Enhancement of zeaxanthin in two-steps by environmental stress induction in rocket and spinach

    Enhancement of zeaxanthin in two-steps by environmental stress induction in rocket and spinach Raquel Esteban, Eva Fleta-Soriano, Javier Buezo, Fatima Miguez, Jos´e M. Becerril, Jos´e I. Garc´ıa-Plazaola PII: DOI: Reference:

S0963-9969(14)00359-7 doi: 10.1016/j.foodres.2014.05.044 FRIN 5286

To appear in:

Food Research International

Received date: Revised date: Accepted date:

29 January 2014 27 May 2014 29 May 2014

Please cite this article as: Esteban, R., Fleta-Soriano, E., Buezo, J., Miguez, F., Becerril, J.M. & Garc´ıa-Plazaola, J.I., Enhancement of zeaxanthin in two-steps by environmental stress induction in rocket and spinach, Food Research International (2014), doi: 10.1016/j.foodres.2014.05.044

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ACCEPTED MANUSCRIPT Enhancement of zeaxanthin in two-steps by environmental stress induction in rocket and spinach

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Running title: Increased levels of zeaxanthin in two-steps.

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Raquel Estebanab*, Eva Fleta-Sorianoa, Javier Buezoa, Fatima Migueza, José M. Becerrila &

Universidad del País Vasco (UPV/EHU),c/Sarriena s/n; apdo. 644 48080 Bilbao,Spain b

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a

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José I. García-Plazaolaa

Institute of Agrobiotechnology, IdAB-CSIC-UPNA-Government of Navarre, E-31192

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Pamplona, Spain

Author for correspondence

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phone: (0034) 948168000; Fax: (0034) 948 232191; e-mail: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT Humans are continuously exposed to oxidative damage risk and in order to counteract it, the

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consumption of antioxidants and carotenoids of plant origin is recommended. Numerous

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studies show the need to include substantial amounts of the carotenoid zeaxanthin (Z) in the

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diet, because its deficiency provokes the development of macular degeneration, which leads to irreversible loss of vision. However, Z is a less-abundant carotenoid in plants, because most of its pool is rapidly converted to the carotenoid violaxanthin (V) via antheraxanthin (A), due

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to its involvement in the operation of the xanthophyll (V+A+Z) cycle. The aim of this paper,

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therefore, was to develop a protocol to enhance the Z content in spinach and rocket through two strategies: first, by applying stress (chilling, high light and drought) in order to enhance

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the total pool of V+A+Z and, secondly, to apply post harvest treatments before consumption

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in order to enhance Z formation. The results showed that high light was the most beneficial stress, increasing the fresh weight production in rocket and showing the highest accumulation

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of V+A+Z and carotenoids in both species. An enhancement of -tocopherol in rocket was, as well, accomplished by the environmental stress induction. Besides, by the second strategy

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(post harvest treatments before consuming, such as boiling and vinegar dressing), both species showed Z enhancement. By combining both approaches in two-steps, the Z content can be enhanced up to 15-fold in spinach and 28-fold in rocket, increasing as a result, the nutritional value of food. Key-words: health, nutritional quality, rocket, spinach, tocopherols, xanthophylls Abbreviations: A antheraxanthin; -toc -tocopherol; Ch chilling; C control; DW Dry weight; Dt drought; HL high light; L lutein; FW fresh weight; Fo basal fluorescence; Fm maximum fluorescence; Fv/Fm maximum quantum yield of PSII; ML medium light; PPF photosynthetic photon flux; PSII photon system II; RWC relative water content; TW turgid

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ACCEPTED MANUSCRIPT weight; V violaxanthin; V+A+Z

the xanthophyll cycle involving the carotenoids

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violaxanthin, antheraxanthin and zeaxanthin; VDE violaxanthin deepoxidase; Z zeaxanthin.

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ACCEPTED MANUSCRIPT 1. INTRODUCTION There is growing evidence that specific dietary components, the so-called

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“nutraceuticals”, often found in plant-based food, as carotenoids and tocopherols (Namitha, &

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Negi, 2010), may prevent diseases and disorders (Davies, 2007). Indeed, α-tocopherol

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(Vitamin E; DellaPenna, & Pogson, 2006), found in green parts of plants, has an important role in protecting membrane lipids from oxidative damage and it has been suggested that is effective avoiding obesity (Lira et al., 2011). Among carotenoids, lycopene and -carotene

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have been shown to be inversely related to the risk of cardiovascular diseases and certain

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cancers (Sharoni et al., 2012), while lutein (L) and zeaxanthin (Z) are believed to function as protective antioxidants both in plants and in human nutrition (Demmig-Adams, & Adams,

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2002), preventing eye diseases (Rao, & Rao, 2007), coronary heart diseases and stroke

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(Chorng, Wong, Kreis, Simpson, & Guymer, 2007). In fact, it has been demonstrated that macular pigments (L and Z) limit retinal oxidative damage by absorbing blue light and

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quenching reactive oxygen intermediates (Beatty, Koh, Henson, & Boulton, 2000). Zeaxanthin cannot be replaced by any other carotenoid in the retina and its levels in blood

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plasma have been negatively correlated to the development of age related macular degeneration (Gale, Hakk, Philips, & Martyn, 2003), which is the cause of blindness in the elderly (Bressler, 2004). The underlying mechanisms are not well understood, but the significance of carotenoids in human diet is considered to be attributable of their antioxidant properties (Linnewiel et al., 2009; Asensi-Fabado, & Munné-Bosch, 2010). An improvement in Z content would thus be a desirable trait to incorporate into crops in order to improve their nutrient intake. Progress in this direction has been made by biofortification (Bouis, & Welch, 2010), either through conventional plant breeding (Stommel, 2001) or through biotechnology, engineering carotenoid biosynthesis (Sandmann, Romer, & Fraser, 2006). However, as pointed out by Mou (2009), breeding efforts for the

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ACCEPTED MANUSCRIPT nutrition and commercialization of transgenic crops would depend on progress in transgene expression, public acceptance, economic and marketing challenges, intellectual property

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issues and risk assessment. Alternatively, the biofortification of crops, understood as a broad

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term, including agronomic practices, and post harvest modifications, may be effective in

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increasing the micronutrient concentration of foods crops to avoid dietary deficiencies. This strategy of tackling micronutrient malnutrition is considered to be among the best investments, as this will generate a high return in the form of socio-economic benefits (The

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Copenhagen Consesus, 2004).

Several factors affect the content of these compounds in food: variety, genotype,

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environmental growing conditions and/or post-harvest treatments (Maiani et al., 2009). In the case of L and Z, plants typically have high levels of L, however, Z is usually found in low

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amounts, due to the dynamic interconversion of Z to the carotenoid V (violaxanthin) via antheraxanthina (A) within minutes to hours of darkening by the enzyme zeaxanthin

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epoxidase. When plants are illuminated, V is again converted to Z by the enzyme violaxanthin deepoxidase (VDE), completing the xanthophyll (V+A+Z) cycle (Yamamoto, Nakayama, &

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Chichestser, 1962).The balance between V and Z is then controlled by periods of darkness and light. Apart from this light-induced inter-conversions on time scale of hours to days, this cycle can also be regulated over a longer time scale (days to weeks) in response to sustained environmental changes, such as low temperature, water deprivation (Demmig-Adams, & Adams, 2006) or increasing light (Niinemets, Kollist, García-Plazaola, Hernández, & Becerril, 2003). Thus, inducing moderate environmental stress during the crop growing period may be a suitable approach to increase some phytochemicals and enhance the nutritional quality of crops, without having an important effect on crop yield. Another main point to consider in the change of the green vegetables profile of phytochemicals, is what occurs at the different phases after the growing period, scilicet, in the

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ACCEPTED MANUSCRIPT postharvest handing, in the vegetables storage and with the consumers´ habits before consumption. Actually, the V+A+Z cycle is active in green vegetables and as a consequence

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of dark storage prior to consumption, the Z content dramatically decreases due to the

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operation of the cycle upon darkness in a short-term (Yamamoto, 1979). In addition, at the

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consumption time, vegetables are traditionally consumed as salad or cooked (generally boiling), which probably may affect phytochemicals stability and carotenoid isomerization (Yang, He, & Zhao, 2013). Thus, studying how to improve the Z content by post-harvest

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approach before cnsumint will complement the strategies for growing vegetables. Taking into account that guaranteeing the presence and accumulation of these

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phytochemicals may help human nutrition and health, the goals of the present work were: (i) to activate the long-term up-regulation of V+A+Z cycle components (enhancing the total pool

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of V+A+Z cycle pigments) in green vegetables during the growth period in the presence of mild (stress that does not induce senescence) environmental stress (chilling, high light and

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drought), (ii) to activate the conversion of V+A+Z cycle (from V to Z) and (iii) to stabilize the Z content after the usual post harvest treatments for consumption. The experiments were

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carried out with spinach (Spinacea oleraceae L.), a leafy vegetable traditionally considered to have high nutritional quality and large amounts of antioxidants (Bergquist, Gertsonn, & Osllon, 2006), and rocket (Eruca sativa Mill.), which has attracted considerable interest recently as a culinary vegetable for salads because of its strong flavour and content of putative health-promoting compounds (Pasini, Verardo, Cerretani, Caboni, & D´Antuono, 2011). Both vegetables are widely accepted by consumers because they are easy to prepare for eating. This paper will attempt to set out a number of simple recommendations in order to achieve the aforesaid goals by manipulating the environmental conditions during the growth period and/or during post-harvest treatments.

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ACCEPTED MANUSCRIPT 2. MATERIALS AND METHODS 2.1. Pre-harvest experimental design: growth conditions and treatments

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Seeds of spinach (Spinacia oleracea L. var. Viroflay, Chenopodioideae) and rocket (Eruca

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sativa Mill. var. Golden line, Brassicaceae) were germinated at 26ºC in darkness for 96 h, in

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a mixture of peat and vermiculite (2:1, v:v) and moistened with deionized water, prior to placement in a growth chamber under controlled conditions (25/18ºC day/night, relative humidity of 60/70% day/night and a photosynthetic photon flux, PPF, of 300 µmol m-2 s-1)

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Ten days after sowing, two seedlings of each species were transplanted to 1.5 l pots (two

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plants per pot and 6 pots per species and per treatment), filled with a mixture of peat and vermiculite (3:1, v: v). Prior to treatments, plants were allowed to grow for 14 days under the same controlled conditions as above. Irradiation from fluorescent lamps provided a PPF of

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300 µmol m-2 s-1 during a 14 h photoperiod with a ramp for illumination at dawn and dark sunset of 30 minutes. These conditions were considered as medium light (ML) for light and

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drought treatments (see details in section 2.1.1 and 2.1.2) and control (C) for chilling treatment (see details in section 2.1.3). The weekly watering regime consisted of watering the

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plants twice with 150-200 ml of distilled water and once with a 150-200 ml of a Hoagland nutritive solution. After 15 days (day 24 of the experiment) under the conditions described above the following treatments were applied: 2.1.1. High Light (HL) treatment: On the day 24 of the experiment, one set of plants of both species (6 pots per species) was exposed to a HL regime (600 µmoles m-2 s-1), the other set continued with the ML regime (300 µmoles m-2 s-1) and we finally harvested the plants on day 42. 2.1.2. Drought (Dt) treatment: We tested the effects of drought stress and subsequent recovery on both species. To this end, in one set of ML plants (3 pots per species), we reduced watering with Hoagland solution to only once a week, from day 36 to day 40 of the

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ACCEPTED MANUSCRIPT experiment, and from day 40 to the end, plants were watered as regular regime until harvest. For comparison, one set of ML plants (3 pots per species) were watered with the regular

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regime indicated above. Finally, we harvested all the plants on day 42.

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2.1.3. Chilling (Ch) treatment: On the day 24 of the experiment, one set of plants of both

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species (6 pots per species) was exposed to chilling stress. First at 12/8ºC day/night, relative humidity of 91/100% day/ night and a PPF of 300 µmol m-2 s-1 until day 35 of the experiment, and later the conditions were hardened to 9/4ºC day/night until harvest on day 38 of the

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experiment. The other set (6 pots per species) continued until harvest with control conditions

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(25/18ºC day/night).

Samples were collected, during the course of the experiment, every 3-4 days throughout the growth period and finally at harvest. For this purpose, 3-5 leaves from each species and

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treatments were randomly selected and analysed for fluorescence. Then, leaf discs (3 mm diameter) were collected from leaves, where fluorescence was measured, frozen in liquid

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nitrogen and stored at -80°C until the carotenoid, chlorophyll and tocopherol analyses were conducted. Sampling was performed after a period of dark incubation (12h) in order to reduce

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the variability and to provide comparable conditions (Tausz, Wonisch, Grill, Morales, & Jiménez, 2003).

2.2. Post harvest experimental design On day 42, ML spinach and rocket shoots were exposed to post harvest treatment in accordance with the way each species is normally consumed (boiling for spinach and vinegar dressing for rocket). Prior to treatments, spinach and rocket plants were dark adapted for 10 minutes and exposed to direct sunlight for 30 min (PPF of 1001 ± 63.3 µmol photons m-2 s-1 for spinach and 760.6 ± 79.4 µmol photons m-2 s-1 for rocket). For the boiling treatment, discs of 5 mm diameter, from leaves exposed to light, were introduced in eppendorfs with water and boiled during 20 minutes. For the dressing, discs of 5 mm diameter, from leaves exposed

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ACCEPTED MANUSCRIPT to light, were introduced in eppendorfs with 500 µl of pure white wine vinegar of 6º acidity during 20 minutes. Both treatments, boiling and vinegar dressing, were followed by

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incubation in the dark for 2h. Following each of the treatments (darkness, illumination,

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vinegar o boiling and darkness) samples were frozen in liquid nitrogen and stored at -80°C

2.3. Plant biomass and relative water content

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until the carotenoid, chlorophyll and tocopherol analyses were conducted.

At harvest, three plants of each species and treatment were randomly selected to determine the

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fresh weight (FW) of the aerial part. The dry weight (DW) in leaves was determined after

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drying the plant material at 80ºC for 2 days. In order to determine the relative water content (RWC), five discs of each species and treatment were taken and the FW was determined, followed by flotation on water for up to 24h. The turgid weight (TW) was then recorded and

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the leaf tissue was subsequently oven dried for 48h at 80ºC. RWC was calculated according to the formula: RWC = (FW-DW)/ (TW-DW) x 100; where FW is the fresh weight, DW is the

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dry weight and TW the turgid weight. 2.4. Fluorescence analysis

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The maximum quantum yield of photosystem II (PSII) reflects the potential quantum efficiency of PSII and is used as a sensitive indicator of plant photosynthetic performance, considering 0.83 as the optimal value (Maxwell, & Johnson, 2000). Values lower than this will be an indicator of stress exposure. This parameter was determined with a portable chlorophyll fluorimeter (Fluorpen FP100, PSI, Brno, Czech Republic) on five dark adapted leaves (overnight) to allow the complete relaxation or oxidation of reaction centres in order to determine basal fluorescence (Fo). Then, a saturation pulse of 3000 µmol photons m-2 s-1 was applied to determine the maximal fluorescence (Fm). The maximal photochemical efficiency of PSII was estimated by the ratio Fv/Fm = (Fm-Fo)/Fm according to Genty, Briantais, & Baker (1989).

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ACCEPTED MANUSCRIPT 2.5. Analytical methods Extraction was made by an electric tissue homogeniser Tearor 985370 (BioSpec,

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Bartlesville, USA) with 1 ml of acetone (100%) with 0.5 g/l of CaCO3, in order to avoid

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acid traces that might change pigment composition. Samples were centrifuged at 16100 g for

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20 minutes at 4ºC and syringe-filtered through a 0.22 μm PTFE filter (Whatman, Maidstone, UK). Extracts were injected (15 l) on a reversed-phase C18 column (Waters Spherisorb ODS1, 4.6 × 250 mm, Milford, MA, USA) HPLC system following the method of García-

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Plazaola, & Becerril (1999) with some modifications (García-Plazaola, & Becerril, 2001).

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The 717 plus autosampler was equipped with a thermostat, which maintains temperature constant at 4°C avoiding pigment degradation or alteration. Photosynthetic pigments were

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measured with a PDA detector (Waters model 996) in the range 250-700 nm. Peaks were

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detected and integrated at 445 nm for carotenoid and chlorophyll content. Pigments were identified by comparing spectral characteristics obtained by the PDA detector and retention

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times with those of standard materials (DHI, Hørsholm, Denmark). Retention times and conversion factors for pigments were the same as those described by García-Plazaola, &

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Becerril (1999, 2001). For tocopherols, detection was carried out with a fluorescence detector (Waters model 474) set to λexc= 295 nm and λem= 340 nm and calibrated with tocopherol standards (Calbiochem, San Diego, CA). Total carotenoids were calculated by the sum of neoxanthin, V, A, L, Z and -carotene. As the discussion and manuscript´s aims focus on the nutritional quality of leafy green vegetables, authors considered that results should be expressed and discussed on a fresh basis. 2.6. Statistical analysis In order to test differences, one-way Analyses of Variance (ANOVAs) were performed, considering time as a fixed factor. Besides, we used ANOVAs, considering

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ACCEPTED MANUSCRIPT treatment as a fixed factor, to test differences between treatments. All data were tested for normality and homogeneity of variances and log-transformed, if necessary. Student–

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Newman–Keul (SNK) tests were used to discriminate between different treatments. In the

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case of V+A+Z of spinach under chilling and total carotenoids in rocket under HL, one piece

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of data was replaced by the mean of the group and 1 df subtracted from Residual (Winer, Brown, & Michels, 1991), in order to standardise variances. All the effects discussed in this

statistical package.

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3. RESULTS AND DISCUSSION

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paper were significant at =0.05. All statistical analyses were performed using the SPSS 19.0

3.1. Step one: mild environmental stress during the growth period.

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The application of the HL regime had a significant effect on the growth of the

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vegetables studied here, with no effect on spinach (Fig. 1A), while increasing the DW of rocket (Fig. 1B). In contrast, chilling stress and drought treatment caused a significant

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decrease in the FW of both species (Fig. 1). The reduction of FW under drought was due to a diminution of RWC up to 40% of the plants (Fig. 2C, D). After rewatering on day 40, both

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species recovered control values of RWC within three days, however, plant growth remained affected. Manipulating environmental factors with the aim of enhancing antioxidants, such as carotenoids and tocopherols, may entail a cost in terms of the drop in production, as has been shown in this study with drought and chilling treatments. This was also the case for lettuce after the exposure to UV radiation (Tsormpatsidis, Henbest, Battey, & Handley, 2010), which caused an inhibition of plant biomass, probably due to the higher metabolic cost of phenolic compounds, which play an important role in human health. On the other hand, the Fv/Fm parameter, which reflects the physiological status of the plant, was not affected by the episodic drought and rewatering (Fig 2A, B), being close to the optimal value (0.83; Maxwell, & Johnson, 2000). Photochemical efficiency was only affected by chilling stress in both

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ACCEPTED MANUSCRIPT species (Fig. 2E, F). Particularly, rocket seems to be a more sensitive crop than spinach, as a reduction of photochemical efficiency was observable from the beginning of the chilling

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treatment (Fig 2F). Low Fv/Fm values are indicative of the bad physiological condition of

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plants (Maxwell, & Johnson, 2000) exposed to chilling stress. Indeed, under these stress

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conditions, the main physiological response of plants is the downregulation of photosynthesis associated with the absence of the relaxation of the xanthophyll cycle being maintained the photochemical system in its dissipative state (Z; Demmig-Adams, & Adams, 2006). This was

eight days under the stress (Fig 3C) This slowest

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pigments was achieved after being

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observed in spinach under the chilling treatment, in which an enhancement of V+A+Z cycle

relaxation of the photochemical efficiency upon darkening is associated with photoinhibion in

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many plants (Demmig-Adams, & Adams, 2006).

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However, this FW decrease observed in some of the treatments applied here may be compensated with better nutritive quality. Such is the case for drought stress that triggers the

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accumulation of antioxidant compounds (carotenoids and tocopherols) in leaves as part of their photoprotection mechanisms. In fact, under our experimental conditions, a moderate

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drought stress was very effective in increasing the V+A+Z (Fig. 3B), the total carotenoids (Fig. 4B) and -tocopherol (Fig. 5) content in rocket, but not in spinach (Fig. 3A and 4A). Besides, the chilling treatment only increased the V+A+Z pool in spinach (Fig. 3C) and it had no effect on the total pools of carotenoids of both species (Fig 4C and D). The results showed that HL treatment was the most effective treatment to increase the total pool of V+A+Z pigments (Fig. 3A, B) and carotenoids (Fig. 4A, B) in both species. These enhancements were mainly due to the new synthesis of V (data not shown), which upon illumination may be interconverted to Z, due to the operation of the xanthophyll cycle. However, the behavior of this trend (enhancement of V+A+Z) was different for each of the species. Indeed, after HL treatment, rocket reached higher values of V+A+Z than spinach (Fig. 3A, B). It has been

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ACCEPTED MANUSCRIPT pointed out that high light stress alters the synthesis of -xanthophylls such as the synthesis of Z from -carotene, being the amount of Z photo-inducible (Demmig-Adams, & Adams,

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1992). As shown in figure 3 (A and B), after 4 days under the stress, the level of V+A+Z

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increased and the highest value was recorded after 11 days exposure to high light for spinach

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and 14 days for rocket, and later both showed a downward trend. This indicates that the optimum concentration of compounds in response to environmental factors for each species and the days of growth under the treatment should be taken in account, in order to prevent the

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destruction of pigments and photodamage, with the final goal, therefore, of optimizing the

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harvest of leafy tissues. Other approaches such as UV-B light source (Li, & Kubota, 2009) and mycorrhizal symbiosis (Baslam, & Goicoechea, 2012), also increased the levels of total

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carotenoids. In the latter, these enhancements were more pronounced when plants were grown

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under a restricted water supply (Baslam, & Goicoechea, 2012). Regarding α-tocopherol (vitamin E), no significant differences were found in spinach

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(data not shown). However, in rocket, an enhancement in α-tocopherol (Fig. 5) was also achieved with both HL and drought treatments. Lizarazo, Fernández-Marín, Becerril, &

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García-Plazaola (2010) also described an accumulation of tocopherols induced by high light in spinach. Plant tissues generally contain low levels of vitamin E compared with seeds and particularly oil seeds (Asensi-Fabado, & Munné-Bosch, 2010). So, this fact gives these treatments an additional nutritional value. The harvest of green leaves in sunlight usually contains increased levels of Z because of the photoconversion from V (Demmig-Adams, & Adams, 1992). However, in dark or low light, Z is usually reconverted to V in the short-term. Thus, if a high Z content is a desirable trait in crops, its natural dynamic conversion to V in the dark, during the period from harvest and storage to consumption, greatly limits the benefits of an enhanced Z pool in the tissues.

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ACCEPTED MANUSCRIPT However, we were able to overcome this limitation and take advantage of the more stable long-term modulation of the violaxanthin cycle, such as the one shown in our results during

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the acclimation of both species to moderate environmental stresses. The optimum

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concentration for each species depends on the duration of the exposure to stress factors.

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3.2. Step two: post-harvest treatments

To maximize the success of this first step (long-term modulation of V+A+Z cycle pigments),

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we were able to control the short-term inter-conversion from V to Z of the V+A+Z cycle sensu stricto with post-harvest treatments before consuming. This control should avoid the

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dark conversion of Z to V, stimulate the conversion of V to Z and finally stabilize the Z pool in plant tissues. In this sense, figure 6 corroborates that the enhancement of total V+A+Z

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cycle pigments achieved during growth conditions can be transformed straight into Z, due to

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post harvest modification according to the way each species is normally consumed and can be performed easily by consumers: (i) illumination followed by boiling for spinach and (ii)

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illumination followed by vinegar dressing for rocket. A direct and short exposure of green leaves to full sunlight (30 minutes) enhanced 4-fold the total Z content by converting V to Z,

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providing the leaves with extra nutritional value. This Z enhancement was accompanied by a reduction in V content during illumination and an increase in A, indicating an effective interconversion of the xanthophylls by de-epoxidation. This was due, therefore, to the operation of the V+A+Z cycle sensu stricto. Furthermore, this newly formed Z can be stabilized by subsequent cooking or dressing (depending on the species). The temperature, during boiling, had a differential effect on the xanthophyll contents in spinach leaves (Fig. 6). The Z concentration was very stable after 20 min of boiling, but V and A were very sensitive and, finally, a complete degradation of V and A occurred due probably to the cooking of vegetables, which promotes to isomerization of carotenoids (Yang et al., 2013). However, carotenoids are relatively stable compared to other antioxidants, as ascorbic acid (Bergquist et

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ACCEPTED MANUSCRIPT al. 2006). In the case of vinegar dressing on rocket, after illumination, the surface acidification of rocket leaves stabilised the xantophyll contents (Z, A, and V). This could be

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due to the activation of the enzyme VDE (deepoxidation of V to Z), which becomes active

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when pH decreased (Yamamoto, 1975) by the vinegar dressing. Both culinary treatments

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(boiling and acidification) prevented the activation of the reverse reaction of the V+A+Z cycle, and thus, after 2 h of dark incubation, the Z content not only was stabilized (Fig. 6), but significantly increased with respect to the previous treatments in both species. However, these

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post harvest modification should be consider carefully because phytochemicals stability is time of processing

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affected by many variables as pH, oxygen, temperature and the

(Harbourne, Marete, Jacquier, O'Riordan, 2013) ) and then influence the nutritional value (Rao, & Rao, 2007) Other studies support the formation of Z in spinach by post harvest

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modifications, using acetic/acid acetate as a buffer (Clausen, Huang, Emek, Sjöholm, & Akerlunk, 2010). Overall, combining both pre- and post- harvest treatments as two steps of a

rocket.

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process of biofortification, the Z content may increase up to 15-fold in spinach and 28-fold in

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4. CONCLUSION

In summary, for the enhancement of phytochemicals, high light was the most beneficial stress, among the treatments applied here: (i) increasing the FW production in rocket; (ii) maintaining plant photosynthetic performance and showing (iii) the highest accumulation of V+A+Z

and carotenoids in both species and -tocopherol in rocket.

Considering a daily requirement of Z in the range of 0.2-0.5 mg/day (Rapp, Maple, & Choik, 2000), the consumption of 300 g of spinach each day (Clausén et al., 2010) is required for reach this quantities. However, following the pre-post-harvest processes applied here, the daily intake of Z can be accomplished with just 20 g of spinach leaves. Modern agriculture

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ACCEPTED MANUSCRIPT has been driven mainly by producing higher yields at lower cost, with little emphasis on the nutritional quality (Sands, Morris, Dratz, & Pilgeram, 2009). By elevating optimal human

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nutrition to a central goal of plant breeding and production of plants-based foods, it has been

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advocated that a new paradigm for agriculture linking farming closely to human health is

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needed (Graham et al., 2007). Our results demonstrate clearly that HL treatment together with household habits could be used strategically to enhance nutritional value. Indeed, rocket seems to be more sensitive than spinach to these changes. However, it should be remembered

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that bioavailability, defined as the fraction of an ingested nutrient that becomes available, is influenced by many dietary and physiological factors (Castenmiller, & West, 1998).

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Nevertheless, the two-step process proposed in this paper with the aim of enriching crops may

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be an excellent approach to improving food quality.

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5. ACKNOWLEDGEMENTS

R. E. received a doctor specialization grant from the UPV/EHU and a JAE-Doctor

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grant from the CSIC. This research was supported by research BFU 2010-15021 from the Ministry of Education and Science of Spain and research project UPV/EHU-GV IT-299-07.

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(A) and rocket (B) subjected to the different treatments (C, HL, Dt, Ch). Values are the mean

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statistically significant differences at =0.05 after SNK.

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Fig. 2 Upper panels show the photochemical efficiency (Fv/Fm) in spinach (A) and rocket

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(B). Closed circles, open circles and grey circles denote ML, HL and Dt treatment respectively. Inset panels (C, D) show changes in the relative water content (RWC) during the

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experiment. Lower panels indicate Fv/Fm in spinach (E) and rocket (F) under chilling. Closed circles and opened circles denote control and Ch treatment respectively. Grey squares indicate

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Fig. 3 Upper panels show xanthophyll cycle pigments (V+A+Z) changes (g/gFW) in spinach (A) and rocket (B) under medium light treatment (ML; closed circles), high light

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(HL; opened circles) and drought (Dt; grey circles). Lower panels indicate V+A+Z pigments changes (g/gFW) in spinach (C) and rocket (D) under control conditions (C; closed circles), and chilling treatment (Ch; opened circles). White arrows indicate the start of the HL and chilling treatments and grey arrows the beginning of the drought treatment. Grey squares indicate the period when the temperature was decreased to 9/4ºC. Symbols represent the means of 3-5 replicates ± S.E. Errors are shown when larger than symbols. An asterisk indicates significant differences at =0.05 between treatments and different letters denote statistically significant differences at =0.05 among time after the SNK test. In order to standardise the variances, one piece of data was replaced by the mean of the group in Spinach chilling and 1df subtracted from Residual (Winer et al., 1991)

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(C) and rocket (D) acclimated to control conditions (C; closed circles), and chilling (Ch;

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(ML; closed circles), high light (HL; opened circles) and drought (Dt; grey circles).White and grey arrows indicate respectively the start of the HL and the drought treatment. Symbols represent the means of 3 replicates ± S.E. Errors are shown when larger than symbols. An

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asterisk indicates significant differences at =0.05 between treatmentsand different letters denote statistically significant differences at =0.05 among time after the SNK test. Fig. 6 Zeaxanthin (bars), violaxanthin (white circles) and antheraxanthin (grey circles) (g/gFW) of spinach (A) and rocket (B) subjected to post-harvest modifications. Values are mean of 5 ±S.E. Different letters denote statistically significant differences at =0.05 after the SNK test.

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A two-step process to enhance carotenoids and tocopherols content in crops is proposed.

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Rocket seems to be a more sensitive crop for manipulating its nutritional value

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Combining preharvest and postharvest approach, Z content can be enhanced.

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