Statement of Foliar Fertilization Impact on Yield, Composition, and

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Agricultural and Environmental Chemistry

Statement of Foliar Fertilization Impact on Yield, Composition, and Oxidative Biomarkers in Rice M. Pinciroli, R. Dominguez-Perles, Angel Abellan Victorio, Valerie BultelPoncé, T. Durand, Jean Marie Galano, F. Ferreres, and A. Gil-Izquierdo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05808 • Publication Date (Web): 19 Dec 2018 Downloaded from http://pubs.acs.org on December 26, 2018

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Journal of Agricultural and Food Chemistry

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Statement of Foliar Fertilization Impact on Yield,

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Composition, and Oxidative Biomarkers in Rice

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Pinciroli, M.,† R. Domínguez-Perles,‡,* Á. Abellán,‡ V. Bultel-Poncé,§ T. Durand,§ J.

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M. Galano,§ F. Ferreres,‡ Á. Gil-Izquierdo.‡

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†

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National University de la Plata, Street 60 and 119, 1900 La Plata, Buenos Aires, Argentina

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‡

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Technology, CEBAS-CSIC, University Campus of Espinardo – Edif. 25, 30100 Espinardo, Spain

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§

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Department of Climate and Agricultural Phenology, Faculty of Agriculture and Forestry Sciences of the

Research Group on Quality, Safety and Bioactivity of Plant Foods. Department of Food Science and

Institut des Biomolécules Max Mousseron, IBMM, UMR 5247, University of Montpellier, CNRS,

ENSCM, Montpellier, France.

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Running title: Phytoprostanes and phytofurans content of Oryza sativa L. upon foliar fertilization

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ACS Paragon Plus Environment


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ABSTRACT: In rice crops, fertilization is a naturalized practice, although inefficient,

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that could be improved by applying foliar fertilization. Phytoprostanes (PhytoPs) and

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phytofurans (PhytoFs) are products of α-linolenic acid peroxidation, useful as

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biomarkers of oxidative degradation in higher plants. The objective was to determine

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the effect of the foliar fertilization on the concentration of PhytoPs and PhytoFs, and its

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relationships with modifications of yield and quality of rice productions. It was

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described that the concentration of biomarkers of stress decreased with the application

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of foliar fertilization, being the response significantly different depending the genotypes

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and compound monitored. Moreover, fertilization did not modify significantly the

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parameters of yield (961.2 g m-2), 1000 whole-grain (21.2 g), and protein content

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(10.7% dry matter). Therefore, this is the first work that describes the effect of

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fertilization on PhytoPs and PhytoFs in rice genotypes and reinforces the capacity of

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these compounds as biomarkers to monitor specific abiotic stress, in this case,

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represented by nutritional stress.

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KEYWORDS:

Oryza

sativa,

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phytoprostanes, phytofurans

foliar

fertilization,

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genotypes,

protein

grain,

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ABBREVIATIONS: ALA, α-linolenic acid; BHA, butylated hydroxy anisole; PhytoPs,

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phytoprostanes; PhytoFs, phytofurans; ROS, reactive oxygen species; SPE, solid phase

34

extraction; UHPLC-ESI-QqQ-MS/MS, ultra-high performance liquid chromatography

35

coupled to electrospray ionization and triple quadrupole mass spectrometry.

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37

INTRODUCTION

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Rice crops play a central role in the population nutrition of more than half world

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population.1 In this regard, given the current growth pattern, the human world

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population could reach 9 billion people by 2050, which would require an augment of the

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agricultural productions.2 To overcome this new situation, plant nutrition research has

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been pointed out as crucial to meet food quality and safety requirements.3 The fact that

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at least 60% of the nowadays cultivated soils globally have several mineral problems,

44

like toxicities or deficiencies, makes plant nutrition-based research a cutting-edge issue

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essential to meet the demand for massive increases in food production.3

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When analyzing the most important constraints associated to agricultural

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production, it is found that, to date, most yield losses and inappropriate nutrient

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concentrations are due to abiotic stress caused by drought, salinity, extreme

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temperatures, and soils acidity, which deteriorate the composition of soils and,

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consequently, the nutritional status of plants. Efficient and sustainable use of natural

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resources, along with appropriate agronomic practices, is key to overcome the

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challenges of the increasing world population and resources shortage. Under such

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challenging situation, enhancing crops yield per unit area is the main goal in the field.4

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In this frame, nitrogen (N) fertilization is a widely adopted practice in conventional

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agriculture. This is of especial relevance due to the management of irrigation water,

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responsible for the status of organic N into soils (concerning both concentration and

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molecular form), as well as the lack of consistent evidences on the optimal timing for its

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application.5 As an alternative, foliar spraying provides nutrients availability at the exact

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time when they are required by plants, reducing the time-lapse between application and

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absorption. Hence, when applied at panicle initiation, foliar spraying provides plants the

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essential nutrients required for obtaining an optimal number of grains per panicle.6

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Besides, Waraich et al. demonstrated that the application of micronutrients, reduces the

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toxicity of reactive oxygen species (ROS), due to an increase in the concentration of

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antioxidant enzymes,6 contributing to the protection and maintenance of the integrity

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and stability of the cell membrane.7

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The production of ROS during photosynthesis is intensified due to a limited use of

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the light absorbed upon photosynthetic electron transport and fixation of CO2.8 These

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radicals are unstable and react rapidly with other compounds in the separate cell

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compartments, causing cellular or tissue damage. To avoid this negligible situation,

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plant cells account with molecular tools to combat excessive ROS, maintaining the

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redox balance within physiological limits. However, biotic and abiotic stress may

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disturb this balance.9

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In many plant species, α-linolenic acid (ALA) constitutes the end point of fatty acid

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biosynthesis that is prone to react with ROS. A non-enzymatic peroxidation of ALA

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takes place that give rise to phytoprostanes (PhytoPs), prostaglandin-like compounds

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found in plants.10 Moreover, recently, a new class of plant oxylipins generated non-

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enzymatically was reported, and called the phytofurans (PhytoFs).11

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Concerning the physiological role in plants, PhytoPs and PhytoFs have been

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noticed as signaling molecules, also responsible for the protection of plant cells against

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negligible reactions triggered under oxidative stress. However, nowadays, remains a gap

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on the impact of management practices on their concentration of such molecules, as

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well as on the modification of their levels as a consequence of plant stress.12-14 Hence,

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nor the extent neither the sense in which fertilization modulates the content of oxylipins

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in plants have been completely explored, as well as their critical impact on plants

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growth and productivity, despite the accurate description of their occurrence in an array

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of plant species, which was recently reviewed in detail by Medina et al.15

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In this context, foliar fertilization could contribute to modify yield and nutritional

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quality, as well as the level of PhytoPs and PhytoFs in plant materials according to the

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genotype. Thus, although the oxylipins’ profile have been set up in different plant

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matrices and farming systems (conventional and ecological), a range of irrigation

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regimes, technological management (plastic cover), and genetic resources,13,16,17 to date

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the influence of a naturalized practice such as fertilization in productive species remains

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underexplored. In this regard, the present study pursues to shed some light on the

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response of PhytoPs and PhytoFs in situations of nutritional stress directly related to

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photosynthesis, at the level of photosystem II (PSII), the intercellular ROS levels, and

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the genetic differential response of the diverse varieties. In parallel, the information

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retrieved upon this work will allow the consideration of rice as a source of PhytoPs and

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PhytoFs in further works in vitro and in vivo devoted at the establishment of

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bioaccessibility and bioavailability of these compounds, as well as their actual

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biological capacity, in agreement with the information suggested so far by other

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authors.18-21

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MATERIALS AND METHODS

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Chemical and Reagents. The PhytoPs 9-F1t-PhytoP, 9-epi-9-F1t-PhytoP, ent-

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16-F1t-PhytoP, ent-16-epi-16-F1t-PhytoP, ent-9-D1t-PhytoP, ent-9-epi-9-D1t-PhytoP, 16-

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B1-PhytoP, 9-L1-PhytoP and the PhytoFs ent-16-(RS)-9-epi-ST-Δ14-10-PhytoF, ent-9-

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(RS)-12-epi-ST-Δ10-13-PhytoF,

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synthesized according to already published procedures and supplied by the Institut des

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Biomolécules Max Mousseron (Montpellier, France).14 Hexane was purchased from

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Panreac (Castellar del Valles, Barcelona, Spain), butylated hydroxyanisole (BHA), and

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Bis–Tris were purchased from Sigma-Aldrich (St. Louis, MO, USA). LC–MS grade

and

ent-16-(RS)-13-epi-ST-Δ14-9-PhytoF,

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were

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solvents were from J.T. Baker (Phillipsburg, NJ, USA). Water was obtained from a

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milli-Q water purification system from Millipore (Bedford, MA, USA). The solid phase

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extraction (SPE) cartridges acquired from Phenomenex (Torrance, CA, USA).

115 116

Field Experiment. The field trial was set up in the Experimental Station ’Julio

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Hirschhorn’, located in La Plata (34º52’S, 57º57’W, 9.8 m of altitude, Buenos Aires,

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Argentina) during the seasons 2016/2017 and 2017/2018. The soil was featured by 3.7%

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of organic matter (Walkey-Black method), 0.2% of total N (micro-Kjeldahl method),

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18.0 ppm of P (Bray Kurtz method Nº1), and pH 6.2, on average. The panel of rice

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cultivars included in the present work: One (1) featured by an average grain protein

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content, which is widely sown by farmers in the production area (‘Guri INTA’), one (1)

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cultivar (‘Nutriar FCAyF’) characterized by having 30% more grain protein, and three

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(3) advanced breeding lines of high grain protein content (‘H458-41-1-1-1’, ‘H475-3-1-

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1-2’, and ‘H484-9-1’) from the ‘Programa Arroz’ of the Agronomy and Forestry

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Faculty (FCAyF).

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The experimental setting was a randomized block design, with 3 replicates. Sowing

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was done manually in dry land, at a rate of 350 seeds m-2 in grooves at 0.20 m, in plots

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of 5 m2. The trials were settled with flood irrigation after 30 days of emergence and

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weeds were controlled with sodium bispyribac. The treatments were Control and 0.3 L

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ha-1 foliar fertilizer (Nutri-Fort®) in panicle initiation. The product was diluted in water

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and applied as a spray with hand sprayed. The experimental plots were sprayed during

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late afternoon, when wind speed was less than 10 km h-1. Nutri-Fort® contains 7.0%

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Nitrogen, 2.0% Phosphorus assimilable, 2.2% soluble Potassium, 0.6% Calcium, 1.0%

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Magnesium, 0.1% Copper, 2.0% Iron, 0.7% Manganese, and ≤0.1% Zinc, and 1.0%

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Boron.

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The climatic conditions during the field experiment set up are summarized in

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Table 1. The trials were sown in November, the anthesis and harvest were in February

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and April of the following, respectively. The temperatures recorded during seedling,

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establishment, and tillering meet the optimum values (25-31ºC), according to previous

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descriptions,22 while during ripening, the temperatures recorded oscillated between 20

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and 29 ºC.

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Samples and Standards Preparation. Harvest and threshing were done

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manually and, afterwards grains were dried in an oven at 40 °C until reaching 14%

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humidity. Dried grains were randomly weighed to record grain weight. Then, they were

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shelled to obtain whole grain. Samples were milled and sieved using Cyclone Mill 0.4

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mm mesh and there were immediately stored at 4 °C and protected from light until

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analysis.

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Determination of Protein Content. The protein content was evaluated on dry

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whole grain flour by micro-Kjeldahl method. Shortly, samples (50 mg) were digested

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with 2 mL of 98% H2SO4 and a mixture of potassium sulfate/copper sulfate catalyst

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(10:1). Then, the mixture was heated until complete digestion of the samples and

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cooled. Distilled water was added and steam distillation of NH3 was carried out by the

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addition 10 mL of 40% sodium hydroxide in water (w/v) and collected in saturated

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boric acid solution (40 g L-1). The acid neutralized by ammonia was evaluated with

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standardized 0.02 M HCl, using as indicator methyl red and bromophenol blue. The

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percentage of nitrogen was calculated using the equation “% Nitrogen = ((mL

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standardized acid – mL blank) x N of acid) x 1.4007) / weight of sample in grams”. The

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F-factor 6.25 was applied for the determination of the protein concentration in rice

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samples.

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Extraction of Phytoprostanes and Phytofurans. The PhytoPs and PhytoFs

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present in brown rice flours were extracted and clean-up by SPE, following the

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methodology previously optimized and validated in our own.15,23,24 Afterwards, samples

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were reconstituted and filtered through a 0.45-µm filter (Millipore, MA USA).

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UHPLC-ESI-QqQ-MS/MS Analysis. Chromatographic separation of PhytoPs

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and PhytoFs was performed using a UHPLC coupled with a 6460 triple quadrupole-

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MS/MS (Agilent Technologies, Waldbronn, Germany), using the analytical column

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BEH C18 (2.1 mm × 50 mm, 1.7 μm) (Waters, Milford, M.A.). The column

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temperatures were 6 °C (both left and right). The mobile phases consisted of Milli-Q-

174

water/ acetic acid (99.99:0.01, v/v) (A) and methanol/acetic acid (99.99:0.01, v/v) (B).

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The injection volume and flow rate were 20 μL and 0.2 mL min−1 upon the following

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linear gradient (time (min), %B): (0.00, 60.0%); (2.00, 62.0%); (4.00, 62.5%); (8.00,

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65.0%); and (8.01, 60.0%). An additional post run of 1.5 min was considered for

178

column equilibration. The spectrometric analysis was conducted in Multiple Reaction

179

Monitoring mode (MRM) operated in negative mode, assigning preferential MRM

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transition for the corresponding analytes. The ionization and fragmentation conditions

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were as follows: gas temperature 325 °C, gas flow 8 L min−1, nebulizer 30 psi, sheath

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gas temperature 350 °C, jetstream gas flow 12 L min−1, capillary voltage 3000 V, and

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nozzle voltage 1750 V, according to the most abundant product ions. Data acquisition

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and processing were performed using Mass Hunter software version B.04.00 (Agilent

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Technologies).24 The quantification of PhytoPs and PhytoFs detected in rice flour was

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performed using authentic standards according to standard curves freshly prepared as

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mentioned in the previous section. The selected reaction monitoring and chemical

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names are referred in Table 2 and the chemical structures are shown in Supplemental

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file 1.

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Statistical Analysis. All assays were developed in triplicate (n=3) and

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concentrations were provided as means ± standard deviation (SD). To assess the

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consequence of the foliar fertilization, an analysis of variance (ANOVA) and a multiple

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range tests (Tukey’s test) were carried out using treatment of fertilization and genotypes

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as sources of variation. It expresses the values of F-ratio and p-value. The F-statistic is a

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ratio of two variances. The analyses were carried out using IBM SPSS statistics 24.0

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(SPSS Inc., Chicago, IL, USA). Significant differences between means were considered

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at p0.05), exception made of the protein content in the variety ‘Guri-INTA’ that

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experienced a significant augment from 8.8 to 9.3% under foliar fertilization.

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When comparing yield, 1000-whole grain weight, and protein content of the five

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genotypes under study, it was found that the average yield under control conditions was

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961.22 g m-2, being not observed statistically significant differences between genotypes

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(p>0.05). This trend was also observed when monitoring the effect of foliar fertilization

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on yield that revealed that, even though the treatment applied increased by 7.2% the

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average yields means of all five genotypes, the differences observed were not

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statistically significant (Table 3). These results are in agreement with most authors that

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have found positive responses to foliar fertilization. Concerning this, the application of

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N concentrated near of panicle initiation was demonstrated as the most efficient

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practice, which favors the formation of a higher number of spikelet per panicle.25 On the

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other hand, Bhuyan et al. reported yield increases of up to 9.3% when applying urea,

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using the technique of foliar fertilizations.26 However, although in this regard, it was

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revealed a clear trend, the present study failed in identifying significant differences as a

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consequence of foliar fertilization in the two seasons that could be due to the application

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of the minimum dose recommended by the manufacturer, which could be insufficient.

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Regarding 1000-whole grain weight, it was observed that foliar fertilization

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induced significant differences between genotypes (Table 3). Hence, the higher value

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corresponded to ‘H475-3-1-1-2’, H458-41-1-1-1’, and ‘H484-9-1’ (23.41 g, on average)

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that surpassed the values recorded for ‘Guri-INTA’ and ‘Nutriar FCAyF’ by 19.00 g, on

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average. These data revealed that grain weight is a phenological feature weakly

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influenced by the environmental conditions. Indeed, rice grains size is physically

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restricted by the size of the hull and its weight, under most conditions, is a very stable

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varietal feature.27 These results were in agreement with Bezerra Barreto et al. that

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studied the effect of three seasons of application of N and four doses of N on five rice

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cultivars.25 Besides, Midorikawa et al. evaluated the effect of the spraying with

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ammonium chloride on the soil surface at a rate of 8 g m-2 on the ‘Niponbare’ cultivar

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and Figueroa studied the effect of Fertideg foliar fertilizer on rice cultivars.28,29 Upon

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none of these works it was reported differences in the weight of the grains due to the

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treatments applied. Possibly, the relative contribution of grains per panicle to grain yield

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is moderate, while that of grain is very low.

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The evaluation of the impact of foliar fertilization on the protein content evidenced

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that only ‘Guri INTA’ responded to the treatment applied increasing protein

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significantly in fertilized plants (by 6.4%, p=0.0273) compared to unfertilized controls

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(Table 3). Nowadays, it is already known that foliar fertilization produces contradictory

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effects regarding protein content in rice grain, since it depends on several factors, such

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as the moment of application and the initial nutrition status according to the soil

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features.5 In this regard, several authors have reported critical differences among

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cultivars featured by an average protein content, as a consequence of foliar fertilization,

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upon field trials developed in soils with similar composition.28 In addition, Bezus et al.

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and Vidal et al., evaluated genotypes featured by high protein content, and did not

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observe a statistically significant response to the nitrogen fertilization applied.30

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Additional studies by the same authors evidenced that the response varies according to

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the genotype, which has been justified because the applied N can be directed to other

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destinations different from grain, as yield or biomass, for which it is necessary to

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evaluate higher doses and other moments of application.30

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Significant differences were observed between cultivars under both no fertilization

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and foliar fertilization conditions (Table 3). Genotypes featured by high protein content

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(‘Nutriar FCAyF’, ‘H458-41-1-1-1’, ‘H475-3-1-1-2’, and ‘H484-9-1’) presented, on

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average, 11.1% dw, while ‘Camba INTA’ revealed a significantly lower content

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(9.1% dw). Thus, from these results, it was noticed that ‘Nutriar FCAyF’ was the

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genotype displaying the highest protein content. Indeed, this variety has been obtained

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and registered by the Rice Program, as a high-protein in grain variety that could

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contribute to improve the nutritional properties of this cereal.

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Total and Individual Phytoprostanes in Rice Grain. The evaluation of the

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abundance of total PhytoPs in the genotypes studied evidenced that their concentration

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decreased as follows: ‘H475-3-1-1-2’ (509.25 ng g-1 dw) > ‘Guri INTA’ (396.92 ng g-1

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dw) > ‘Nutriar FCAyF’ (385.23 ng g-1 dw) > ‘H484-9-1’ (346.43 ng g-1 dw) > ‘H458-

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41-1-1-1’ (309.16 ng g-1 dw). In this regard, recently, Pinciroli et al. evaluated the

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concentration of total PhytoPs in an array of rice varieties by studying 14 genotypes.31

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Upon this work, the highest concentration of total PhytoPs in whole grain flour

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corresponded to the variety ‘Arborio’ and the lowest to ‘Itape’, with the values 9.51 and

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32.89 ng g-1 dw, respectively. Concerning additional plant materials, and in order to get

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a rational understanding on the level of these compounds in rice, it is required to notice

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the abundance of total PhytoPs in other plant materials. In this regard, to date, the total

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PhytoPs level has been described in green olives (6.0-87.9 ng g-1 fw), vegetable oils

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(1.9-119.2 ng mL-1), wines and must (134.1-2160.0 y 21.4-447.1 ng mL-1, respectively),

283

and in 24 species of macroalgae (0.1-113.8 ng g-1 dw),17,32-34 in three types of raw

284

‘Pecan’ nuts showed the highest PhytoPs concentration, with a total of 7.8 ng g-1 fw,

285

followed by ‘Macadamia’ (6.5 ng g_1 fw) and ‘Walnut’ (5.5 ng g-1 fw).35 In almond

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kernels, the total PhytoPs concentration was in the range of 40.0-238.0 ng g-1 fw,16

287

banana passion fruit shell samples (23180 ng g-1 dw),36 in gulupa shell

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(67.6 ng g-1 dw), and gold berry 21659.8 ng g-1 dw.37 The comparison of the level of

289

PhytoPs in the various matrices allowed to describe that the total amount in rice grains

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is in the range of most vegetable foods (being surpassed only by tropical fruits of high

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water content and scarce consumption rate), which revealed this matrix as an especially

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interesting source of PhytoPs in the diet, given the high naturalization in the rice and

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rice flour diets.

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Apart from total PhytoPs, when profiling the individual compounds within this

295

oxylipins class, it was observed the presence of seven individual PhytoPs: 9-F1t-PhytoP,

296

ent-16-F1t-PhytoP + ent-16-epi-16-F1t-PhytoP, 9-epi-9-F1t-PhytoP, ent-9-epi-9-D1t-

297

PhytoP, ent-9-D1t-PhytoP, 16-B1-PhytoP, and 9-L1-PhytoP (Tables 4 and 5). In respect

298

to the abundance of the individual PhytoPs present in concentrations higher than the

299

limit of quantification, they could be ordered according to lowering average

300

concentrations under control conditions as follows: 16-F1t-PhytoP+ent-16-epi-16-F1t-

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PhytoP (213.25 ng/g dw) > 9-F1t-PhytoP (125.57 ng g-1 dw) > ent-9-epi-D1t-PhytoP

302

(22.66 ng g-1 dw) > 16-B1-PhytoP (12.61 ng g-1 dw) > 9-L1-PhytoP (8.69 ng g-1 dw) > 9-

303

epi-9-F1t-PhytoP (3.44 ng g-1 dw) > ent-16-epi-16-F1t-PhytoP (3.08 ng g-1 dw) (Tables 4

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and 5).

305

These results were in agreement with previous studies on the level of PhytoPs in a

306

range of rice flours, upon which was stated the predominant occurrence of 16-B1-

307

PhytoP (52.79 ng g-1 dw), ent-9-D1t-PhytoP (28.98 ng g-1 dw) and 9-L1-PhytoP (27.04

308

ng g-1) in rice bran of the indica subspecies.16 In addition, when evaluating the

309

information available in the literature on the presence of individual PhytoPs in edible

310

and non-edible plant material, it was stated that ent-16-F1t-PhytoP and ent-16-epi-16-

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F1t-PhytoP were also the most abundant compounds in dry melon leaves (1160.0 ng g-1)

312

and aged wine (133.8 ng mL-1).13,33 On the other hand, in rice grains, 9-F1t-PhytoP

313

ranked second, while this PhytoP has been described as the most abundant in olive oil

314

(5.2 ng mL-1),12 almonds (42.4 ng g-1),16 macroalgae (152.6 ng g-1),34 and in grape musts

315

(32.7-13.5 ng g-1).33 The PhytoPs 16-B1-PhytoP and 9-L1-PhytoP resulted in low in

316

carbonic maceration wine (4.11 ng mL-1, on average) and macroalgae (0.10 ng g-1, on

317

average),33,34 while ent-9-epi-9-D1t-PhytoP has been described in low concentrations

318

olive oil and almonds,12,16 and reported as almost absent in melon leaves.13 It is

319

important to notice that certain agronomic and industrial procedures may enhance the

320

PhytoPs content of fruits and derived vegetable oils. In this way, it may be possible to

321

“functionalize” plant products with higher concentrations of these compounds, which

322

are readily bioavailable and absorbed by the human body.18

323

The relative abundance of the individual PhytoPs in the plant materials evaluated so

324

far is of special relevance and indicates the challenge of getting a further insight in the

325

biological function of specific classes of PhytoPs, upon dedicated in vivo assays in the

326

frame of animal experimental models and clinical trials. So, it is of special relevance the

327

works developed by Barden et al. that measured, by using GC-MS, the content of F1-

328

PhytoPs in human plasma and urine samples after consuming flaxseed oil for four

329

weeks.19 This nutritional trial allowed them to describe an increase of the plasma

330

concentration of F1-PhytoPs; however, the pharmacokinetic curve and the physiological

331

effects on the human body remain underexplored,19 and require further characterization

332

in the near future, suggesting that the assessment of new food matrices deserves to be

333

developed to identify optimal dietary sources of these compounds.

334

15



335

Total and Individual Phytofurans in Rice Grain. When profiling PhytoFs in

336

the five varieties of rice included in the present work, the 3 PhytoFs monitored (ent-16-

337

(RS)-9-epi-ST-Δ14-10-PhytoF, ent-9-(RS)-12-epi-ST-Δ10-13-PhytoF, and ent-16-(RS)-

338

13-epi-ST-Δ14-9-PhytoF) were found in levels higher than the limit of detection of the

339

method (Table 6).

340

The concentration of PhytoFs decreased as follows: ent-16-(RS)-9-epi-ST-Δ14-10-

341

PhytoF (19.18 ng g-1 dw) > ent-16-(RS)-13-epi-ST-Δ14-9-PhytoF (10.48 ng g-1 dw) >

342

ent-9-(RS)-12-epi-ST-Δ10-13-PhytoF (3.46 ng g-1 dw) (Table 6). This decreasing order

343

coincides with that reported by Pinciroli et al. upon the analysis of different types of

344

flour and rice varieties under matching agro-climatic conditions.31 Moreover, these data

345

are also in agreement with Yonny et al. that observed ent-16-(RS)-9-epi-ST-Δ14-10-

346

PhytoF as the most abundant PhytoF in dry melon leaves, while ent-16-(RS)-13-epi-ST-

347

Δ14-9-PhytoF was not detected.13 However, when considering the diverse concentration

348

of total PhytoFs in the separate varieties assessed it was found the following decreasing

349

order of abundance: ‘Guri INTA’ (38.01 ng g-1 dw) > ’H458-41-1-1-1’ (37.11 ng g-1

350

dw) > ‘Nutriar FCAyF’ (35.92 ng g-1 dw) > ‘H475-3-1-1-2’ (35.24 ng g-1 dw) > ‘H484-

351

9-1’ (19.34 ng g-1 dw) (Table 6).

352

To date, there is limited information on the occurrence of PhytoFs in plant materials

353

and vegetable foods, concerning both profile and quantitative presence. Among few

354

articles providing information on this issue, Pinciroli et al. revealed differences between

355

flour types and rice varieties featured by concentrations ranging between 0.06 ng g-1dw

356

(for white grain flour obtained of the variety ‘Quebracho FA’) and 27.70 ng g-1 dw

357

(bran rice of the cultivar ‘Guri INTA’).31 Moreover, Cuyamendous et al. and Yonny et

358

al. reported the occurrence of these compounds in pine, nuts, chia, flaxseeds, and melon

359

leaves in concentrations ranging from 0.30 to 4400.00 ng g-1 dw.13,38 However, it has to

16


Page 16 of 34

Page 17 of 34


360

be noticed that the concentration of PhytoFs in the plant material evaluated has been

361

reported in levels significantly lowers relatively to those described for PhytoPs, which

362

could be related with the specific physico-chemical features of rice and rice flour,

363

accounting for very low water content.

364 365

Influence of Foliar Fertilization in Phytoprostanes and Phytofurans

366

Concentration. When analyzing the effect of foliar fertilization on the concentration

367

of PhytoPs and PhytoFs it was revealed that this practice modified their concentration

368

significantly, exception made of ent-9-epi-9-D1t-PhytoP that remained unaltered in

369

comparison with no treated controls. Overall, upon this work, it was noticed that foliar

370

fertilization causes a decrease of the concentration of plant oxylipins in the five

371

genotypes evaluated.

372

Similarly, according to diverse authors, PhytoPs and PhytoFs are generated in plant

373

species as a consequence of non-enzymatic peroxidation reactions catalyzed by free

374

radicals (ROS).32 Actually, biotic and abiotic stresses alter the balance between the

375

generation of ROS and the scavenging capacity, which entails the disturbance of the

376

redox homeostasis in cells.38 In this frame, probably, foliar fertilization could contribute

377

to prevent crops from a situation of nutritional stress. Indeed, increasing evidence

378

suggests that a proper mineral-nutrient status of plants contribute to enhance plants

379

resistance to environmental stress factors.8 In addition, according to Cakmak (2002),

380

fertilization increases the antioxidant defense mechanisms, resulting in a reduced photo-

381

oxidation of the chloroplast pigments and the stability of cell membranes.3 In this

382

regard, this author suggested that the improvement of the nutritional status of plants,

383

regarding the mineral nutrients Zn, K, Mg, and Bo, might contribute to lower the

384

production of ROS and thereby, to minimize the activity of NADPH oxidases, while

17



385

maintaining the photosynthetic transport of electrons.3 This is further supported by the

386

fact that micronutrients integrate the isoforms of the set of enzymes responsible for

387

detoxification ROS like CuZn-SOD, Mn-SOD, and Fe-SOD.7 These antioxidants

388

neutralize ROS contributing to maintain the integrity of the chloroplast membrane in

389

plants.6 This protection from the oxidative damage generated by the adequate supply of

390

minerals could be the cause of the lower generation of oxylipins.

391

Hence, the quantification of individual PhytoPs in grain of the five rice genotypes,

392

grown under control and foliar fertilization conditions, revealed significantly different

393

concentrations in control plants relatively to those fertilized. In this regard, resorting to

394

the analysis of variance, it was observed that foliar fertilization is enclosed to significant

395

and varied effects on the levels of diverse plant oxylipins. Table 4 shows the mean

396

concentrations of the compounds (9-epi-9-F1t-PhytoP, ent-9-epi-9-D1t-PhytoP, ent-9-

397

D1t-PhytoP, ent-16-F1t-PhytoP + ent-16-epi-16-F1t-PhytoP) that did not show significant

398

foliar fertilization x genotypes interaction in the two seasons. Indeed, these exhibited

399

equal modifications of their concentrations in all genotypes, while the compounds

400

featured by a significant foliar fertilization x genotypes interaction (9-F1t-PhytoP, 16-B1-

401

PhytoP, 9-L1-PhytoP, ent-9-(RS)-12-epi-ST-Δ10-13-PhytoF, ent-16-(RS)-13-epi-ST-Δ14-

402

9-PhytoF) are referred in Table 5.

403

In respect to the compounds that presented no significant foliar Fertilization x

404

genotype interaction (Table 4), the foliar fertilization decreased significantly (p

Statement of Foliar Fertilization Impact on Yield, Composition, and

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