspersion date (PH) (15, 7, 5, 3 or 1) on both green and red lettuce. Three green and three red lettuces were randomly treated for each elicitor at each treatment day. Hence, a total of 30 lettuces (15 green and 15 red) per elicitor were collected at the end of the experiment. Lettuce samples were harvested 60 days after being transplanted and subsequently lyophilized (Labconco 6 Freezone, Labconco Corporation, Kansas City, MO, USA), homogenized, sieved through a mesh (0.42 mm), and vacuum stored at −80 °C. Extraction and Quantification of Polyphenols. Polyphenols were extracted as described by Moreno-Escamilla et al.16 with slight modifications. Fifty-milligram samples were placed in a centrifuge tube, to which 10 mL of 80% methanol in water was added and later sonicated at 40 kHz for 30 min in the dark. The extract was subsequently centrifuged (2000g) for 30 min at 4 °C, and its supernatant was collected. The residues were re-extracted under the same settings, and both supernatants were combined. These combined methanolic extracts were stored at 80 °C until further analysis. Total phenolic content (TPC) was determined using the method described by Singleton and Rossi17 with slight modifications. Two hundred fifty microliters of methanolic extract was mixed with 1 mL of 7.5% sodium carbonate and incubated at room temperature for 3 min. Folin−Ciocalteu’s reagent (1.25 mL, 1:10 v/v) was added to the mixture, incubated at 50 °C for 15 min, and cooled to room temperature. Absorbance was read at 760 nm in a microplate reader (Microplate Spectrophotometer, Bio Rad, Mexico). Gallic acid was used as a standard. Results are expressed as mg of gallic acid equivalents (GAE)/g of dry sample. Total flavonoids (TF) were determined as described by Zhishen et al.18 with modifications. An aliquot (31 μL) of methanolic extract was mixed with 2 mL of water and 125 μL of 5% NaNO2 and incubated at room temperature. After 5 min, 125 μL of 10% AlCl3 was added and mixed thoroughly. Two milliliters of 0.5 M NaOH was added to the samples after 3 min. The sample was then incubated at room temperature for 30 min. Absorbance was read at 510 nm in a microplate reader. Catechin was used as the standard, and results were expressed as milligrams of catechin equivalents (CE)/g of dry sample. Extraction and Quantification of Anthocyanins. Total anthocyanin (TAN) content was measured as described by Lee et al.19 with slight modifications. Extracts were obtained by mixing 50 mg of freeze-dried red lettuce samples with 20 mL of methanol/water/ acetic acid (85:15:0.5, v/v/v). All samples were vortexed for 30 s, sonicated for 5 min, kept at room temperature for 20 min, and vortexed again for 30 s. The extracts were then centrifuged for 10 min

at 2000g. The pH of the samples (2 mL) was adjusted to 1.0 and 4.5 using potassium chloride/0.025 M HCl and 0.4 M sodium acetate buffer, respectively. Absorbance at 520 and 700 nm was measured on a microplate after 30 min of incubation at room temperature. Absorbance (A) was calculated using eq 1

A = (A 520 nm − A 700 nm)pH 1.0 − (A 520 nm − A 700 nm) pH 4.5.

(1)

Total anthocyanin content (mg of cyanidin 3-rutinoside/g) was calculated using eq 2. Results are expressed as mg of cyanidin 3rutinoside/g of dry sample.

Total anthocyanin =

A × 449.2 × 25 × 1000 26900 × 1

(2)

Extraction and Quantification of Ascorbic Acid. Ascorbic acid (ASC) content in lettuce samples was determined as described by Moreno-Escamilla et al.16 with slight modifications. ASC was extracted from green and red lettuces samples by sonicating 50 mg of freezedried samples with 5 mL of metaphosphoric acid (5%) for 20 min in the dark and later centrifuging at 2000g for 10 min at room temperature. The supernatant collected was subsequently used to quantify ASC by mixing 300 μL of supernatant with 200 μL of 6.65% trichloroacetic acid and 75 μL of DNPH reagent (2 g dinitrophenylhydrazine, 230 mg of thiourea, and 270 mg of CuSO4·5H2O in 100 mL of 5 M H2SO4). The mixture was then incubated for 3 h at 37 °C before adding 0.5 mL of 65% H2SO4. Two hundred fifty microliters of this mixture was placed in a microplate where absorbance was measured at 520 nm. ASC was used as a standard, and results are expressed as mg ASC/g of dry sample. Extraction and Quantification of Carotenoids. Total carotenoids (CAR) in lettuce were determined using the method described by López-Cervantes et al.,20 with slight modifications. Fifty milligrams of the sample were mixed with 10 mL of acetone, sonicated for 20 min, and centrifuged at 2000g for 10 min. The supernatant was collected, and the residue was re-extracted two more times under the same conditions. The supernatants were combined and a total of 250 μL taken and placed on a microplate to be read at 474 nm. Total carotenoids were determined using eq 3:

mg β‐carotene/mg sample = (A × V × DF × 10)/(g × E1% cm)

(3)

where A = absorbance, V = volume (30 mL), DF = dilution factor, g = grams of sample, and E1 % cm = the specific extinction coefficient of β-carotene, which is 2500. Results are expressed in mg β-carotene/g of dry sample. Identification and Quantification of Tocopherols. Tocopherol identification and quantification were performed as described by Sánchez-Machado et al.21 Tocopherols were extracted using 200 mg of freeze-dried green or red lettuce samples and placed in a centrifuge tube. Two hundred microliters of catechol (20% w/v in methanol) and 5 mL of KOH (0.5 M in methanol) were added and vortexed for 20 s. The tubes were then heated at 80 °C for 15 min, and homogenized with a 15 s vortex every 5 min. Once cooled, 1 mL of distilled water and 5 mL of hexane were added. The tubes were again vortexed for 1 min and centrifuged for 2 min at 425 g. Three milliliters of the upper phase were taken and placed in another tube to be evaporated with nitrogen. The residue was dissolved in 3 mL of HPLC mobile phase and filtered (0.45 μ membrane filter). All samples were injected into a PerkinElmer model 200 series HPLC equipped with a diode array detector (DAD) and an autosampler. A C18 Luna column (250 × 4.6 mm), 5 μm particle size (Phenomenex, Torrance, CA, USA), was used. Fifteen microliters was injected under isocratic conditions using methanol/acetonitrile 20:80 (v/v) as mobile phase at 25 °C at a flow rate of 1.0 mL/min for 20 min. Tocopherols were identified and quantified at 208 nm. Identification of tocopherols was done taking into account retention time, coinjection with standards, and UV spectra. Tocopherols were quantified using pure standard compounds (α-tocopherol, δ-tocopherol, and γ-tocopherol). The results are 5245

DOI: 10.1021/acs.jafc.7b01702 J. Agric. Food Chem. 2017, 65, 5244−5254

Article

Journal of Agricultural and Food Chemistry

Table 1. Phytochemical Content in Green Butterhead Lettuce Treated with Different Elicitors at Different Preharvest Daysa TPCb

treatment PH day 15

PH day 7

PH day 5

PH day 3

PH day 1

AA SA MJ HP C AA SA MJ HP C AA SA MJ HP C AA SA MJ HP C AA SA MJ HP C

37.61 32.41 33.41 33.37 35.57 37.74 35.91 31.96 35.41 32.04 34.09 31.81 31.65 31.24 30.57 34.84 33.26 31.41 34.08 33.77 32.31 32.21 31.02 29.66 31.51

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

5.44 4.33 3.70 1.77 1.99 1.91 1.81 1.56 1.21 1.31 1.10 0.87 1.48 0.80 0.38 0.72 2.11 3.02 2.38 2.73 2.53 0.22 2.89 0.12 1.95

TFc a a a a a a a b ab b a a ab b b a a a a a a a a a a

21.39 18.54 21.69 22.16 22.92 21.25 21.01 21.38 36.66 14.62 38.36 19.63 26.28 28.68 17.82 31.26 16.67 15.96 19.38 25.91 34.79 19.22 30.79 18.49 26.82

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.04 2.15 3.94 2.72 2.24 0.80 0.38 1.25 1.71 2.23 0.52 2.03 1.66 2.01 0.48 2.88 0.50 1.52 0.73 2.36 4.09 1.58 0.43 2.36 5.53

ASCd a a a a a b b b a c a c b b c a c c c b a b a b ab

3.58 3.85 3.67 3.98 3.47 3.67 3.45 3.03 4.13 4.03 3.35 3.81 4.06 3.98 4.03 3.72 3.95 3.95 4.32 4.89 3.42 3.60 4.34 3.89 4.47

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.19 0.12 0.04 0.11 0.19 0.28 0.27 0.25 0.25 0.28 0.08 0.16 0.14 0.25 0.27 0.23 0.32 0.09 0.42 0.22 0.13 0.09 0.34 0.08 0.53

CARe b ab ab a b ab ab b a a b ab a a a b b b ab a c bc ab abc a

3.38 2.82 2.88 3.26 3.08 3.19 3.52 3.22 2.49 3.02 3.34 2.61 2.94 2.87 2.83 2.62 2.56 2.57 3.22 2.93 3.05 2.31 2.49 2.99 2.35

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.36 0.10 0.60 0.47 0.38 0.30 0.11 0.19 0.35 0.15 0.44 0.21 0.28 0.55 0.15 0.19 0.26 0.34 0.19 0.45 0.67 0.17 0.16 0.47 0.39

a a a a a ab a ab b ab a a a a a a a a a a a a a a a

a Values represent the mean ± SD of three measurements. All values are given on a dry weight basis. Different letters indicate differences between the elicitor and the control (distilled water) in the same preharvest day of application. (Tukey’s test, P < 0.05). TPC: total phenolic content. bmg GAE/ g; TF, total flavonoids. cmg CE/g; ASC, ascorbic acid. dmg ASC/g; CAR, carotenoids. emg β-carotene/g. AA, arachidonic acid; C, control; HP, Harpin protein; MJ, methyl jasmonate; PH, preharvest; SA, salicylic acid.

expressed in mg of each tocopherol/g of sample and total tocopherols/g of dry sample. Antioxidant Activity. The antioxidant activity of methanolic extracts of green and red lettuce was measured using three different methods: DPPH, TEAC, and FRAP. DPPH and TEAC were measured as described by Thaipong et al.22 DPPH analysis involved taking 50 μL of sample being mixed with 200 μL of DPPH radical (190 μM in methanol) in each well of a 96-well plate. Absorbance was measured at 515 nm for 10 min on a microplate reader. The inhibition percentage of radical scavenging activity was calculated using eq 4:

Inhibition = (Abs blank − Abs sample)/Abs blank × 100

methods. In all cases, the results were expressed as milimol Trolox equivalent/g of dry sample. Identification of Phenolic Compounds by HPLC-ESI-QTOFMS. Identification of polyphenols was carried out with the help of HPLC-ESI-QTOF-MS and by following the protocol described by Pellati et al.23 with slight modifications. An HPLC 1200 Series system (Agilent Technologies, Palo Alto, CA, USA) equipped with a vacuum degasser, an autosampler, and a quaternary pump was used to identify polyphenols. Separation was performed at 25 °C using a rapid resolution high definition (RRHD) reverse phase C-18 analytical column (2.1 × 50 mm, 1.8 μm particle size; ZORBAX Eclipse Plus) protected with a guard cartridge of the same packing. The mobile phase consisted of formic acid (0.1%) in Milli-Q deionized water (A) and acetonitrile (B). A volume of 1 μL (methanolic extract) was injected at a flow of 0.4 mL/min. The gradient used was the following: 0−4 min, 90% A, 4−6 min, 70% A, 6−8 min, 62% A, 8−8.5 min, 40% A, and 8.5−9.5 min, 90% A. The column was re-equilibrated for 2 min before each injection. MS were obtained using a quadrupole time-offlight (QTOF) mass spectrometer (G6530BA, Agilent Technologies, Palo Alto, CA, USA). ESI-MS measurements were done by negative ionization with the gas temperature set at 340 °C, drying gas flow of 13 L/min, nebulizer of 30 psi. The MS was recorded in the range of 100 to 1700 m/z. Statistical Analysis. A pool of all individuals for each treatment (3 lettuces) was made to minimize the variability among individual samples. All analyses were carried out in triplicate. Values are expressed as the mean ± standard deviation (SD). Two-way ANOVA and Tukey analyses were performed to determine statistical differences (p < 0.05) between elicitors and the days of application. Data values were analyzed using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA).

(4)

where Abs blank is the absorbance of DPPH (or ABTS) at 10 min, and Abs sample is the absorbance of the radical plus the sample. The TEAC method was carried out using the ABTS radical. The radical cation was prepared in 50 mL of 0.1 M saline phosphate buffer (PBS, pH 7.4, 0.15 M KCl) by mixing ABTS salt (7 mM, final concentration) with potassium persulfate (2.45 mM final concentration). This solution was kept in the dark at room temperature for 12−16 h before use. After incubation, the ABTS•+ solution was diluted with saline phosphate buffer to obtain an absorbance of 0.700 ± 0.1 at 734 nm. For the reaction, 285 μL was mixed with 12 μL of the sample or blank (PBS) in a 96-well plate. The reaction was measured every 30 s for 6 min at 734 nm with a microplate reader. Inhibition percentage of radical scavenging activity was calculated using eq 4. Reducing power was measured using FRAP according to the method of Moreno-Escamilla et al.16 One hundred and eighty microliters of FRAP reagent (0.3 M acetate buffer (pH 3.6), 10 mM TPTZ−HCl (2,4,6-tripyridyl-s-triazine; 40 mM HCl), and 20 mM ferric chloride 10:1:1, v/v/v, heated at 37 °C for 30 min were mixed with 24 μL of sample into each well of a 96-well plate. Absorption was measured at 595 nm every 60 s for 30 min. Standard curves using Trolox were used to calculate antioxidant activity with the three 5246


Article


Table 2. Phytochemical Content in Red Butterhead Lettuce Treated with Different Elicitors at Different Preharvest Daysa TPCb

treatment PH day 15

PH day 7

PH day 5

PH day 3

PH day 1


53.34 51.17 44.81 49.70 39.89 42.19 44.08 46.51 47.98 46.05 48.23 46.24 44.54 36.65 42.37 44.25 45.83 44.71 40.94 42.11 45.07 45.21 48.58 48.97 44.21

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.12 0.88 3.31 2.11 2.68 2.01 1.19 3.82 1.88 4.14 2.36 1.69 0.75 3.92 4.00 2.10 1.54 2.22 3.88 3.69 3.70 8.02 7.24 4.26 4.35

TFc a a bc ab c a a a a a a a a ab a a a a a a a a a a a

36.56 38.26 31.10 33.96 30.64 33.88 34.63 33.89 36.34 36.15 38.98 40.98 41.14 34.48 41.97 37.03 41.21 36.31 37.46 37.22 45.48 35.45 51.69 51.80 43.65

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

TANd

1.16 3.94 0.94 0.04 1.65 1.81 3.95 3.00 1.56 5.67 1.70 5.06 2.94 0.74 2.02 4.18 1.88 4.59 2.60 1.37 3.05 2.14 3.51 5.30 2.14

a a b ab b a a a a a a a a a a a a a a a a b a a ab

3.85 4.99 2.57 3.25 2.95 4.07 4.07 4.09 5.27 4.27 3.84 4.66 2.75 3.12 3.06 3.61 3.82 4.41 3.71 4.27 4.09 3.91 4.85 5.91 4.46

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.22 0.19 0.30 0.25 0.18 0.28 0.58 0.55 0.27 0.25 0.13 0.44 0.33 0.71 0.23 0.28 0.15 0.48 0.73 0.20 0.08 0.69 0.35 0.63 0.65

ASCe a a b b b b b b a ab ab a b b b a a a a a b b ab a b

6.05 5.59 5.72 4.90 4.36 5.26 5.43 5.20 5.34 6.11 4.54 6.10 4.60 4.52 5.02 5.38 5.10 6.65 5.50 5.12 5.53 6.89 5.94 7.08 5.18

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

CARf

0.26 0.35 0.92 0.33 0.39 0.58 0.27 0.58 0.29 0.42 0.40 0.15 0.45 0.23 0.19 0.18 0.31 0.53 0.47 0.55 0.32 0.21 0.30 0.79 0.46

a ab ab ab b a a a a a b a b b b b b a ab b b a ab a b

6.16 6.15 6.91 6.73 5.59 6.88 6.35 7.18 6.80 6.38 7.71 7.54 6.60 6.61 7.07 5.89 6.71 4.97 5.47 6.75 5.42 5.65 6.46 5.86 6.24

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.17 0.25 0.64 0.59 0.06 0.11 0.36 0.49 0.25 0.25 0.11 0.39 0.57 0.59 0.06 0.62 0.37 0.40 0.50 0.57 0.16 0.35 0.30 0.46 0.33

a a a a b a a a a a a a a a a ab a b ab a b ab a ab ab

a Values represent the mean ± SD of three measurements. All values are given on a dry weight basis. Different letters indicate differences between the elicitor and the control (distilled water) in the same preharvest day of application. (Tukey’s test, P < 0.05). TPC: total phenolic content. bmg GAE/ g; TF, total flavonoids. cmg CE/g; TAN, total anthocyanins. dmg of cyanidin 3-rutinoside/g; ASC, ascorbic acid. emg ASC/g; CAR, carotenoids. fmg β-carotene/g. AA, arachidonic acid; C, control; HP, Harpin protein; MJ, methyl jasmonate; PH, preharvest; SA, salicylic acid.

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RESULTS Effect of Elicitors on Polyphenol Content. Table 1 shows the total phenolic content (TPC) of green lettuce treated with elicitors at different preharvest days. Values were higher than those previously reported for green lettuce.8,24 Only AA and SA induced a significant increase of TPC when applied on PH days 5 and 7. Lettuces treated with AA and SA on PH day 5 showed a small increase in TPC of 11% and 4%, respectively, compared to that of the control of that day. Application of the same treatments (AA and SA) on PH day 7 increased TPC content by 17% and 12%, respectively. Table 2 shows changes in the TPC content of red lettuces after being treated with elicitors. The values obtained in this experiment fall below those previously reported for red lettuce.25 Elicitors applied on PH day 15 showed the strongest effect with a significant increase in TPC of lettuce treated with AA, SA, and HP (33%, 28%, and 24%, respectively), compared to that of the control. Elicitors did not affect TPC content when applied on any other PH days. In the case of MJ, there was a small, nonsignificant increase. Table 1 shows the TF content in green lettuce, which is higher than those reported in other studies.8 None of the elicitors applied on PH day 15 had a significant effect. However, TF concentration levels changed when elicitors were applied on the rest of the days, especially in PH days 5 and 7. Application of HP, MJ, AA, and SA on PH 7 induced a significant increase of 150%, 45%, 45%, and 43% in TF, respectively. In the same way, application of AA, HP, MJ, and SA on PH day 5 significantly increased TF content by 115%, 60%, 47%, and 10% respectively. Table 2 shows TF content in red lettuce. SA

and AA induced increases of 24% and 19%, respectively, when they were applied at PH day 15. Since no anthocyanins were detected in green lettuce samples, TAN content is only reported in red lettuce samples (Table 2). In agreement with TPC and flavonoids, the highest effect of elicitors on TAN content was observed on PH day 15. SA and AA induced a significant increase of 70% and 30%, respectively, when applied on this PH day. For the rest of the PH days, only SA (52%) and HP (32%) had a significant increase compared to that of the control. Effect of Elicitors on Other Phytochemicals. Table 1 shows ASC content in green lettuce. Of all elicitors, HP, applied on PH day 15, was the only one able to significantly increase ASC content (13%). The rest of the treatments showed no significant increases. Table 2 shows the ASC content in red lettuce. The major impact on ASC content was obtained with AA applied on PH day 15, where a significant increase of 38% was observed. Other significant increases on other days of application were observed. SA (21%) and MJ (29%) increased the ASC content of red lettuce when applied on PH days 5 and 3, respectively, and SA (33%) and HP (36%) had significant increases when they were applied at PH day 1. However, these increments were lower than those obtained by application on PH day 15. Table 1 shows CAR values of green lettuce treated with elicitors. From an analysis of this table, it is possible to assert that the elicitors did not cause a significant increase in carotenoid content. Table 2 shows CAR content in red lettuce. Red lettuce showed higher CAR content than green lettuce. The maximum effect of elicitors over CAR content was 5247


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Table 3. Tocopherol Content (mg/g of Dry Sample) in Butterhead Lettuce Treated with Different Elicitors at Different Preharvest Daysa green lettuce α-tocopherol

treatment PH day 15

PH day 7

PH day 5

PH day 3

PH day 1


0.19 0.24 0.26 0.26 0.22 0.21 0.23 0.24 0.21 0.22 0.17 0.20 0.21 0.20 0.19 0.21 0.18 0.20 0.16 0.28 0.22 0.16 0.15 0.25 0.14

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.01 0.01 0.04 0.01 0.07 0.06 0.01 0.02 0.01 0.03 0.01 0.03 0.03 0.03 0.01 0.03 0.00 0.02 0.02 0.04 0.07 0.06 0.06

a a a a a a a a a a a a a a a a a a a a a a a a a

γ-tocopherol 1.30 1.14 1.09 1.29 0.92 1.01 1.09 1.11 1.21 1.12 1.09 1.07 1.13 1.01 1.13 1.15 1.25 1.59 1.23 1.48 1.18 1.19 1.21 1.96 1.82

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.05 0.08 0.10 0.08 0.13 0.04 0.02 0.03 0.11 0.09 0.11 0.18 0.11 0.17 0.16 0.15 0.13 0.14 0.09 0.14 0.15 0.06 0.20 0.16 0.06

red lettuce total tocopherols

a a ab a b b a a a a a a a a a b b a b a b b b a a

1.49 1.39 1.35 1.56 1.14 1.22 1.32 1.35 1.42 1.34 1.27 1.27 1.34 1.21 1.33 1.36 1.44 1.80 1.40 1.76 1.41 1.36 1.37 2.21 1.97

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.06 0.08 0.12 0.09 0.17 0.05 0.02 0.04 0.12 0.11 0.11 0.21 0.12 0.21 0.19 0.18 0.14 0.18 0.09 0.16 0.17 0.07 0.19 0.18 0.07

a a ab a b a a a a a a a a a a b b a b a b b b a a

α-tocopherol 0.32 0.34 0.24 0.83 0.32 0.65 0.59 0.57 0.52 0.46 0.57 0.42 0.45 0.56 0.75 0.87 0.95 0.53 0.97 1.24 0.55 0.52 0.64 0.87 0.27

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.02 0.01 0.05 0.02 0.05 0.05 0.07 0.03 0.04 0.03 0.03 0.01 0.02 0.08 0.03 0.04 0.07 0.08 0.04 0.08 0.08 0.03 0.05 0.01

b b c a b a b b b c b c c b a c b d b a c c b a d

γ-tocopherol 0.85 0.75 0.58 0.97 0.83 1.03 0.80 0.82 0.99 0.93 0.99 0.89 0.84 1.14 1.04 1.23 1.07 0.98 1.14 1.24 0.94 1.02 1.14 1.33 0.77

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.02 0.07 0.03 0.01 0.02 0.05 0.08 0.06 0.05 0.06 0.10 0.05 0.06 0.07 0.03 0.04 0.10 0.05 0.03 0.14 0.12 0.11 0.10 0.02

b c d a b a a a a a a b b a a a c c b a a a a a b

total tocopherols 1.17 1.10 0.82 1.81 1.16 1.68 1.40 1.40 1.52 1.39 1.56 1.31 1.30 1.70 1.80 2.10 2.02 1.52 2.12 2.48 1.35 1.55 1.79 2.21 1.04

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.05 0.05 0.08 0.04 0.01 0.06 0.10 0.14 0.07 0.09 0.09 0.13 0.04 0.05 0.09 0.05 0.08 0.18 0.13 0.06 0.06 0.21 0.14 0.16 0.03

b b c a b a b b b b b bc c a a b b c b a c bc b a d

Values represent the mean ± SD of three measurements. All values are given on a dry weight basis. Different letters indicate differences between the elicitor and the control (distilled water) in the same preharvest day of application. (Tukey’s test, P < 0.05). AA, arachidonic acid; C, control; HP, Harpin protein; MJ, methyl jasmonate; PH, preharvest; SA, salicylic acid. a

Effect of Elicitors on Antioxidant Activity. Elicitors had an impact on the antioxidant activity of green and red lettuces. As expected, antioxidant activity was higher in red pigmented lettuces in comparison with green.26 In green lettuce samples, antioxidant activity, measured by the three methods, significantly increased when elicitors were applied on PH day 7 and to a lesser degree on PH day 5 (Figure 1a−c). When assayed by the DPPH method, antioxidant activity was highest on samples treated on PH day 7, with an increase of 81%, 22%, 18%, and 16% for HP, MJ, SA, and AA, respectively, while at PH day 5 20% and 11% increments were observed for HP and SA, respectively. The TEAC method showed a significant increase in antioxidant activity of samples treated on PH day 7 for all elicitors with an increase of 34%, 33%, 27%, and 25% for AA, HP, MJ, and SA, respectively. In the same way, the FRAP technique showed a significant increase for samples treated with SA and AA (22% and 18%, respectively) on the same date. Red lettuce antioxidant activity is shown in Figure 2a−c. Its antioxidant activity, measured with DPPH (16% for AA) and TEAC (29%, 22%, 21%, and 10% for SA, MJ, HP, and AA, respectively), showed a significant increase when elicitors were applied on PH day 15, with this behavior being more evident for DPPH where all the treatments produced a significant increase. On the rest of the days, only HP showed an effect on PH day 1 for DPPH (16%) and TEAC (20%). No significant differences were observed when the FRAP assay was used. Effect of Elicitors on Phenolic Profile. An HPLC-ESIQTOF-MS analysis was carried out in samples of green lettuce treated with elicitors on PH day 7 and samples of red lettuce

observed by application at PH day 15, when all treatments exerted a significant increase. MJ showed the highest effect (23% increase), followed by HP, AA, and SA with 20%, 10%, and 10% increases, respectively. Applying elicitors on different PH days affected the content of tocopherol in lettuce. α-, δ-, and γ-tocopherols were analyzed by HPLC. γ-Tocopherol and α-tocopherol were detected in all samples, whereas δ-tocopherol was not (Table 3). αTocopherol was not affected by elicitors in green lettuce samples. γ-Tocopherol showed a significant increase when samples were treated with AA (41%), HP (40%), and SA (23%) on PH day 15. This caused an increase in total tocopherol content of 27%, 24%, and 19% in green lettuce treated with AA, SA, and HP, respectively. Regarding red lettuce, α-tocopherol increased significantly by treatment with HP on PH day 15 (159%) and AA on PH day 7 (41%). Values of α-tocopherol increased when treated on PH day 1 by 222%, 137%, 103%, and 92% for HP, MJ, AA, and SA, respectively. HP treatment on PH day 15 caused γ-tocopherol to experience a significant increase (16%) in red lettuce. On the other hand, application of all elicitors only 1 day before harvest (PH day 1) raised γ-tocopherol content by 72%, 48%, 32%, and 22% for HP, MJ, SA, and AA, respectively. The rest of the treatments showed no significant increase. Total tocopherols were increased in red lettuce by HP treatment on PH day 15 (56% increment) and by AA application on PH day 7 (20% increment). HP had a stronger effect on total tocopherols when applied on PH day 1 (112% increment). 5248


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Figure 1. Antioxidant activity determined with different techniques in green butterhead lettuce after elicitation. TEAC (A), DPPH (B), and FRAP (C). The asterisk symbolizes a significant increase in comparison with that in the control.

Figure 2. Antioxidant activity determined with different techniques in red butterhead lettuce after elicitation. TEAC (A), DPPH (B), and FRAP (C). The asterisk symbolizes a significant increase in comparison with that in the control.

treated by the same elicitors on PH day 15. Figure 3 shows the chromatograms of green lettuce (A), red lettuce (B), and the mass spectra of tentatively identified compounds (C). Polyphenols were tentatively identified based on their Rt, UV, accurate mass weight, and fragmentation pattern similar to those of other compounds previously described in lettuce.25 The results are shown in Table 4. Peak b had a negative molecular ion [M − H]− at m/z 311.1 and a fragment at m/z 179.08, probably corresponding to caffeoyltartaric acid, according to Llorach et al.25 Peak c showed an [M − H]− at m/z 703.3 with a fragment [M − H]− at m/z 353.1, tentatively identified as a chlorogenic acid dimer.27 Peak d showed an [M − H]− at m/z 353.1 with a fragment at m/z 191.1 similar to those reported for chlorogenic acid.25 Peak e showed an [M − H]− at m/z 473.1 and a fragment at m/z 311.1, and was tentatively identified as chicoric acid.25,27,28 Peak f showed an [M − H]− at m/z 473.1 and was tentatively identified as mesodicaffeoyltartaric acid (m/z 473.1) and isochlorogenic acid (m/ z 515.2) according to previously reported results.25,28 Our results suggest that application of all elicitors may promote activation of the caffeic acid pathway leading to the production of these subsequent products in both green and red lettuce. Peak g, only detected in red lettuce, showed an [M − H]− at m/z 461.1 and was tentatively identified as luteolin-7-glucoside,

which has been recently identified in red lettuce.25,29 Peaks a and h showed [M − H]− at m/z 133.0 and 505.2, respectively. However, they were not identified and thus are reported as unknown. Finally, peak i showed an [M − H]− at m/z 515.22 and was tentatively identified as isochlorogenic acid, according to previously reported results.25,27 When samples treated with elicitors were analyzed, it was possible to observe that the same peaks appeared in all samples. However, the abundance of each peak varied depending on the elicitor used. Table 4 presents the percentage of increase (+) or decrease (−) of each peak in elicitor-treated samples compared with those of the control. SA had the highest effect on the increase of identified polyphenols in green and red lettuce samples that were analyzed. Green lettuce showed an increase of its caffeoyl tartaric acid and isochlorogenic acid contents when treated with SA, AA, and HP. Chlorogenic acid was also increased in green lettuce with SA treatment, while chicoric acid was increased with HP and AA. In red lettuce, SA and HP treatments had the highest impact on the phenolic profile, increasing the content of caffeoyl tartaric acid (only SA), chlorogenic acid, chicoric acid, luteolin-7-glucoside, and isochlorogenic acid. MJ only showed an increase in chicoric acid, and AA only influenced luteolin-7-glucoside content. 5249


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Figure 3. HPLC chromatogram with UV detection at 320 nm of green control lettuce (A) and red control lettuce (B). (a) unknown, (b) caffeoyltartaric acid, (c) chlorogenic acid dimer, (d) chlorogenic acid, (e) chicoric acid, (f) meso-dicaffeoyltartaric acid, (g) luteolin-7-glucoside, (h) unknown, and (i) isochlorogenic acid. ESI negative ion mass spectra of phenolic compounds detected in green and red lettuce (C). 5250


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Table 4. Chromatographic, UV, and Mass Spectra Information for the Tentative Identification of Polyphenols and Their Percentage of Variation in Green and Red Lettuce Treated with Different Elicitorsa green lettuce

red lettuce

peak

tR (min)

[M − H]−

proposed molecule

AA

SA

MJ

HP

AA

SA

MJ

HP

a b c d e f g h i

0.34 1.02 1.49 2.67 4.00 4.15 4.35 4.60 4.70

133.05 311.11 707.33 353.16 473.11 473.11 461.1 505.2 515.22

unknown caffeoyl tartaric acid chlorogenic acid dimer chlorogenic acid chicoric acid meso-dicaffeoyltartaric acid luteolin-7-glucoside unknown isochlorogenic acid

−3.5 7.5 −9.1 −42.9 18.2 −28.6 ND −20.0 40.0

5.9 42.5 54.5 −28.6 −9.1 −21.4 ND 26.7 137.5

−5.9 −10.0 −47.3 −57.1 0.0 −7.1 ND −20.0 0.0

11.8 12.5 −9.1 −52.4 36.4 −28.6 ND 33.3 50.0

150.0 −30.4 −7.1 −22.5 0.0 −31.4 20.0 12.5 0.0

155.6 13.0 28.6 −5.0 76.5 0.0 50.0 37.5 55.6

150.0 −4.3 −7.1 −34.0 41.2 −8.6 0.0 0.0 −5.6

163.9 −4.3 14.3 −5.0 29.4 −14.3 10.0 −12.5 22.2

a

All treatments were applied at PH day 7 in green lettuce and PH day 15 in red lettuce. AA, arachidonic acid; C, control; HP, Harpin protein; MJ, methyl jasmonate; ND, not detected; PH, preharvest; SA, salicylic acid.

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DISCUSSION Polyphenols are secondary metabolites present in plants. They play an important role in response to biotic and abiotic stress that can be induced by environmental factors and other elicitors.30 In this research, a significant increase of total phenols in green and red lettuce samples was observed. This increase depended on two factors: elicitor used and time of application before harvest. In all cases and in agreement with previous results, red lettuce showed higher TPC content than green lettuce.25,31 No effect on TPC was noticeable when elicitors were applied on PH days 1 and 3. This lack of effect can be explained considering that the plant needs a certain amount of time to respond to elicitor-induced stress, as reported by Kim et al.,24 who observed an increase in TPC in romaine lettuce treated with MJ only after 7 days after treatment. The increase in TPC induced by elicitor application on other PH days could be explained considering that the addition of elicitors, either biotic or abiotic, triggers the activation of several plant responses such as acidification of cytosol, an increase of Ca2+ flux, activation of NADPH, and production of ROS, among others.32 The use of elicitors for the increase of TPC had been widely used. AA showed a 11% increment of TPC values in butterhead lettuce;8 MJ showed a 25% increase in romaine lettuce,24 and SA increased TPC in broccoli approximately 20%.9 Harpin protein has been reported to increase 9% TPC in lettuce.11 There is evidence that suggests that the biotic or abiotic elicitors applied to vegetables act as second messengers that activate transcription factors to synthesize enzymes related with the production of secondary metabolites as a defense tool for the plant.6 The upregulation of these enzymes after elicitation has been reported in tomato after Harpin protein elicitation.10 Kim et al.24 reported that PAL, responsible of the phenylpropanoids biosynthesis, is activated after elicitation with MJ in romaine lettuce. These results suggest that the treatment of plants with elicitors can activate secondary metabolite enzymes leading to an increase of these compounds in the plant. The increase in TF content elicited in green lettuce by AA, SA, and HP agrees with the behavior observed for TPC (Table 1). TF content in green lettuce is higher compared to that in previous studies.8 TF content in red lettuce was higher compared to that in green lettuce. These results are in agreement with previous studies.25,31 A 31% increase of TF after elicitation with MJ has been reported in broccoli.9 Wang et al.33 observed a 10% increase of TF in melon after elicitation

with HP. The increase in TF could be related to the activation of chalcone synthase, a key enzyme in the synthesis of flavonoids by coupling ρ-coumaryl-CoA and malonyl-CoA resulting in a chalcone.34 Campos et al.35 reported that SA increased the activity of this enzyme in bean. The activation of this enzyme could be related to an overproduction of enzyme mRNA, leading to protein translation and thus to a possible increase of flavonoid synthesis as reported by Schenk et al.36 The increase in TF values after elicitation could be related with ROS production. When ROS concentration increased and destabilized the plant homeostasis, some flavonoids (i.e., quercetin) provided stress protection by acting as ROS scavengers, as well as chelating metals that generate ROS via the Fenton reaction.37 Anthocyanins are responsible for the red color in several fruits and vegetables. Our results are in agreement with previously published data showing that anthocyanins are only found in red pigmented vegetables. However, our results showed higher TAN values than those reported for other red lettuce varieties.38,39 Increase of anthocyanins has been observed after elicitation with MJ in blackberries,40 SA in carrots,41 and HP in Impatiens.42 These results are in agreement with our results in red lettuce. The increment of anthocyanins is related to the activation of several defense responses, which include the activation of defense transcription factors that activate enzymes related to phytochemicals, including the activation of UPGT (UDP-glucose, flavonoid-O-transferase), a key enzyme for anthocyanin synthesis, as observed in Arabidopsis by Shan et al.43 ASC content in control lettuce was high in both green and red lettuce compared to previous studies performed on other lettuce varieties.8,25 Elicitor treatment on green lettuce did not influence ASC content. Nonetheless, red butterhead lettuce showed a significant increase in ASC content. AA and jasmonic acid in butterhead lettuce increased ASC values by 217% and 211%, respectively, as reported by Złotek et al.8 SA (25%) and MJ (14%) had the ability to increase vitamin C in broccoli.9 Authors have proposed that this increase in ASC content after elicitor treatment could be caused by the activation of LGalLDH (L-galactono-1,4-lactone dehydrogenase), a key enzyme in the production of ASC.6 Studies suggest that the activation of this enzyme is related to ROS generated during the oxidative burst due to stress conditions.44 In the presence of ROS, the cell triggers the transcription of L-GalLDH mRNA that can be translated to protein which acts in the 5251


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Journal of Agricultural and Food Chemistry mitochondria, using oxidized cytochrome c as electron receptor to produce ascorbic acid.45 CAR content in control samples was in the range of those reported for green lettuce by Kim et al.24 Slight increases in CAR content were observed in green lettuce treated with elicitors on PH day 7 (Table 1). In red lettuce, SA, MJ, and HP applied on day 15 increased the content of carotenoids (Table 2). Złotek et al.8 report a 45% carotenoids increase in butterhead lettuce after AA treatment. MJ was used as the elicitor in romaine lettuce, observing a 33% increase in carotenoids.24 Saini et al.7 report a 23% and 20% increment in carotenoid content in Moringa olifeira leaves after elicitation with SA and MJ, respectively. Our results are in agreement with those of these researchers and may be explained considering the fact that elicitation may increase the activity of lycopene βcyclase, the main enzyme in carotenoid synthesis, due to the plant−elicitor interaction.7 This increase of CAR content in lettuce could be also linked to the overproduction of ROS in the plant after elicitation.46 There is little information regarding the effect of elicitors over tocopherol content in plants. Saini et al.7 reported a 154% and 187% increment in α-tocopherol in Moringa olifeira leaves after elicitation with MJ and SA, respectively. Our result confirmed that elicitors enhanced phytochemical content such as tocopherols. The observed increase in specific and total tocopherols after elicitor treatment may be explained considering the fact that elicitors may interact with the cells of plants resulting in overexpression and activation of gammatocopherol methyl transferase (γ-TMT), responsible for the synthesis of these molecules.6,7 Because these molecules are essential in the protection of lipids in the cell membrane due to ROS production, after eliciting, it can be assured that αtocopherol, in cooperation with other antioxidants, limits the extent of lipid peroxidation by reducing the lipid peroxyl radicals to its corresponding hydroperoxides, transforming them in an essential step to cell protection.47 Antioxidant capacity showed good correlations (values not shown) with TPC and TF. In agreement with phytochemical content, green lettuce showed the highest antioxidant activity when treated with elicitors on PH day 7, while red lettuce showed the highest antioxidant activity when treated on PH day 15. Polyphenol profiles in all elicitor-treated lettuce showed changes when compared with those of the control. Elicitors are related to the activation of several defense responses in plants due to the interaction of a pathogen (virus, bacteria) or in the presence of a defense signal (phytohormones). In this study, phenolic acid levels increased after elicitor treatment in both green and red lettuce samples. The rise in chlorogenic acid, caffeoyl tartaric acid, and isocholorogenic acid could be related to a possible activation of PAL, the regulating enzyme in the metabolism of phenylpropanoid.48 After the activation of PAL, the metabolic pathway continues until the production of caffeic acid which can couple with either quinic or tartaric acid to produce other polyphenols.49 In the present study, we did not identify caffeic acid. However, caffeolyltartaric acid, a chlorogenic acid dimer and isochlorogenic acid, all of them derived from caffeic acid, showed the highest increase during elicitor treatment, suggesting that PAL activity may be enhanced. Also, caffeic acid derivates, such chlorogenic acid, has been related to antifungal activities as well as their capacity to reduced disease through reinforcement of defense barriers in response of any stressful environment.8 Chicoric acid also

increased in lettuce after elicitation. Even though the synthesis pathway of this molecule is not known, it is suggested that it could be synthesized via the shikimic acid/phenylpropanoid pathway, just like other phenolic acids such as rosmarinic acid or chlorogenic acid.50 AA and HP increased chicoric acid in green lettuce, whereas SA, MJ, and HP increased their content in red lettuce. These findings suggest that elicitors probably activate enzymes mainly associated with the biosynthesis of phenolic acids. Elicitation produced changes in the phytochemical content and antioxidant activity in green and red lettuce. Changes were dependent on the elicitor used as well as the time of elicitor application. Two-way ANOVA (Table 5) shows that some Table 5. Two-Way ANOVA for Phytochemical Content and Preharvest Day of Green and Red Butterhead Lettucea green lettuce

red lettuce

phytochemical

elicitor

day

elicitorday

elicitor

day

elicitorday

TPC TF ASC CAR TAN

** **** **** NS ND

*** **** **** ** ND

NS **** **** ** ND

* NS ** NS ****

** **** **** **** ****

** **** **** **** ****

****(ρ < 0.0001), ***(ρ < 0.0010), **(ρ < 0.100), *(ρ < 0.0500). TPC, total phenolic content; TF, total flavonoids; ASC, ascorbic acid; CAR, carotenoids; TAN, total anthocyanins; NS, not significant.

a

phytochemicals showed elicitor−day of treatment interactions. Mostly quantitative differences were observed: SA and HP produced the strongest effects, while MJ had a minimal impact in the phytochemical content. Nonetheless, antioxidant activity increased in both green and red lettuce. Elicitor application day also affected the plant response. After a global analysis of the effect of all elicitors on the phytochemical content changes, it was established that the best application days for green and red lettuce were PH day 7 and PH day 15, respectively. We suggest that because a higher number of phytochemicals were modified compared with the rest of the treatments, in most of the cases these days showed the highest increases on phytochemical content in comparison with that on the rest of the PH days. Elicitors affected mainly the content of phenolic acids, indicating that they may interact with the main enzymes associated with TPC synthesis.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +52-656-688-1800 ext. 1464. E-mail: [email protected]. ORCID

Joaquín Rodrigo-García: 0000-0002-0997-5811 Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS We are grateful to InnoBio Hidroponia Inc. for providing the greenhouse and hydroponic facilities for experimentation. ABBREVIATIONS USED ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6- sulfonic acid; AA, arachidonic acid; ASC, ascorbic acid; CAR, carotenoids; DPPH, 2,2-diphenyl-1-picryl-hydrazylhydrate; FRAP, ferric 5252


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Journal of Agricultural and Food Chemistry reduction antioxidant power; γ-TMT, gamma-tocopherol methyltransferase; HP, Harpin protein; L-GalLDH, L-galactono-1,4-lactone dehydrogenase; MJ, methyl jasmonate; PAL, phenylalanine ammonia lyase; TAN, total anthocyanins; TF, total flavonoids; TPC, phenolic compounds; PH, preharvest; ROS, reactive oxygen species; SA, salicylic acid; TEAC, Trolox equivalent antioxidant capacity; TPTZ, 2,4,6-tripyridyl-striazine; UPGT, UDP-glucose, flavonoid-O-transferase

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