Abstract This study was conducted with the objective of testing the hypothesis that tomato fruits from organic farming accumulate more nutritional compounds, such as phenolics and vitamin C as a consequence of the stressing conditions associated with farming system. Growth was reduced in fruits from organic farming while titratable acidity, the soluble solids content and the concentrations in vitamin C were respectively +29%, +57% and +55% higher at the stage of commercial maturity. At that time, the total phenolic content was +139% higher than in the fruits from conventional farming which seems consistent with the more than two times higher activity of phenylalanine ammonia lyase (PAL) we observed throughout fruit development in fruits from organic farming. Cell membrane lipid peroxidation (LPO) degree was 60% higher in organic tomatoes. SOD activity was also dramatically higher in the fruits from organic farming. Taken together, our observations suggest that tomato fruits from organic farming experienced stressing conditions that resulted in oxidative stress and the accumulation of higher concentrations of soluble solids as sugars and other compounds contributing to fruit nutritional quality such as vitamin C and phenolic compounds.

Citation: Oliveira AB, Moura CFH, Gomes-Filho E, Marco CA, Urban L, Miranda MRA (2013) The Impact of Organic Farming on Quality of Tomatoes Is Associated to Increased Oxidative Stress during Fruit Development. PLoS ONE 8(2): e56354. https://doi.org/10.1371/journal.pone.0056354 Editor: Hany A. El-Shemy, Cairo University, Egypt Received: August 27, 2012; Accepted: January 14, 2013; Published: February 20, 2013 Copyright: © 2013 Oliveira et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding was provided by BNB-Fundeci, CAPES-REUNI, and CNPq/INCT – Frutos Tropicais. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction The consumption of fruit and vegetables has been associated with lower risk of chronic human health problems like cardiovascular diseases, cancer, hypertension and diabetes type two due to their high contents in dietary bioactive compounds, the so-called phytochemicals, endowed with protective properties [1]; [2]; [3]; [4]; [5]. Until recently, the health benefits of fruits and vegetables have been attributed to the antioxidant properties of the phytochemicals they provide. However, nowadays, the routine consumption of antioxidant supplements has become highly controversial as studies have demonstrated that they can actually be harmful [6]. Besides the antioxidant theory, there are now alternative theories about the way phytochemicals induce protective mechanisms in consumers. For instance, it has been shown that a large range of secondary metabolites in fruit and vegetables as phenolic compounds [7] act as elicitors that activate Nrf2, a transcription factor that binds to the antioxidant response element in the promoter region of genes coding for enzymes involved in protective mechanisms [4]. Supplementation of processed tomato products, containing lycopene, has shown to lower biomarkers of oxidative stress and carcinogenesis in healthy and type II diabetic patients, and prostate cancer patients, respectively. The mechanisms of action involve protection of plasma lipoproteins, lymphocyte DNA and serum proteins against oxidative damage, and anticarcinogenic effects, including reduction of prostate-specific antigen, up-regulation of connexin expression and overall decrease in prostate tumor aggressiveness [5]. As stressed in a recent survey of literature, environmental factors represent a powerful lever to increase the concentrations in phytochemicals [8]. Among all the factors that seem effective in enhancing the concentrations in phytochemicals in fruits and vegetables, stress emerges as especially promising. This makes sense considering that all stresses, either biotic or abiotic, are conducive to oxidative stress in plants [9] and that oxidative signaling controls synthesis and accumulation of secondary metabolites [10]; [11]; [12]. Thereby, it may be hypothesized that cropping systems that allow plants to undergo (moderate) stress such as organic farming result in products with higher concentrations in phytochemicals resulting of low mineral availability and, therefore of the diversion of carbon skeletons from protein synthesis [13]. Indeed, several studies have demonstrated that fruits and vegetables from organic farming generally are endowed with enhanced nutritional properties [14]; [15]; [16]; [17]. A recent comparative study showed that organic tomato juice has a higher phenolic content and hydrophilic antioxidant activity when compared to conventional tomato juice [17]. Organic tomatoes from Felicia, Izabella and Paola varieties had higher vitamin C and carotenoid contents which were more pronounced when expressed on fresh matter than on dry matter [14]. Organic strawberries present higher antioxidant concentrations and have been shown to inhibit the proliferation of human colon (HT29) and breast (MCF-7) cancer cells more effectively than conventional ones [15]. The hypothesis that oxidative stress is involved in enhanced concentrations in phytochemicals of fruits and vegetables from organic farming has rarely been tested to our knowledge [18]. The objective of this paper is to contribute to fulfilling this gap by comparing not only the concentrations in compounds contributing to quality in fruits from organic and conventional farming, but also by measuring indicators of oxidative stress, namely the activities of antioxidant enzymes, superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT), the concentration in ascorbate (AsA) and cell membrane lipid peroxidation (LPO) degree. The study was conducted on tomatoes which are climacteric fruits, representing a relevant source of vitamins C and E and other phytochemicals such as carotenoids and polyphenols [19]. We focused in this study on phenolic compounds and the activity of phenylalanine ammonia-lyase (PAL) because this enzyme controls a rate-determining step of the biosynthetic pathway of phenolic compounds in plants and is well-known to be induced by environmental stress [20]; [21]; [22]; [23].

Materials and Methods Fruit Material Organic and conventional tomato fruits (Solanum lycopersicum cv. Débora) cultivated organically and conventionally were obtained from local producers of the district of Crato-Ceará State at northeastern part of Brazil (7°14′03′′S and 39°24′34′′W). The organic and conventional farms were within 1.5 km from each other, therefore with similar environmental conditions and plants were planted in rows 1 m apart with 0.4 m between plants. The soil was representative of this region of Brazil and classified as humic yellowish latosol with medium phosphate and potassium levels, medium-textured clay and 0–20 cm top layer with pH (H 2 O) 6.2; cation exchange capacity of (cmolc.dm−3) Ca2+2.0; Mg2+0.8; K+0.2; Al3+0.0; P (mg.dm−3) 0.6 and organic matter (g.kg−1) 2.6. As reported by the growers, the organic cultivation system used a compost of animal manure (10 t.ha−1), legume cocktail and sugar cane bagasse incorporated into soil just before sowing; and every 10 days, 0.5% Bordo mix (a mixture of copper (II) sulfate (CuSO 4 ) and slaked lime) was used preventively as a fungicide. In the conventional system, the pesticide FASTAC®100 was applied at 0.01% when needed and inorganic fertilizer was used as recommended by the Brazilian agricultural development department at the following rates: 100 kg.ha−1 for nitrogen, 400 kg.ha−1 for P 2 0 5 and 80 kg.ha−1 for K 2 0. Organic and conventional tomatoes were evaluated at three developmental stages: immature (green colored), physiologically mature (breaker), and at the harvesting stage (red). Fruits were harvested manually from 30 plants for each system, then washed in tap water and carefully selected to insure good uniformity in maturity and size. After cleansing and selection, fruits from each treatment were divided into samples made up of four replicates each consisting of twelve fruits. Fruit pericarp was ground and homogenized using a domestic food processor and then stored at −20°C for further analysis. Fruit Quality Parameters Weight was measured on a semi-analytical scale (TECNAL, São Paulo-Brazil) as whole fruits were individually weighed and results expressed in grams (g). Size measurements were made with a handheld pachymeter (ZAAS Precision, São Paulo-Brazil) and expressed in centimeters (cm). Soluble solids (SS) content was determined by refractometry as described by AOAC [24] using a digital refractometer (ATAGO N1, Kirkland-USA) with automatic temperature compensation. The results were expressed in °Brix (concentration of sucrose w/w). The pH was measured using an automatic pHmeter (Labmeter PHS-3B, São Paulo-Brazil) as recommended by AOAC (24). Titrable acidity (TA) was evaluated following AOAC (24) by using an automatic titrator (Mettler-Toledo DL12, Columbus-USA). Results were expressed as % of the predominant acid for each species. Antioxidants The total antioxidant activity (TAA) was determined using the ABTS method as described by Re and others [25], which measures the ability of lipophilic and hydrophilic antioxidants to quench a 2,2′-azino-bis 3-ethylbenzthiazoline-6-sulphonic acid (ABTS*+, Sigma) radical cation. Before the colorimetric assay, the samples were subjected to a procedure of extraction in 50% methanol and 70% acetone as described by Larrauri and others [26]. The radical solution was formed using 7 mM ABTS*+ and 140 mM potassium persulfate, incubated and protected from light for 16 h. Once the radical was formed, the reaction was started by adding 30 µL of extract in 3 mL of radical solution. Absorbance was measured (734 nm) after 6 min, and the decrease in absorption was used to calculate the TAA. A calibration curve was prepared and different Trolox (Sigma) concentrations (standard trolox solutions ranging from 100 to 2000 µM) were also evaluated against the radical. Antioxidant activity was expressed as Trolox equivalent antioxidant capacity (TEAC), µmol Trolox. g −1 FW (fresh weight). The total phenolic content was measured by a colorimetric assay using Folin–Ciocalteu reagent (Sigma) as described by Obanda and Owuor [27]. Before the colorimetric assay, the samples were subjected to a procedure of extraction in 50% methanol and 70% acetone as described by Larrauri and others [26]. Absorbance was measured at 700 nm, gallic acid (Acros Organics) was used as the standard and results were expressed as galic acid equivalents (GAE) mg.kg−1 FW. Anthocyanins and yellow flavonoids were extracted and determined as described by Francis [28]. The absorbance of the filtrate was measured at 535 nm and at 374 nm for total anthocyanin and for yellow flavonoid content using absorption coefficients of 98.2 and 76.6, respectively. The results were expressed as mg. kg−1 FW. Total vitamin C was determined by titration with Tillman solution (0.02% 2,6 dichloro-indophenol, DFI from Sigma) described by Strohecker and Henning [29]. The results were expressed as mg. kg−1 FW. Enzymes Samples of two grams of pulp were homogenized in an ice-cold extraction buffer (100 mM potassium-phosphate buffer pH 7.0+0.1 mM EDTA). The homogenate was filtered through a muslin cloth and centrifuged at 3300×g for 40 min. The supernatant fraction was used as a crude extract for antioxidant enzyme activity assays. All the procedures were performed at 4°C. The total protein content was determined according to Bradford [30]. Catalase (CAT, EC 1.11.1.6) activity was measured according to Beers and Sizer [31]. The decrease in H 2 O 2 (Merck) was monitored through absorbance at 240 nm and quantified by its molar extinction coefficient (36 M−1 cm−1). The results were expressed as µmol H 2 O 2 . min−1. mg−1 P (protein). Ascorbate peroxidase (APX, EC 1.11.1.1) activity was assayed according to Nakano and Asada [32]. The reaction was started by adding ascorbic acid and ascorbate oxidation was measured through absorbance at 290 nm. Enzyme activity was measured using the molar extinction coefficient for ascorbate (2.8 mM. cm−1) and the results expressed in µmol H 2 O 2 . min−1. mg−1 P, taking into account that 1 mol of ascorbate is required for the reduction of 1 mol H 2 O 2 . Superoxide dismutase (SOD, EC 1.15.1.1) activity was determined by spectrophotometry, based on the inhibition of the photochemical reduction of nitroblue tetrazolium chloride (NBT, Sigma) [33]. The absorbance was measured at 560 nm and one unit of SOD activity (UA) was defined as the amount of enzyme required to cause a 50% reduction in the NBT photo-reduction rate. Thus, results were expressed as UA. mg−1 P. Phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) activity was assayed with samples extracted by a modified version of the method developed by Mori and others [34]. Pulp samples (1 g) were homogenized for 3 min at 4°C with (2 mL) 0.1 M Tris-HCl buffer solution (pH 8.0), 1 mM EDTA and 0.5 g PVP, and then centrifuged at 5000×g for 20 min. The supernatant was the extract used to determine the PAL activity which was assayed using an assay modified from that of El-Shora [21]. The reaction mixture contained 100 mM Tris-HCl buffer (pH 8.4), 40 mM L-phenylalanine and 100 µL of enzyme to a total volume of 880 µL. The reaction was stopped by adding 6 M HCl and the A 290 of the clear solution was measured. The PAL activity was expressed as µmol of trans-cinnamic acid. mg −1 P. Lipid Peroxidation Degree The thiobarbituric acid reactive substances (TBARS) assay measures lipid peroxidation degree through determination of hydroperoxides and aldehydes such as malondialdehyde (MDA). MDA reacts with thiobarbituric acid (TBA, 1∶2) to form a fluorescent adduct MDA-(TBA) 2 using a modified version of the method developed by Zhu and others [35]. Pulp samples (0.5 g) were homogenized in 5 mL of 0.1% trichloroacetic acid (TCA) and centrifuged at 3300×g at 4°C for 20 min. The supernatant (750 µL) was collected and added to 3 mL 0.5% TBA in 20% TCA and incubated at 95°C for 30 min. Following incubation, the tubes were immediately cooled in ice bath and centrifuged at 3000×g for 10 min. Absorbance at 532 nm was measured and corrections were made for unspecific turbidity by subtracting the absorbance at 600 nm. TBARS are expressed as MDA equivalents, calculated using an extinction coefficient of 155 mmol.cm−1 and expressed as nmol. g−1 FW. Leaf Relative Chlorophyll Content The relative levels of total chlorophyll were estimated with a portable chlorophyll meter (SPAD-502 Minolta, Osaka, Japan). Measurements were performed on four of the youngest fully expanded leaves on five different plants and results were expressed as SPAD values. Statistical Analysis All results are expressed as means ± standard error (SE). Analysis of variance, followed by multiple comparisons of means was performed using SISVAR version 5.1. Means were compared using Tukey’s test at α = 0.05.

Conclusions Our work clearly demonstrates that tomato fruits from organic farming have indeed a smaller size and mass than fruits from conventional growing systems, but also a substantially better quality in terms of concentrations in soluble solids and phytochemicals such as vitamin C and total phenolic compounds. Until recently, the focus has been mainly on yield rather than on gustative and micronutritional quality of fresh plant products. This might be all right for staple food, but, as far as fruits and vegetables are concerned, it may be argued that gustative and micronutritional quality matter more than energy supply. Our observations suggest that, at least for fruit and vegetable production, growers should not systematically try to reduce stress to maximize yield and fruit size, but should accept a certain level of stress as that imposed by organic farming with the objective of improving certain aspects of product quality. More research is needed in the future to better understand the links between stress and oxidative stress, on one side, and oxidative stress and secondary metabolism in fruits, on the other side. Also the physiological mechanisms behind the positive effect of organic farming on fruit quality will require additional studies to be conducted.

Author Contributions Conceived and designed the experiments: MRAM LU EG-F. Performed the experiments: ABO CFHM CAM. Analyzed the data: ABO MRAM. Contributed reagents/materials/analysis tools: MRAM EG-F. Wrote the paper: ABO MRAM.