Impact of Salicylic Acid Content and Growing Environment on

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Food and Beverage Chemistry/Biochemistry

Impact on phytoprostane and phytofuran (stress biomarkers) content of salicylic acid and growing environments on Oryza sativa L. M. Pinciroli, R. Dominguez-Perles, M. Garbi, Á. Abellán, C. Oger, T. Durand, J.M. Galano, F. Ferreres, and A. Gil-Izquierdo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04975 • Publication Date (Web): 02 Nov 2018 Downloaded from http://pubs.acs.org on November 2, 2018

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Journal of Agricultural and Food Chemistry

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Impact on phytoprostane and phytofuran (stress biomarkers)

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content of salicylic acid and growing environments on Oryza

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sativa L.

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Pinciroli, M.,† Domínguez-Perles, R.,‡,* Garbi, M.,† Abellán, A.,‡ Oger C.,§

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Durand. T.,§ Galano, J.M.,§ Ferreres, F.,‡ Gil-Izquierdo, A.‡,*

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†

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Universidad Nacional de la Plata. Calle 60 y 119 (1900) La Plata, Buenos Aires, Argentina

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‡

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and Technology, CEBAS-CSIC, Campus de Espinardo 25, 30100 Espinardo, Spain

Cátedra de Climatología y Fenología Agrícola, Facultad de Ciencias Agrarias y Forestales

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

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

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ENSCM, Montpellier, France

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Running title: Salicylic acid impact on the rice level of PhytoPs and PhytoFs

1 ACS Paragon Plus Environment


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ABSTRACT

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Phytoprostanes (PhytoPs) and phytofurans (PhytoFs) are oxylipins synthesized by

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non-enzymatic peroxidation of α-linolenic acid. These compounds are biomarkers

18

of oxidative degradation in plant foods. In this research, the effect of the

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environment and the supplementation with salicylic acid (SA) on PhytoPs and

20

PhytoFs was monitored resorting to UHPLC-ESI-QqQ-MS/MS on seven japonica

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rice genotypes. The plastic cover and the spray application with 1 and 15 mM of

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SA produced a reduction in the concentration of most of these newly established

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stress biomarkers (9-F1t-PhytoP, ent-16-F1t-PhytoP, ent-16-epi-16-F1t-PhytoP, 9-

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D1t-PhytoP, 9-epi-9-D1t-PhytoP, 16-B1-PhytoP, 9-L1-PhytoP, ent-16(RS)-9-epi-ST-

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

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Δ14-9-PhytoF) by 60.7%, on average. The modification observed in the level of

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PhytoPs and PhytoFs differed according to the specific oxylipins and genotype

28

demonstrated a close linkage between the genetic features and the resistance to

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abiotic stress, in some extent mediated by the sensitivity of plants to the plant

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hormone SA that participates in the physiological response of higher plants to stress.

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Thus, in plants exposed to stressing factors, SA contribute to modulate the redox

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balance, minimizing the oxidation of fatty acids and thus, the syntheis of oxylipins.

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These results indicated that SA could be a promising tool for the manage of the

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thermotolerance of rice crop. However, it remains necessary to study the

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mechanism of action of PhytoPs and PhytoFs in biochemical processes related to

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the defense of plants and define their role as stress biomarkers through a non-

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enzymatic pathway.

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KEYWORDS: Rice; Air temperature; Oxidative stress; Agronomical practices;

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Biomarkers


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ABBREVIATIONS: BHA butylated hydroxy anisole; PhytoPs phytoprostanes; PhytoFs

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phytofurans; ROS reactive oxygen species; SA Salicylic acid; SPE solid phase extraction;

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UHPLC-ESI-QqQ-MS/MS ultra-high performance liquid chromatography coupled to

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electrospray ionization and triple quadrupole mass spectrometry.



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INTRODUCTION

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Rice (Oryza sativa L.) is a tropical or subtropical crop featured by high adaptability to

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diverse environmental conditions.1 High temperature impairs grain ﬁlling, leading to loss

48

of yield for crops, such as rice through decreases in grain size, number, and quality. Thus,

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considering recent climate change, an understanding of the physiological processes

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responsible for these effects deserves to be further evaluated.2 To date, studies on japonica

51

varieties of rice developed in Japan have provided information on the optimum air

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temperature for ripening that ranges between 20 and 25 °C,2-3 while air temperature above

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30 °C causes deleterious effects on yield and nutritional quality, as well as to modify

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physicochemical features of the grain.4-5 Indeed, in the last decades, it has been described

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varietal differences regarding the sensitivity of rice plants to high temperatures.4

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Under normal physiological conditions, reactive oxygen species (ROS) are

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continuously produced in plants as a consequence of the cellular metabolism, especially

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in the frame of the photosynthetic process.6 Most of these conditions either increase of

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light-driven electron transport or restrict CO2 availability, augmenting ROS production

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in the chloroplast, associated to the Photosystems I and II, as well as in peroxisomes,

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associated to photorespiration.6 These radicals are very unstable and react rapidly with

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other groups or substances leading to cell or tissue injury.

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One of the main targets of the attack of free radicals is the membrane lipids, which

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results in the initiation of the enzymatic lipid peroxidation.7 In higher plants, ROS react

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with α-linolenic acid (ALA) in cell membranes, fundamentally, giving rise to

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phytoprostanes (PhytoPs). On the other hand, higher oxygen pressure has as a

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consequence the formation of an additional peroxidation products named phytofurans

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(PhytoFs) as a consequence of further oxidation after the primary cyclization of ALA.8


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Salicylic acid (SA) is a plant hormone responsible for the control of diverse

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processes, namely: germination, cell growth, floral induction, absorption of ions,

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respiration, rate of transpiration, net photosynthesis, stomatal conductance, flowering,

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senescence, response to abiotic stress, and essential in thermogenesis, as well as in

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resistance to diseases, while induces changes in leaf anatomy and chloroplast ultra-

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structure.9-10. Besides, this signaling hormone induces hypersensitive response and the

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expression of pathogenesis-related genes, as well as systemic resistance in plants,11

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closely related with plant defense by regulating ROS (H2O2 and O2.-) levels, as well as

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enzymatic (ascorbic acid peroxidase, superoxide dismutase, catalase, glutathione-S-

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transferase) and non-enzymatic antioxidants systems.12-13. The information reported so

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far on the physiological role of SA implies that could also contribute to the plants

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resistance against stressing factors by improving the mechanical barrier.14 On the other

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hand, SA induces the synthesis of phenylalanine ammonia lyase (PAL). The activation of

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this enzyme by SA seems to contribute to modulate PAL gene expression and activation.14

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The application of SA with agronomical purposes increases the endogenous content of

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SA and the expression of genes codifying to critical redox-regulating proteins (NPR1 and

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PR), responsible, for instance, for the regulation of the expression of TOP2 gene, closely

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related to the plant resistance against abiotic stress.15 However, the data available turn

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into intriguing the effect of exogenous SA on resistance in plants against biotic and abiotic

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stress.16

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In respect to the relationship between salicylic acid and PhytoPs/PhytoFs, although

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the biological functions of these oxylipins before their conversion to jasmonic acid are

91

less understood, to date, it has been described their contribution to a number of biological

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activities that include the activation of diverse enzymes, the regulation of defense genes,

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and the accumulation of antimicrobial compounds in plants.17 The pathways in which



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PhytoPs and PhytoFs are involved are distinct from the responses induced by jasmonate.

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In this regard, recently it has been demonstrated that the oxylipins are generated before

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the accumulation of SA, in response to the activation of MPK3 and MPK6 induced by the

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guard cell lipoxygenase LOX1.18

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In the light of the information available, this research seeks to deepen the current

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understanding of the interrelation of environment (open field and plastic cover) widely

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managed in the local area and the application of salicylic acid with the qualitative and

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quantitative presence of new and powerful markers of abiotic stress (PhytoPs and

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PhytoFs) in higher plants, by advanced chromatographic and spectrometric analyses in

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one well characterized genotype (‘Yerua’) and six advanced lines (R/03-5x desc/04-52-

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1-1’, ‘R/03-5x desc/04-45-1-1’, ‘Amaroo x desc /08-1-1-1-2’, ‘R/03-5x desc/04-27-3-1’,

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‘R/03-5x desc/14-1-1-1’, and ‘H489-5-1-2’) belonging to the japonica rice variety. This

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genetic diversity were obtained from the rice breeding program of the National University

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“de la Plata” (Argentina), which are implanted in the local area due to their agronomic

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features within a group of genotypes of the commercial type length-width.

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MATERIALS AND METHODS

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Chemical and reagents. The standards of PhytoPs and PhytoFs were synthesized

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as previously described,8,19-23 and provided by the Institut des Biomolécules Max

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Mousseron (IBMM, Montpellier, France). N-hexane was purchased from Panreac

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(Barcelona, Spain), butylated hydroxyanisole (BHA), bis–tris, and SA were

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obtained from Sigma–Aldrich (St. Louis, MO, USA) and all LC–MS grade solvents

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used to complete the analytical tasks described in the present work were from J.T.

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Baker (Phillipsburg, NJ, USA). Water was treated in a milli-Q water purification

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system from Millipore (Bedford, MA, USA). The solid-phase extraction (SPE)


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cartridges used (Strata X-AW, 100 mg/3 mL) were from Phenomenex (Torrance,

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CA, USA).

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Plant samples and crop management. The field trial was set up in the experimental

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field ‘Julio Hirschhorn’ (34º52'S, 57º57'W, 9.8 m of altitude, la Plata, Buenos Aires,

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Argentina). This field experiment included the widespread variety in the producing area

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‘Yerua PA’ (Yerua), and the advanced lines ‘R/03-5x desc/04-52-1-1’ (L1), ‘R/03-5x

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desc/04-45-1-1’(L2), ‘Amaroo x desc /08-1-1-1-2’ (L3), ‘R/03-5x desc/04-27-3-1’ (L4),

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‘R/03-5x desc/14-1-1-1’ (L5), and ‘H489-5-1-2’ (L6), all them belonging to the subspecie

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japonica. The rice varieties were sourced by the Rice Breeding Program of the Agriculture

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and Forestry Sciences Faculty, of the National University “de la Plata”. The experimental

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design included randomized blocks with 3 repetitions. Dry-land seeding was done

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manually at a rate of 350 seeds m-2 in 0.20 m lines within plots of 5 m2. The assay was

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conducted applying flood irrigation for 30 days after emergence. Weeds were controlled

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with bispiribac sodium. Three experimental conditions were included in the assay; no SA

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application (SA0, control condition), application of 1 mM SA (SA1), and application of

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15 mM SA (SA15). These treatments were sprayed 10 and 17 days after anthesis. The SA

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solutions were prepared in distilled water with 50 ppm Tween-20 surfactant. The plots

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were sprayed at late afternoon hours, under wind conditions of less than 10 km hr-1.

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Regarding the environmental conditions, the arable area evaluated was exposed to

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two diverse growing condition: crop covered with plastic of 100 μm thickness (EP)

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between the second SA application and harvest and open crop (E). Temperature values

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were recorded by an XR440 Pocket TM data logger (Pace Scientific Inc. Charlotte, NC,

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USA) every 30 minutes during the ripening. Two temperature sensors (PT940, Pace



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Scientific Inc.) were located in the canopy, at the panicles height (0.90 m of the ground)

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in both EP and E crop systems.

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Each rice variety samples were collected and threshers manually at maturity

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according to standard of industrial processes. The kernels were dried in an oven at 40 °C,

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until constant (14.0%) de humidity. Paddy grain samples (100 g) were flaked to obtain

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brown rice that was ground to a fine powder by milling in a Cyclone Mill and passing the

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powder through 0.4 mm mesh; afterwards samples were stored at refrigeration, until

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analysis.

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Extraction and UHPLC-ESI-QqQ-MS/MS analysis of rice phytoprostanes and

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phytofurans. To obtain the PhytoPs and PhytoFs extracts from rice flours it was applied

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tye methodology reported by Pincirolli et al.24 The extracts obtained were cleaned up by

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SPE as previously described.25. The quantification of the extracted plant oxylipins was

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achieved using freshly standard curves of authentic standards.

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The Chromatographic resolution of the PhytoPs and PhytoFs previously extracted

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was done by applying the instrumentation and methodology described by Collado-

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Gonzalez et al. and Domínguez-Perles et al.25,26 The identification and quantification of

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the individual PhytoPs and PhytoFs was developed applying Multiple Reaction

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Monitoring (MRM) in negative mode, assigning quantification and confirmation MRM

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transition for each analyte (Table 1).

163 164

Statistical analysis. All extractions were performed in triplicate (n=3) and results

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were presented as means ± SD. The statistical analysis was completed using Statgraphics

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Centurion XVI statistic software (StatPoint Technologies Inc., Warrenton, VA, USA) by


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which one-way analysis of variance (ANOVA) and multiple range test (Tukey’s test)

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were developed. Significant differences were set up at p ‘R/03-5x desc/04-45-1-1’ (L2) (420.4 ng g-1 dw) > ‘R/03-5x desc/04-27-3-1’ (L4)

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(368.0 ng g-1 dw) > ‘R/03-5x desc/14-1-1-1’ (L5) (257.6 ng g-1 dw) > ‘Amaroo x desc

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/08-1-1-1-2’ (L3) (180.2 ng g-1 dw).



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These levels were inferior than the observed in different rice genotypes belonging to

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the subspecies indica, as they ranged between 509.3 and 309.2 ng g-1 dw.24 However,

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other authors have found modifications in the concentration of total PhytoPs according to

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the variety. In this regard, Barbosa et al. (2015), upon a study including 24 species of

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macroalgae, found the highest values in the species Saccharina latissima and the lowest

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in Asparagopsis armata (13.8 and 0.1 ng g-1 dw, respectively).27 Additionally, when

197

studying 11 diverse almond cultivars exposed to an array of hydric stress growing

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condition, Carrasco-Del Amor et al. (2015) described maximum values in the cv.

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‘Blanqueta’ and minimus in the cv. ‘Garriges’, with values of 238.1 and 40.3 ng g-1,

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respectively.28 Finally, in 2017, Pinciroli et al. studied 14 genotypes of rice, stating the

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maximum level of PhytoPs in the subespiecies ‘Itape’ (variety japonica), with values of

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9.2, 32.9, and 67.1 ng g-1 for white grain flour, brown grain flour, and bran flour,

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respectively.24 In this work, upon profiling PhytoPs in rice flour from the seven varieties

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under study, independently on the growing environmental condition (open field or plastic

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cover) and salicylic treatment, it was observed the following decreasing average

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concentration of individual PhytoPs: ent-16-F1t-PhytoP + ent-16-epi-16-F1t-PhytoP

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(333.8 ng g-1 dw) > 9-F1t-PhytoP (172.5 ng g-1 dw) > 9-epi-9-F1t-PhytoP (164.0 ng g-1

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dw) > 9-D1t-PhytoP (30.5 ng g-1 dw) > 16-B1-PhytoP (PP6) (6.4 ng g-1 dw) > 9-L1-PhytoP

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(4.5 ng g-1 dw) > 9-epi-9-D1t-PhytoP (4.1 ng g-1 dw) (Table 2). Indeed, this trend was also

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observed when evaluating the isolated efect of the salicylic acid treatments on these plant

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oxylipins (Fig. 1).

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When comparing the abundance of the separate PhytoPs in the variety ‘Yerua’ and

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the six advanced genotypes evidenced that, the overall analysis, developed independently

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on the environmental and salicylic acid treatments effects, evidenced that, on average,

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‘Yerua’ displayed the highest concentration of 9-F1t-PhytoP, ent-16-F1t-PhytoP + ent-16-


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epi-16-F1t-PhytoP, 9-epi-9-F1t-PhytoP, 9-epi-9-D1t-PhytoP, 9-D1t-PhytoP, 16-B1-PhytoP,

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and 9-L1-PhytoP (353.3, 803.8, 445.1, 6.3, 75.3, 17.7, and 11.8 ng g-1 dw, respectively).

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These values surpassed the level of the variety exhibiting the second higher concentration,

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the advanced genotype L1, from 7.9 to 46.3%, and the advanced genotypes L2 to L6,

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which remained in similar lower levels, from 46.3 to 86.2% (Table 2).

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The comparison of the results obtained with other plant foods suggests that F1-

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PhytoPs series is also the most abundant in dry melon leaves Sweet Ball cv. Early Spring

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cv. (2740.0 and 2340.0 ng g-1, respectively),29 in aged wine and grape musts (210.7 and

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18.0 ng mL-1, on average, respectively),30 in olive oil (8.1, on average),31 in almonds (90.6

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ng g-1, on average),28 and in macroalgae (2.7 ng g-1, on average).27

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In addition to PhytoPs, the evaluation of the array of rice grains on the occurrence of

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plant oxylipins was also focused on PhytoFs. The average concentration of the individual

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PhytoFs in the separate genotypes of rice evidenced, the following decreasing levels: ent-

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16(RS)-9-epi-ST-Δ14-10-PhytoF (PF1) (9.7 ng g-1 dw) > ent-16(RS)-13-epi-ST-Δ14-9-

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PhytoF (PF3) (5.5 ng g-1 dw) > ent-9(RS)-12-epi-ST-Δ10-13-PhytoF (PF2) (2.5 ng g-1 dw)

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(Fig. 2). These values matched with that reported by Pinciroli et al. (2017) in an array of

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diverse rice flours obtained from varieties belonging to japonica and indica subspecies.24

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Again, when comparing the concentration of PhytoFs in the range of rice grain under

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study, it was noticed ‘Yerua’ was featured by the highest content of ent-16(RS)-9-epi-ST-

235

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

236

PhytoF (21.0, 5.2, and 12.0 ng g-1 dw, respectively), while the advanced genotypes L1,

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L2, and L6 presented lower concentrations (10.1, 2.5, and 5.8 ng g-1 dw, respectively).

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The remaining genotypes L3, L4, and L5 were found in similar lower levels with average

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the concentrations 5.6, 1.5, and 2.9, ng g-1 dw, respectively (Table 2).



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In all cases, significant differences between genotypes were revealed (Table 2).

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These results are fairly consistent with previous reports available in the literature that

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indicate that there are genetic varietal differences in response to high critical

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temperatures,4,32 according to their ability to regulate redox homeostasis in cells.

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Pinciroli et al. (2017) upon the study of diverse genotypes of rice discerned the lower

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concentration of total PhytoFs, on average, of the 14 genotypes 0.7, 4.4, and 10.7 ng g-1

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dw, regarding white grain flour, brown grain flour, and bran flour.24 Furthermore,

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PhytoFs have been also studied in few additional plant matrices. In this regard,

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Cuyamendous et al. (2016) reported the occurrence of these compounds in nuts and

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vegetable oils ranging between 0.30 and 9.00 ng g-1. In the same way, Yonny et al. (2016)

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described their occurrence in leaves of melon in which are found at the concentration

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2355.0 ng g-1 dw.29, 33

252 253

Environmental effect on the level of phytoprostanes and phytofurans in rice. The

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average temperatures during ripening were 20.3 and 21.8 °C in the E and EP systems,

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respectively, while the results retrieved revealed plastic covered providing less stressful

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growing conditions to the genotypes assessed. Rice is a tropical or subtropical species

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that in the experimental procedure described in the present work was grown under

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suboptimal conditions, in terms of thermic supply. The warm environment provided by

259

the plastic cover was found favorable for the development of all genotypes. Heat stress

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entails a myriad of cellular events in plants when temperature augment beyond a threshold

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level during a lapse of time that causes irreversible damage to plants.34,35 In this regard,

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previous studies demonstrated that optimal air temperature for rice ripening, in plants

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belonging to japonica varieties, ranges from 20 to 25 °C, while higher or lower

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temperatures may reduce significantly grain yield.3,36 Hence, so that damages by high


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temperatures turn out to be severe, these must last in the time. For instance, a period of

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20 days with temperatures above 27 °C, during ripening, seems to be enough to decrease

267

grain production, grain weight, and protein content, as well as to augment chalkiness.37

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On the other hand, 15 days at 30 °C has been demonstrated as a growing environmental

269

condition capable to reduce significantly rice yield and appearance quality, while change

270

grain shape.38,39 Besides, higher temperatures (35 °C for 5 days) cause damage in the

271

phenological phases and infertility of the spikelets in rice plants.36 In our study, high

272

temperatures were recorded during a 8 and 6 hours in cover plastic (EP) and opem field

273

(E) environments, respectively (Supplemental file 1). These conditions caused no harmful

274

effect, contrary to what was expected accordingly to the information available in the

275

literature.

276

When evaluating the differential response of rice genotypes to the environment in

277

crops set up under EP or E conditions, concerning the level of individual PhytoPs and

278

PhytoFs, it was found that, overall, EP growing rice reduced significantly (p 225.1

Negative

327.1 > 283.2Z

Negative

327.1 > 225.1Y

Negative

327.2 > 272.8

Negative

327.2 > 171.0

Negative

325.2 > 307.2

Negative

325.2 > 134.9

Negative

325.2 > 307.3

Negative

325.2 > 134.7

Negative

307.2 > 223.2

Negative

307.2 > 235.1

Negative

307.2 > 185.1

Negative

307.2 > 196.7

Negative

343.9 > 209.0

Negative

343.9 > 201.1

Negative

344.0 > 300.0

Negative

344.0 > 255.9

Negative

343.0 > 171.1

Negative

343.0 > 97.2

Phytoprostanes PP1Z

PP2

PP2

PP3

PP4

PP5

PP6

PP7

9-F1t-PhytoPX Ent-16-F1t-PhytoP

1.712

Ent-16-epi-16-F1t-PhytoPX 9-epi-9-F1t-PhytoP

1.583

1.785

9-epi-9-D1t-PhytoP

2.022

9-D1t-PhytoP

1.791

16-B1-PhytoP

2.620

9-L1-PhytoP

3.079

Phytofurans PF1

PF2

PF3

Ent-16(RS)-9-epi-ST-Δ14-10PhytoF



1.501

0.906

1.523

Z PP1, 9-F -PhytoP; PP2, ent-16-F -PhytoP + ent-16-epi-16-F -PhytoP; PP3, 9-epi-9-F -PhytoP; PP4, 1t 1t 1t 1t 9-epi-9-D1t-PhytoP; PP5, 9-D1t-PhytoP; PP6, 16-B1-PhytoP; PP7, 9-L1-PhytoP, PF1, ent-16(RS)-9-epiST-Δ14-10-PhytoF; PF2, ent-9(RS)-12-epi-ST-Δ10-13-PhytoF; and PF3, ent-16(RS)-13-epi-ST-Δ14-9PhytoF. Quantification transition. Y confirmation transition. X coeluting diasteroisomers quantified together

609



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610 Table 2. Average values of each phytoprostane and phytofuran (ng g-1 dw) in rice (Oryza sativa L.) genotypes Genotype

Phytoprostanes

Phytofurans

PP1 y

PP2

PP3

PP4

PP5

PP6

PP7

PF1

PF2

PF3

‘Yerua’

353.3 ± 33.3 a

803.8 ± 67.3 a

445.1 ± 73.5 a

6.3 ± 0.7 a

75.3 ± 9.6 a

17.7 ± 1.3 a

11.8 ± 0.8 a

21.0 ± 3.2 a

5.2 ± 0.5 a

12.0 ± 1.3 a

L1

238.0 ± 25.5 b

732.3 ± 65.0 a

396.7 ± 34.8 a

5.8 ± 0.8 b

54.0 ± 4.1 a

9.5 ± 1.1 b

7.1 ± 0.5 b

11.9 ± 1.3 b

3.3 ± 0.3 b

6.9 ± 0.5 b

L2

91.9 ± 10.8 c

222.6 ± 27.6 b

70.6 ± 7.4 b

4.1 ± 0.4 c

23.1 ± 1.3 c

4.6 ± 0.4 c

3.6 ± 0.2 c

9.4 ± 0.7 c

2.1 ± 0.3 c

5.4 ± 0.2 c

L3

70.4 ± 13.1 c

58.6 ± 7.8 c

31.9 ± 5.1 b

2.5 ± 0.3 d

11.4 ± 1.1 c

3.4 ± 0.6 c

2.0 ± 0.2 e

6.8 ± 1.1 d

1.7 ± 0.3 c

3.7 ± 0.4 d

L4

43.2 ± 16.1 c

186.2 ± 36.9 cb

84.8 ± 27.7 b

3.0 ± 0.4 d

15.8 ± 2.7 c

2.9 ± 0.6 c

2.3 ± 0.3 de

5.2 ± 0.8 d

1.5 ± 0.2 c

2.8 ± 0.3 de

L5

71.8 ± 18.8 c

124.5 ± 17.4 cb

40.4 ± 6.9 b

3.4 ± 0.4 cd

13.4 ± 1.6 c

2.4 ± 0.3 c

1.7 ± 0.2 e

4.9 ± 0.8 d

1.4 ± 0.2 c

2.3 ± 0.3 e

L6

107.3 ± 20.7 c

209.0 ± 23.8 b

78.5 ± 3.5 b

3.9 ± 0.4 c

20.2 ± 2.2 c

4.0 ± 0.4 c

3.2 ± 0.2 cd

8.9 ± 0.6 c

2.1 ± 0.2 c

5.1 ± 0.2 c

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p-value (ANOVA) y

PP1, 9-F1t-PhytoP; PP2, ent-16-F1t-PhytoP + ent-16-epi-16-F1t-PhytoP; PP3, 9-epi-9-F1t-PhytoP; PP4, 0.49-epi-9-D1t-PhytoP; PP5, 9-D1t-PhytoP; PP6, 16-B1-PhytoP; PP7, 9-L1-PhytoP,

PF1, ent-16(RS)-9-epi-ST-Δ14-10-PhytoF; PF2, ent-9(RS)-12-epi-ST-Δ10-13-PhytoF; and PF3, ent-10.66(RS)-13-epi-ST-Δ14-9-PhytoF. genotypes within a column followed by distinct letters are significantly different at p

Impact of Salicylic Acid Content and Growing Environment on

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