NMR-Based Metabolomics Approach To Study the Influence of

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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NMR-Based Metabolomics Approach To Study the Influence of Different Conditions of Water Irrigation and Greenhouse Ventilation on Zucchini Crops Ana Cristina Abreu,† Luis Manuel Aguilera-Sáez,† Araceli Peña,‡ Mar García-Valverde,‡ Patricia Marín,‡ Diego L. Valera,‡ and Ignacio Fernández*,† Department of Chemistry and Physics, Research Centre CIAIMBITAL and ‡Department of Engineering, Research Centre CIAIMBITAL, University of Almería, Ctra. Sacramento, s/n, 04120 Almería, Spain

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S Supporting Information *

ABSTRACT: This study describes the approach of 1H NMR metabolomic profiling for the differentiation of zucchini produced under different conditions of water irrigation (desalinated seawater −0.397 dS/m, 0.52 €/m3 vs groundwater −2.36 dS/m, 0.29 €/m3) and ventilation (surface area of the vent openings/greenhouse area was 15.0% for one sector and 9.8% for the other). Overall, 72 extracts of zucchini (Cucubirta pepo L. cv Victoria) under four different conditions were regularly analyzed during the spring-summer cycle from April to July 2017. We have found that zucchini plants irrigated with desalinated seawater increased the zucchini production yield, presented fruits with higher concentration of glucose, fructose, and vitamin B3, and displayed an increased antioxidant activity. On the contrary, plant groundwater irrigation produced the increment of sucrose level that could rise the sweetness perception of the fruits. Finally, the ventilation variable produced a higher concentration of trigonelline, histidine, and phenylalanine but only on those zucchinis irrigated with groundwater. KEYWORDS: zucchini, NMR spectroscopy, metabolomics, multivariate data analysis

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INTRODUCTION The region of Almería located at the east side of Andalucía (Spain) benefits a warm climate (3000 h yr−1 of sunshine) and an extensive area of protected cropping (31 034 ha),1 of which 86% are under biological control production. This specific region of Spain supplies vegetables to Europe throughout the winter season due to its intensive greenhouse horticulture production. It has been well documented that the greenhouses in Almería have driven the demographic and socio-economic development in the province for decades. Season after season, their production and commercial value have been the central core of the province’s economy. In addition, they provide significant export value and have contributed to the largest share of the international agri-food trade within Andalusia.2 The development of intensive horticulture in Almería in the last years is partly based on few essential factors: good climatic characteristics, high water, and nutrient-use efficiency and mulching sandy soil. The favorable climate, furthermore, allows for much lower energy consumption than for other growing areas. For example, greenhouse production in Almería requires 22-times less energy than in The Netherlands.3 In addition, the continuous incorporation of new technological innovations has resulted in an improvement in the efficiency of this production system with high profit yields.4 Nevertheless, agricultural production in the greenhouses in this region has to deal with two main problems: excessively high temperatures and limited availability of water resources. Controlling the greenhouse climate during summer is a problem of increasing importance in Mediterranean climate areas, specifically in the Spanish southeast. Temperature of plants is a key parameter affecting the quantity and quality of crop production.5 A good management of the climate control equipment © XXXX American Chemical Society

(ventilation, evaporative cooling, and shading) could attenuate crop physiological stress situations, thus having a positive effect on the final yield and quality of the product.6 Natural ventilation is a process that contributes strongly to heat exchange between the conditions inside and the environment outside. As a result, proper design of the greenhouse characteristics affecting ventilation might improve climate control, energy efficiency,2,7 and help to control humidity and gas concentrations for most of the cropping cycles.8 It is therefore an agronomic practice that is energy-friendly, low-cost, and requires little maintenance. A reduced ventilation causes high indoor humidity, favoring attacks by fungal diseases, which traditionally require the application of phytosanitary products. Water scarcity is one of the most serious global challenges of our time and is an increasing problem particularly in arid and semiarid regions. In the context of increasing the supply for agricultural irrigation, desalinated seawater has been recognized as one of the alternative sources of water.9 Despite the large investment in the southeastern Spain to build seawater desalination plants devoted to agricultural irrigation purposes, its use is among the lowest acceptance level among farmers not only due to its price and the need for additional fertilization, but also due to the perception that it would negatively affect the yield and crop quality.9 In fact, irrigation with poor quality water may cause an excessive accumulation of salts in the root zone of soil.10 Received: May 16, 2018 Revised: June 15, 2018 Accepted: July 13, 2018

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DOI: 10.1021/acs.jafc.8b02590 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Scheme of greenhouse distribution with zucchini crops growing under lower (−V) and higher (+V) ventilation, and with groundwater (GW) or desalinated seawater (DW). The surface area of the vent openings/greenhouse area was 15.0% for sector “+V” and 9.8% for sector “−V”. The description of several chemical parameters for both waters are described in Table S1. The greenhouse was located at the UAL-ANECOOP Foundation’s Innovation and Technology Centre (36° 51′ N, 2° 16′ W; 87 m elevation) in the province of Almería, Spain. tension during the process of construction. The roof closing consists of flexible plastic sheets between two wire screens, with the sheeting extending to the sidewalls of the structure. The cover consists of the following two parts: the “raspa”, which is the intersection of the two sides of the roof of the module at its highest part, and the “amagado”, which is the intersection of the bottom sides of the roof between the adjacent modules where rainwater gutters are installed.2 The maximum height of the greenhouse is at the crest and ranges between 3.0 and 4.2 m (4.0 m in our trial greenhouse), which forms the “raspa”. The “amagado” attaches the cover screens to the soil by cables and iron forks that allow for placement of the rainwater drain pipe. The height of the “amagado” was 3.2 m. The greenhouse was permanently divided into two sectors by an interior plastic wall. The ventilation surface, that is, surface area of the vent openings/greenhouse area, was 15.0% for sector (+V) and 9.8% for sector (−V). The roof vents were fitted with insect-proof screens with a thread density of 10 × 20 threads cm−2 (36.0% porosity). The greenhouse was equipped with side vents in all its perimeter and three roof windows. The side vents on sector + V have twice the surface area than sector −V, whereas those located on the roof are identical. The greenhouse was covered with TRIPLAST three-layer coextrusion greenhouse film (PE-EVA-PE) of 0.2 mm thickness (PlastimerMorera and Vallejo Industrial, Almería, Spain). The manufacturer describes the technical characteristics of the cover as diffuse colorless, 200 μm thickness, 85% transmittance to visible light, 50% transmittance to diffuse light, and 8% transmittance to infrared light. The design of the experiment included six different patches on each sector of the greenhouse (+V and −V), in which zucchini plants were alternatively irrigated with two types of water, groundwater (GW) or desalinated seawater (DW) (in triplicate), as shown in Figure 1. The desalinated seawater was purchased from the seawater desalination plant from Carboneras, Almería (0.397 dS/m, 0.52 €/m3), whereas the groundwater was obtained from a water well called El Jabonero ́ at Almería province (2.36 dS/m, 0.29 €/m3). Both located at Nijar, waters were treated with the same nutrition complements. The analyses of several chemical parameters for both waters are described in Table S1 (Supporting Information). Zucchini fruits were harvested successively every 3−4 days, from April to July 2017. The weight, diameter, and length of fruits were estimated directly after harvest. Samples for NMR analysis were taken randomly, five fruits of different sizes from each one of the 12 patches, with an overall of 72 zucchini fruits per harvest. Sample Preparation. The five zucchinis collected from each patch were pooled together to one mixed sample, washed, peeled, cut in pieces, grinded, and freeze-dried to avoid unwanted enzymatic reactions. Forty milligrams of previously washed, freeze-dried, and powdered zucchini samples was extracted using 1 mL of a mixture of CH3OH-d4 and D2O KH2PO4 buffer (pH 6.0) in a ratio of 50:50 (v/v) containing the sodium salt of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP, 0.01% w/w) and sodium azide (NaN3, 90 μM) as an enzyme inhibitor. The extractions were performed by vortexing the mixtures for 30 min (600 rpm), followed by centrifugation (5 min, 13 500 rpm). Five-hundred microliters of the supernatants was transferred to oven-dried 5 mm NMR tubes.

This study aims to understand how crop quality changes under different ventilation conditions and by using groundwater or desalinated seawater for plant irrigation. The zucchini (Cucurbita pepo L.), a variety of summer squash of the Cucurbitaceae family, is easily grown in the agricultural and climatic conditions of Almería, and it is part of the everyday food of their inhabitants.11 The last campaign (2016/2017) reached a production of 350 000 tons of zucchini in Almería’s region.12 It was our aim to evaluate if the differences on ventilation strength and the use of desalinated seawater for plant irrigation has significant impact not only in the production point of view, but also focusing on the overall zucchini metabolome, that is, to understand if organoleptic or nutritional properties of zucchini may decrease under specific environmental conditions. Nowadays, NMR metabolomics is a powerful technique for determination of natural compounds in complex mixtures of plant or fruits extracts that do not need chromatographic purification or laborious and time-consuming extractions.13−16 NMR profiling allows researchers to analyze important changes in the metabolome of a biological system derived from cellular activities and to monitor the change on their levels according to genetic or environmental factors.17 NMR metabolomics have been applied on apples, for example, in the impact of internal browning after different pre- and postharvest methods,18 in the differentiation of organic and conventionally grown tomatoes,19 or in the evaluation of postharvest storage in Citrus melon20 or in avocados.21 To the best of our knowledge, NMR has never been applied on zucchini extracts, and therefore, we describe herein a NMR-based metabolomic approach to identify, for the first time, the effects of seawater desalination and ventilation on zucchini metabolome and to characterize discriminating metabolites associated with each cultivation parameter.

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MATERIALS AND METHODS

Plant material and Greenhouse Experiment Setup. The crop considered was zucchini (Cucurbita pepo L. cv Victoria) in a spring− summer cycle from April to July 2017. Zucchini is an annual plant with compact growth, a pentagonal stem section, and indeterminate and creeping growth types. The planting framework tends to have distances of 150−200 cm between rows and 60−150 cm between plants. Plant density was 1 pl/m2, in total 1515 plants. The experiments were performed in a greenhouse with pitched roof, commonly known as “raspa y amagado”, with a floor surface of 1935 m2 (45 × 43 m2) and located at the UAL-ANECOOP Foundation’s Innovation and Technology Centre (36° 51′ N, 2° 16′ W; 87 m elevation) in the province of Almería, Spain. In the southeast of Spain, these greenhouse structures are the most popular. Almería greenhouses are characterized by the flexibility of the structural elements, which are composed of individual wires or braids that are subject to initial B


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Figure 2. Expansions of a characteristic 1H NMR spectrum obtained at 600 MHz from zucchini CD3OD/D2O KH2PO4 buffer (50:50, v/v) extract. Abbreviations: ala, alanine; arg, arginine; asn, asparagine; asp, aspartate; Chl, chlorogenate; FA, fatty acids; fru, fructose; GABA, 4-aminobutyrate; gln, glutamine; glc, glucose; His, histidine; isoleu, isoleucine; leu, leucine; myo-ins, myo-inositol; Hpl, p-hydroxyphenyllactate; NI, nonidentified; Phe, phenylalanine; suc, sucrose; thr, threonine; Trp, tryptophan; Tyr, tyrosine; val, valine. NMR Experiments. NMR spectra were recorded at 293 ± 0.1 K on a Bruker Avance III 600 spectrometer operating at a proton frequency of 600.13 MHz using a 5 mm QCI quadruple resonance pulse field gradient cryoprobe. The samples were measured, without rotation and using 4 dummy scans prior to 32 scans. Acquisition parameters were set as follows: size of fid = 64 K, spectral width = 20.5 ppm, acquisition time = 2.73 s, relaxation delay = 10 s, receiver gain = 57, FID resolution = 0.25 Hz, and mixing time = 10 ms. A presaturation pulse sequence (Bruker 1D noesygppr1d) was used to suppress the residual water signal via irradiation of the H2O frequency during the recycle and mixing time delays. This pulse sequence is the most used in metabolomics.22 The resulting spectra were automatically phased, baseline-corrected, and calibrated to the TSP signal at 0.0 ppm. The spectrometer transmitter was locked to CH3OH-d4 frequency. Acquisition and processing of spectra were carried out with TOPSPIN software (version 3.1). The multiplicities observed were labeled as s = singlet; d = doublet; dd = doublet of doublets; ddd = doublet of doublet of doublets; t = triplet; and m = multiplet. 1 H−1H total correlation spectroscopy (TOCSY), 1H−13C heteronuclear single quantum coherence (HSQC), and 1H−13C heteronuclear multiple bonds coherence (HMBC) spectra were recorded using standard Bruker sequences. The TOCSY spectrum was obtained applying a relaxation delay of 1.5 s, spectral width in both dimensions of 8403.36 Hz, and a receiver gain of 114. TOCSY spectrum was processed using sine-bell window function (SSB = 2.0). The HSQC spectrum was acquired using a relaxation delay of 1.0 s, spectral width of 7812.50 Hz in F2 and 37 729.66 Hz in F1. Quadratic sine window function (SSB = 2.0) was applied for the HSQC spectrum. The HMBC spectrum was recorded with the same parameters used in the

HSQC spectra except for 7500.00 Hz of spectral width in F2. The delays of coupling evolution were optimized for 1JcH = 145 Hz and n JCH = 8 Hz for HSQC and HMBC experiments. Oxygen Radical Absorbance Capacity (ORAC) Assay. The ORAC analyses were carried out on a CYTATION microplate reader, from Bio-Tek Instruments, Inc. (Winooski, VT), using black 96-well polystyrene microplates. Fluorescence was read through the top, with an excitation wavelength of 485 nm and an emission filter of 520 nm. The oxygen radical absorbance capacity was determined as described by Lucas-Abellán et al.23 with several modifications. The reaction was carried out in 10 mM KH2PO4 buffer (pH 7.4) and a final reaction mixture of 200 μL. Fluorescein (150 μL, 1 μM) and the zucchini extracts (25 μL, 1:100 diluted) were placed in the wells of the microplate. The mixture was preincubated for 30 min at 37 °C, before rapidly adding the AAPH solution (25 μL; 250 mM) using a multichannel pipet. The microplate was immediately placed in the reader and the fluorescence recorded every 1 min for 80 min. The microplate was automatically shaken prior to each reading. Eight calibration solutions using Trolox (12.5, 25.0, 50.0, 100.0, and 200.0 μM) as antioxidant were also used in each assay. A blank was performed using KH2PO4 buffer solution instead of the antioxidant solution. All reaction mixtures were prepared in triplicate, and at least three independent assays were performed for each sample. To avoid the temperature effect, only the inner 60 wells were used for experimental purposes, while the outer wells were filled with 200 μL of distilled water. The results were expressed as relative fluorescence with respect to the initial reading. The area under the fluorescence decay curve (AUC) was calculated according to Lucas-Abellán et al.23 Statistical Analyses. NMR spectra were bucketed using AMIX 3.9.12 (Bruker BioSpin GmbH, Rheinstetten, Germany). Buckets C


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Journal of Agricultural and Food Chemistry were obtained by integrating 0.04 ppm intervals and were normalized by scaling the intensity of individual peaks to the total intensity recorded in the region from δH 0.2 to 10.0 ppm. Regions of δH 5.08− 4.60 and 3.36−3.28 ppm were excluded from the analysis because of the presence of the residual signals of H2O and CH3OH, respectively. Principal component analysis (PCA) and orthogonal projection to patent structures (OPLS) models were scaled to Pareto, while partial least squares discriminant analysis (PLS-DA) models were scaled to unit variance. Subsequent multivariate data analysis was performed using SIMCA-P software (v. 14.0, Umetrics, Sweden). ORAC data were subject to statistical analysis by ANOVA. Least significant difference (LSD) test was used to compare means, and significance was accepted at P = 0.05 level.

Table 1. Peak Assignment of Metabolites Identified on Zucchini CD3OD: D2O KH2PO4 Buffer (50:50, v/v) Extracts by NMR metabolite 1. valine 2. isoleucine 3. leucine 4. threonine 5. alanine 6. n.i 7. n.i 8. gaba 9. arginine 10. glutamine 11. succinate 12. malate

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RESULTS AND DISCUSSION Metabolite Profiling on Zucchini Fruits. Zucchini (Cucurbita pepo L.) has a high nutritional value and a low number of calories.24 It contains several bioactive compounds such as β-carotene, phenolic compounds, flavonoids, vitamins, etc.25 Only few studies could be found in the literature that focus on the identification of compounds from the zucchini fruit, most of them based on mass spectrometry methodologies. The known metabolites are limited to those ascertained by Adlercreutz and Mazur on isoflavones,26 by Appendino on triterpenes,27 by Sicilia et al. on lignans,28 and by Koike and co-workers29 on cucurbitosides A to E and F to M. The most recent study on zucchini composition is the one described by the FernándezGutiérrez group on the characterization of the polyphenolic fraction of three zucchini varieties.30 To obtain a comprehensive view of the metabolome of our own zucchinis, samples were extracted with a solvent system based on methanol and phosphate buffer (50:50, v/v). Figure 2 shows expansions of the three main regions of a typical 1H NMR spectrum of a zucchini extract. The region from δH 0.5 to 3.2 ppm contains mainly signals from amino acids and organic acids. With respect to amino acids, alanine, threonine, valine, leucine, isoleucine, arginine, aspartate, asparagine, and γ-aminobutyrate (GABA) were found and quantified in the different replicates. Organic acids such as acetate, acetoacetate, citrate, and succinate were also identified. The region from δH 3.2 to 5.5 ppm allowed researchers to identify the signals of carbohydrates, mostly glucose (both α- and β- configurations) and fructose, but also sucrose and myo-inositol. The downfield region of δH 5.5 to 10.0 ppm, known as the aromatic region, comprised relatively weak signals, which were attributed to aromatic moieties from amino acids, such as phenylalanine, histidine, tyrosine and tryptophan, nucleosides, and nitrogenous bases, for example, adenosine, uridine, and uracil. Interestingly, diagnostic signals of niacin, a naturally occurring vitamin B complex,31 were also identified in this region at δH 8.97 and 8.59 ppm. Trigonelline, which was derived from the methylation of the nitrogen atom of niacin, was also identified. Besides niacin, other vitamins have been reported in zucchini (including vitamin A, C, and E).24 However, these compounds were not identified, mostly due to its low concentration, but also, in case of vitamin C (ascorbic acid), because its signals appear in regions highly overlapped. The complete list of metabolites identified in our zucchini extracts is provided in Table 1, and for each signal, chemical shift (δ, ppm), multiplicity, and coupling constants (J, Hz) are also reported. All assignments were based on the analysis of homo- and heteronuclear 1D and 2D NMR experiments (1H-NOESY, 1H−1H TOCSY, 1H−1H COSY, 1H−13C edited HSQC, and 1H−13C HMBC), together with the use of the

13. citrate 14. aspartate 15. asparagine 16. 17. 18. 19. 20.

acetate acetoin ethanolamine choline myo-inositol

21. 22. 23. 24. 25. 26. 27. 28.

α-glucose β-glucose sucrose fructose fumarate uracil adenosine uridine

29. histidine 30. tyrosine 31. phydroxyphenyllactate 32. phenylalanine 33. tryptophan 34. formate 35. trigonelline 36. niacin 37. chlorogenate

chemical shifts (ppm), multiplicity, and coupling constants (Hz) 1.06 (d, J = 7.1 Hz), 1.01 (d, J = 7.1 Hz) 1.03 (d, J = 7.1 Hz), 0.96 (t, J = 7.3 Hz) 0.99 (d, J = 6.4 Hz), 0.97 (d, J = 6.4 Hz) 1.34 (d, J = 6.6 Hz) 1.49 (d, J = 7.2 Hz) 1.185 (s) 1.229 (s) 1.90 (m), 2.30 (t, J = 7.2 Hz), 3.01 (t, J = 7.3 Hz) 1.72 (m), 1.91 (m) 2.14 (m), 2.46 (m) 2.43 (s) 2.40 (dd, J = 15.4, 9.4 Hz), 2.69 (dd, J = 15.4, 3.3 Hz), 4.28 (dd, J = 9.4, 3.3 Hz) 2.71 (d, J = 16.4 Hz), 2.54 (d, J = 16.4 Hz) 2.64 (dd, J = 17.4, 9.3 Hz), 2.81 (dd, J = 17.4, 3.6 Hz) 2.82 (dd, J = 16.9, 8.0 Hz), 2.95 (dd, J = 16.9, 4.2 Hz) 1.92 (s) 2.22 (s) 3.12 (t, J = 5.3 Hz) 3.21 (s) 3.24 (t, J = 9.4 Hz), 3.47 (dd, J = 2.8; 9.9 Hz), 4.01 (t, J = 2.8 Hz) 5.19 (d, J = 3.7 Hz) 4.59 (d, J = 7.9 Hz) 5.41 (d, J = 3.9 Hz) 4.08 (m) 6.53 (s) 7.52 (d, J = 7.6 Hz), 5.76 (d, J = 7.6 Hz) 6.02 (d, J = 5.5 Hz), 8.24 (s), 8.35 (s) 5.86 (d, J = 8.2 Hz), 5.90 (d, J = 4.7 Hz), 7.93 (d, J = 8.2 Hz) 7.94 (d, J = 1.1 Hz), 7.12 (d, J = 1.1 Hz) 7.19 (d, J = 8.5 Hz), 6.86 (d, J = 8.5 Hz) 7.24 (d, J = 8.6 Hz), 6.85 (d, J = 8.6 Hz) 7.33 (m), 7.34 (m), 7.40 (m) 7.19 (m), 7.26 (m), 7.31 (s), 7.47 (d, J = 8.2 Hz), 7.73 (d, J = 8.2 Hz) 8.47 (s) 4.45 (s), 8.10 (dd, J = 8.0, 6.1 Hz), 8.86 (d, J = 6.1 Hz) 8.88 (d, J = 8.0 Hz), 9.15 (s) 8.97 (d, J = 2.2 Hz), 8.59 (dd, J = 4.9; 1.7 Hz), 8.28 (ddd, J = 7.9; 2.2, 1.7 Hz), 7.52 (dd, J = 7.9; 4.9 Hz) 6.47 (d, J = 15.9 Hz), 7.70 (d, J = 15.9 Hz)

NMR database BBIOREFCODE 2 from Bruker Biospin, public NMR databases such as COLMAR,32 HMDB, and literature data.30,33 Production and Quality Indicators for Zucchini Fruits. As mentioned earlier, the average crop yield of Almería (Spain) is variable and depends on a number of factors such as crop type, climatic zone, irrigation water quality, structure type, ventilation surface, climate control system, and crop management, among others. Zucchinis from each cultivation patch and grown under different ventilations and water-type irrigation were harvested at the ripe stage of development every 4 days from April to July 2017 (in overall six different days). The purpose D


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Figure 3. Production and quality indicators for zucchini growth (June 2017, 36° 51′ N, 2° 16′ W; 87 m elevation, Almería, Spain) under different conditions, irrigation with groundwater (GW) or desalinated seawater (DW) and low (−V) or high (+V) ventilation: (a) evolution of zucchini weight (kg) along the six harvests; (b) average length (cm) and (c) diameter (mm) for the collected zucchinis; (d) total and (e) specific zucchini production (kg/plant) along the six harvests. Mean ± SD values are presented. For the statistical analysis, a two-letter system (x1 x2) was applied to define the differences on production/size/weight of zucchini fruits between the two conditions of ventilation (x1) and between the two types of water (x2) as statistical significant (x1,2 = b, P < 0.05), or not (x1,2 = a, P > 0.05).

yield increase. In this situation, the high commercial value from the agricultural production could cover the cost of the seawaterdesalination and be profitable for farmers. Multivariate data analysis to determine metabolic changes on zucchini fruits promoted by water-type irrigation. The quality of the zucchini is intimately linked to its organoleptic and nutritional properties, which are, in turn, a function of its metabolic composition. With the NMR metabolite characterization on hand, we decided to evaluate the influence of watertype irrigation (groundwater or desalinated seawater) and different greenhouse ventilation intensities on zucchini metabolome and to identify biomarkers on zucchini associated of such external conditions. The main purpose of an exploratory data analysis, such as PCA, is to reveal the major trends in the 1H NMR data and the possible analytical or biological confounder variables.34 Two types of plots were generated: (1) the PCA scores plot that groups similar samples together based on the input data and (2) PCA loadings plot that indicates which areas of the spectra are contributing the most to the variation between the groups. The PCA scores and loadings plots for the 72 1H NMR spectra collected from zucchini extracts obtained from different cultivation parameters are shown in Figure 4. The scatter plot of PC1 versus PC3 (Figure 4a), which explains a 62.4% and 8.5% of the total variance, respectively, shows the separation of

of the study is to understand the potential of these different agronomic practices to increase crop yield and fruit quality in a metabolically point of view. Production rates of the plants growth in each patch and quality of the zucchini in terms of weight, diameter, and length were evaluated, and the results are shown in Figure 3. Zucchinis from the first harvest showed inferior weight (P < 0.05) than the others, possibly due to an earlier ripening stage. However, no visual trend can be noticed on size/weight among the zucchinis from plants irrigated with different types of water or under different ventilation intensities. In terms of production per plant, no statistically significant differences (P > 0.05) were found in the production of zucchini for the two ventilation systems analyzed; however, they did appear for the two types of water (Figure 3d). The kilograms of zucchini significantly increased when desalinated seawater was used for plant irrigation (P < 0.05) in every collected day (Figure 3e), which accounted for a 57% of the overall zucchini production. Specifically, the commercial production of zucchini with desalinated seawater (5.60 kg m−2) was higher than that obtained with groundwater (4.23 kg m−2). Irrigation with desalinated seawater generally produces higher yields than groundwater, as it is well-known. Removal of the salt excess, such as sodium content, which is not essential for the growth of most plant species and can present specific toxic effects, may lead to a E


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It is known that some organic acids undergo major changes during ripening, and their decrease is accepted to occur alongside with a decrease in the starch content together with an increase in sugar concentrations.35,36 In fact, the sugar/acid ratio is often used to give a technological characterization of vegetable ripeness.35 In our case, the sugars/acids ratios in zucchini extracts obtained for the six harvests were calculated using the sum of the NMR quantifications (mg/g extract) obtained for the major sugars (glucose, fructose, and sucrose) and organic acids (malate and fumarate) and are shown in Figure 5a. The sugars/acids ratios for zucchinis from second to sixth harvests (Figure 5b) were found to be of ∼8.6 whether using desalinated seawater or groundwater plant irrigation (P > 0.05). For example, in tomatoes, a value of about 7.5 is usually accepted as a beneficial sugar/acid ratio.37 Because of their lower ripening stage, zucchini fruits from the first harvest were excluded in the following statistical analyses to focus on the metabolic changes imposed exclusively by the different cultivation parameters. Relatively, to samples clustering according to the type of water used for zucchini plants irrigation, Figure 6a shows a PCA scores plot with PC1 and PC4 explaining 44.4% and 5.6% of total variance, respectively. Groundwater scores dots (gray squares in Figure 6a) are mainly located in the left quadrants, which represent a clear trend in the data. To improve the discrimination associated with the type of water and to identify the most discriminant variables, a partial least squares discriminant analysis (PLS-DA) model was applied to the 1H NMR data (Figure 6b), being the type of water, the class ID. PLS-DA model is useful to understand how the X variables are influenced by a Y categorical variable. It has been widely applied for qualitative identification of food, drug, and agricultural products16,38 since it takes advantage of its high-efficiency resolving ability and intensification of intergroup differences. As expected, the PLS-DA scores plot (Figure 6b) of the first two latent variables showed a slight increase of clustering compared with the previous PCA scores plot (Figure 6a). The discrimination between the two types of water is observed regardless of ventilation intensity. Figure 6c shows the contributions plot of PLSDA model, highlighting the main buckets and their assigned metabolites that are ascribed for desalinated seawater (upper bars) or groundwater (down bars). On one hand, by analyzing the discriminating loadings for each type of water, it is possible to observe that zucchini fruits irrigated with desalinated seawater showed a higher amount of α-, β-glucose, fructose, fumarate, uracil, adenosine, and niacin. On the other hand, discriminating buckets for zucchini fruits irrigated with groundwater were assigned to metabolites such as sucrose, myo-inositol, uridine, asparagine, aspartate, alanine, glutamine, chlorogenate, and trigonelline.

Figure 4. (a) PC1/PC3 PCA scores and (b) loadings plots obtained for 1H NMR data of a total of 72 zucchini CD3OD/D2O KH2PO4 buffer (50:50, v/v) extracts harvested at six different days (June 2017, 36° 51′ N, 2° 16′ W; 87 m elevation, Almería, Spain). Scaling was done to Pareto.

zucchini extracts from the first harvest (black circles) with respect to the others. The loadings plot (Figure 4b) affords the 1 H NMR buckets that are ascribed to the different groups of samples. Buckets that are far away from the origin have a strong influence on the model, and those closer to the center provide a weaker influence. The position of objects in a given direction in a scores plot is influenced by the variables (buckets) lying in the same direction in the loadings plot. The analysis of the discriminating loadings allowed to conclude that the first harvested zucchinis display a lower ripening stage compared to the others since they present a lower amount of sugars, such as α- and β-glucose and fructose, and an increase quantity of organic acids (e.g., malate). This fact agrees with the zucchini quality indicators (weight) previously reported in Figure 3a, which were significantly lower on this harvest.

Figure 5. (a) Sugars/acids ratios for zucchinis obtained from plant irrigation with desalinated seawater (DW) or groundwater (GW) for each harvest date. (b) Means ± SD of sugars/acids ratios for zucchinis from 2nd to 6th harvests and from each cultivar (DW or GW). F


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Figure 6. (a) PC1/PC4 PCA scores, (b) PLS-DA scores (with 6 PCs, R2 = 0.79, Q2 = 0.71), and (c) contribution plots obtained for 1H NMR data of zucchini CD3OD/D2O KH2PO4 buffer (50:50, v/v) extracts from plant irrigated with groundwater (gray squares, down bars) or desalinated seawater (black circles, upper bars). The models include 60 zucchini samples (excluding the 1st harvest samples) and were scaled to Pareto and unit variance for PCA and PLS-DA models, respectively. PLS-DA model was validated by permutation test. Upper and down bars correspond to buckets associated with desalinated seawater (DW) and groundwater (GW), respectively. Abbreviations: aden, adenosine; ala, alanine; asn, asparagine; asp, aspartate; fru, fructose; gln, glutamine; glc, glucose; myo-ins, myo-inositol; suc, sucrose; Trign, trigonelline; urac, uracil; urid, uridine.

For other crops, such as tomato, a positive relationship between the overall tomato taste/quality and the salinity of irrigation water, especially on potassium salts, has been reported.39,40 In the case of zucchini, its size is more relevant as a quality indicator than for flavor since it has a very light and delicate flavor. Therefore, it is unknown how the variation of sugars and organic acids profile on zucchini influences its organoleptic properties. However, an analogy can be made with zucchini relatives such as the winter squash and melon. For winter squash (also from Cucurbitaceae family), fruit quality involves the complex interplay of flavor, texture, and appearance, depending on the fruit content on carotenoids (for color), starch (for texture), and sugars (for perceived sweetness, for which sucrose is considered the most important predictor).41 Perceived sweetness was reported to be important for consumer acceptability in taste panels and to influence perception of overall squash flavor.42 For melon, the most important properties for organoleptic quality and consumer acceptance are also the sucrose levels, besides the aroma

To be sure that the discriminating NMR spectral areas were not influenced by the noise or any baseline effect, the quantification of 13 discriminating metabolites was performed with the help of an internal standard (TSP, see sample preparation section) and is represented as boxplots in Figure 7 and listed in Table S2 of the Supporting Information. On each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. Interestingly, each type of water used in plant irrigation influenced the concentration of different sugars on zucchini: desalinated seawater-irrigation increased the levels of glucose and fructose, while for groundwater an increase on sucrose and myo-inositol was noticed. It was not possible to quantify myoinositol and succinate since their signals are in overlapped regions and are difficult to integrate and, therefore, to quantify. Two main factors usually determine fruit’s characteristic flavor: the correct sugar/acid balance and the production of aroma volatile compounds.37 The effects of water desalination on the quality of the fruits depends on several factors and are controversial. G


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Figure 7. Quantification boxplots of 13 discriminating metabolites identified on zucchini extracts (CD3OD/D2O KH2PO4 buffer, 50:50 v/v, excluding the 1st harvest samples) obtained from plants irrigated with desalinated seawater (DW, light gray) or groundwater (GW, dark gray). Boxplot shows distribution of all individuals, median, first quartile below the median, third quartiles above the median, and the mean represented by a cross. Error lines show the lowest and highest values of the data.

profile.43 In this sense, groundwater-irrigated zucchini fruits may give a higher sweetness perception since they display higher quantity of sucrose. Nevertheless, the amount of glucose and, in less extension, of fructose, decrease for these zucchini fruits. Besides sugars, organic acids play an important role as flavor enhancers and on the perception of sweetness since they give to the vegetables their “acidic taste.” In our samples, the concentration of the major organic acid found, fumarate, increases with desalinated seawater irrigation, whereas succinate decreased. To what extent these changes on organic acid profile influence the organoleptic properties of zucchini are also unknown.

Relatively, to amino acids content, groundwater-irrigated zucchini showed higher level of certain amino acids (asparagine, aspartate, glutamine, and alanine), suggesting a high protein turnover. For tomato, for example, free amino acid content of tomato fruit pericarp increases markedly during ripening transition.44 About the differences observed on the level of certain nucleosides (adenosine and uridine) and the nitrogenous base uracil observed among zucchini fruits, their regulation is a complicated and an extremely important part of plant function since these compounds are essential to plant metabolism and contain a large reservoir of the plant’s nitrogen and phosphorus. Moreover, it is interesting to examine that H


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The antioxidant activity is used as an indicator of the ability of the product in reducing oxidative stress and is related to the overall fruit content on the above-mentioned phenolics, vitamins, and carotenoids.45 The antioxidant activity of the zucchini extracts was measured by ORAC method in 96-wells microtiter plates to understand the influence of water-type irrigation of zucchini plants on the antioxidant activity of their fruits. Figure 8 shows the antioxidant activity, expressed as Trolox equivalent (TE, μM) per mg of dried sample, of zucchini extracts obtained from the second to sixth harvests. This activity seems to be higher (P < 0.05) when plant irrigation was performed with desalinated seawater from second to fourth harvests, and similar (P > 0.05) for the last two harvests. Also, in accordance, a higher amount of niacin (vitamin B3, Figure 7, Tables S2) was quantified on zucchini fruits from plants irrigated with desalinated seawater. Additionally, niacin has long been used for the treatment of lipid disorders and cardiovascular diseases.31 Minor concentration compounds belonging to the already mentioned classes of compounds could also contribute to this antioxidant activity, but they were not detected by NMR, either because they appeared in overlapped regions of the spectra or at concentrations below the NMR detection limit or, in case of carotenoids, because the extraction solvent mixture of choice was not the most appropriate. Nevertheless, the presence of several phenolic compounds (e.g., luteolin, quercetin, and kaempferol derivatives),30 carotenoids (lutein, β-carotene),45 and vitamins (vitamin A, B, C, and E)24 on zucchini have been already reported.

Figure 8. Antioxidant activity measured by ORAC methods for CD3OD/D2O KH2PO4 buffer (50:50, v/v) extracts of zucchini fruits from 2nd to 6th harvests from plants irrigated with desalinated seawater (DW) or groundwater (GW). The results are expressed as Trolox equivalent (TE, μM) per mg of dried sample. Bars with ∗ indicate significant different antioxidant activities between zucchinis from plant irrigation with DW or GW (P < 0.05).

conversion of niacin into trigonelline was higher in groundwater-irrigated zucchini fruits. Influences of Water-type Irrigation on Antioxidant Activity of Zucchini Fruits. The antioxidant/antiradical, anticarcinogenic, anti-inflammatory, antiviral, antimicrobial, and analgesic activities of zucchini have been exploited in traditional folk medicine to treat colds and alleviate aches.25 Variation in water-type irrigation can affect zucchini nutritional value.

Figure 9. (a) PC1/PC2 PCA scores plot obtained for 1H NMR data of zucchini extracts from plant cultivation under low (light gray triangles) and high (dark gray triangles) ventilation intensities, showing no visible discrimination. (b) OPLS scores plot (1 + 4 components; R2 = 0.90, Q2= 0.5) and (c) S-plot obtained for 1H NMR data (region δH 10.0−7.0 ppm) of zucchini extracts from plants irrigated with groundwater (GW). Scaling was done to Pareto for both models. PCA model included 60 zucchini samples (excluding the 1st harvest samples), while OPLS model included 24 samples (GW-derived extracts excluding those from 1st harvest but also 4th harvest to obtain a predictive valid model with Q2-value ≥ 0.5). I



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Multivariate Data Analysis To Determine Metabolic Changes on Zucchini Fruits Promoted by Ventilation Intensity. The next step was to explore if different intensities on the greenhouse ventilation influence zucchini metabolome, besides water-type plant irrigation. The PC1/PC2 PCA scores plot is represented in Figure 9a and colored according to ventilation intensity (from light gray−low (−V) ventilation to dark gray−high (+V) ventilation). No visible discrimination was observed among zucchinis grown with different ventilation intensities. Nevertheless, by applying an orthogonal projection to latent structures (OPLS) model to 1H NMR data of zucchini extracts irrigated only with groundwater and by selecting exclusively the aromatic region (δH 10.0−7.0 ppm, Figure 9b), a clear discrimination could be observed among zucchinis extracts grown under different ventilation intensities. The OPLS technique is an extension of the supervised PLS regression method that produces models of clearer interpretation. In this respect, OPLS removes the so-called structured-noise from a given data set and decomposes the factor data matrix X into two blocks corresponding to the variations correlated and noncorrelated to the response Y. OPLS-based methods were performed with mean centering as data pretreatment. A particularly useful tool that compares the variable magnitude against its reliability is the S-plot obtained by the OPLS model, which is represented in Figure 9c. This plot showed the most relevant variables aimed to sample differentiation between the two ventilation intensities: in particular, buckets at δH 9.16, 8.88, 8.84, 8.12, 7.92, 7.40−7.32, and 7.12 ppm were the most characterizing of the high ventilated crops and those located at δH 8.36, 8.24, 7.52, and 7.24 ppm accounted for less ventilated crops. Overall, it allowed to conclude that by increasing ventilation intensity, zucchinis irrigated with groundwater presented a higher amount of trigonelline and also of amino acids such as histidine and phenylalanine. Trigonelline and amino acids were previously found to appear in higher concentrations in zucchini extracts from plants irrigated with groundwater (Figure 7). Thus, interestingly, an increased ventilation associated with groundwater plant irrigation promoted a synergic effect on amino acids and trigonelline increment. To conclude, with this study, we have found that plants irrigated with desalinated seawater (0.397 dS/m, 0.52 €/m3) showed an increased zucchini production yield when compared with plants irrigated with groundwater (2.36 dS/m, 0.29 €/m3), presented fruits with higher concentration of glucose, fructose, and vitamin B3, and displayed an increased antioxidant activity. Thus, this water supply guarantees optimal plant growth and may enhance the quality of their fruits and their commercial applications. On the contrary, plant groundwater irrigation produced the increment of sucrose level that could rise the sweetness perception of the fruits. Different ventilation conditions (9.8 and 15% of surface area of the vent openings/greenhouse) produced no relevant changes on the zucchini metabolome, although when only the aromatic region of the spectra is considered, it is deduced that an increased ventilation associated with groundwater plant irrigation promoted higher concentrations of trigonelline, histidine, and phenylalanine. The organization of a taste panel for zucchini fruits will be essential to correlate the metabolic changes of zucchini reported in this study with their organoleptic properties, which will attempt to fill the general lack of information on the subject. In addition, evaluations of the degrees Brix are planned to correlate this wellestablished value with the specific sugars profile. These studies are currently undergoing in our laboratories.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b02590. Chemical analysis of desalinated seawater and groundwater; quantification values of 13 discriminating metabolites (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Ignacio Fernández: 0000-0001-8355-580X Funding

This research has been funded by the National R+D+i Plan Projects AGL2015−68050-R and CTQ2017−84334-R of the Spanish Ministry of Economy and Competitiveness, Junta de Andalucía project number P12-FQM-2668, and ERDF funds. We thank Prof. Francisco J. Moyano López (University of Almería) for the helpful discussions and assistance regarding the ORAC assays. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED aden, adenosine; ala, alanine; arg, arginine; asn, asparagine; asp, aspartate; Chl, chlorogenate; FA, fatty acids; fru, fructose; GABA, 4-aminobutyrate; gln, glutamine; glc, glucose; His, histidine; isoleu, isoleucine; leu, leucine; myo-ins, myo-inositol; Hpl, p-hydroxyphenyllactate; NI, nonidentified; Phe, phenylalanine; suc, sucrose; Trign, trigonelline; thr, threonine; Trp, tryptophan; Tyr, tyrosine; urac, uracil; urid, uridine; val, valine

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REFERENCES

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