Variation of polyphenolic composition, antioxidants and physiological characteristics of dill (Anethum graveolens L.) as affected by bicarbonate-induced iron deficiency conditions

Industrial Crops & Products 126 (2018) 466–476

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Variation of polyphenolic composition, antioxidants and physiological characteristics of dill (Anethum graveolens L.) as affected by bicarbonateinduced iron deficiency conditions Hanen Waslia,b, Nahida Jelalic, Artur M.S. Silvad, Riadh Ksourib, Susana M. Cardosod,

T

⁎

a

Université de Tunis El Manar, Faculté des Sciences de Tunis, 2092, Tunis, Tunisie Laboratoire des Plantes Aromatiques et Médicinales, Centre de Biotechnologie de Borj-Cédria, BP 901, 2050, Hammam Lif, Tunisie c Laboratoire des Plantes Extremophiles, Centre de Biotechnologie de Borj-Cédria, BP 901, 2050, Hammam Lif, Tunisie d QOPNA/LAQV-REQUIMTE & Department of Chemistry, University of Aveiro, 3810-193, Aveiro, Portugal b

A R T I C LE I N FO

A B S T R A C T

Keywords: Fe deficiency Anethum graveolens Photosynthetic performance Secondary metabolites Antioxidant activity UHPLC-DAD-ESI-MSnanalysis

This study aimed to investigate the effects of bicarbonate-induced iron deficiency on growth, photosynthetic performance, secondary metabolite content, and related antioxidant capacity in seedlings of Anethum graveolens (L.) grown under controlled conditions in the presence of iron (+Fe) or in induced-iron deficient (+Fe + Bic) media. The main results have shown that dill shoot and plant biomass production significantly decreased under Fe deficiency conditions, whereas that of roots was not affected by such constraint. Moreover, Fe deficiency resulted in a significant reduction of chlorophyll and Fe concentration. Interestingly, when grown under Fe deficiency conditions, A. graveolens was able to increase its shoot iron use efficiency (FeUE). In addition, levels of individual and of total phenolic compounds were shown to be raised in roots as well in leaves. This fact was accompanied by an increased antioxidant activity, as measured through distinct in vitro methods (ABTS%+, O2%−, HO%, FRAP and ORAC). The overall data suggests that A. graveolens was able to maintain plant growth and to preserve adequate chlorophyll synthesis under iron-limiting conditions, probably due to its better Fe-use efficiency. The species was also able to modulate secondary metabolites metabolism and its antioxidant capacity, in order to minimize the deleterious effects of reactive oxygen species.

1. Introduction Iron (Fe) is micronutrient which is essential for plant major metabolic processes and the energy-yielding electron transfer reactions of respiration and photosynthesis (Aisen et al., 2001). Despite its abundance in the Earth’s crust, this element is not readily available for plants under oxygenated conditions. The major factor disturbing acquisition of Fe by plants is the soil pH. In fact, high pH makes Fe, which is mainly present in its oxidized form in well aerated soils, less available thus leading the concentration of free Fe in soil solution far below that required for optimal growth (Bavaresco and Poni, 2003). To cope with Fe acquisition, plants have extended two main strategies: strategy I, observed in dicotyledons and non-graminaceous plants and based on the reduction of Fe3+ to Fe2+ that is then transported into the cell by a

specific transmembrane transporter (IRT1); and strategy II, in grasses, where particular Fe(III) chelators (phytosiderophores) are exuded and subsequently transported inside the cell (Curie and Briat, 2003). In addition to these biochemical mechanisms, strategy I also involves physiological, developmental and metabolic mechanisms intended at adapting the plant to altering levels of available Fe resources. In the Mediterranean area, including Tunisia, calcareous soils are frequent (Jelali et al., 2010). This soil particularity can compromise the growth and development of several Tunisian edible crops, which commonly face Fe deficiency-induced chlorosis. At the cellular level, Fe deficiency induces plant oxidative stress, mainly by impairing the electron transport chain functionality both in mitochondria and chloroplasts (Murgia et al., 2015). Therefore, the disruption of the chloroplast, mitochondria, peroxisomes, endoplasmic

Abbreviations: CID, collision-induced dissociation; Chl, chlorophyll; DAD, diode array; ESI–MS, electrospray ionization–mass spectrometry; LC, liquid chromatography; MS, mass spectrometry; MSn, tandem mass spectrometry; UHPLC, ultra high-performance liquid chromatography; ROS, reactive oxygen species; TFC, total flavonoid content; TPC, total phenolic content; 1,4-DCQA, 1,4-di-O-caffeoylquinicacid; CA, caffeic acid; FQA, feruloylquinic acid; I3Glu, isorhamnetin-3-O-glucuronide; Q3Glu, quercetin-3-O- glucuronide; Q3R, quercetin-3-O-rutinoside ⁎ Corresponding author. E-mail address: [email protected] (S.M. Cardoso). https://doi.org/10.1016/j.indcrop.2018.10.007 Received 23 June 2018; Received in revised form 27 September 2018; Accepted 1 October 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.

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Fe availability, the phenolic profile, along with the effect of some biological properties in terms of antioxidant effectiveness. To our knowledge, there are no previous literature dealing with the physiological behavior of dill in responses to Fe deficiency, or the impact of such deficiency on phenolic compound accumulation and their related biological activities. Such information will be crucial for a better understanding of the physiological and biochemical behavior of this species in response to Fe deficiency, along with a better valorization of this underutilized species in a global goal of new Fe-efficient genotypes screening programs.

reticulum, and plasma membranes metabolism (DelRío et al., 2006; Foyer and Noctor, 2000) raises the production of reactive oxygen species (ROS), such as superoxide (O2%−), hydrogen peroxide (H2O2) and hydroxyl radicals (HO%) (Chou et al., 2011). ROS are highly reactive and lead to cell damage, a process known to be involved in several plant diseases, as well as in senescence (Falleh et al., 2008). In plant cells and tissues, ROS can react with biological molecules such as DNA, proteins or lipids, thus generating mutations and membrane damage (Navarro et al., 2006). To limit damages and face oxidative stress, plants usually respond through improved synthesis of several secondary metabolites biogenerated via the phenylpropanoid pathway such as non-enzymatic system. Polyphenols are among the most active secondary metabolites implicated in response to Fe deficiency in strategy I plants. These metabolites are considered potential Fe chelators owing to their chemical structure. Indeed, due to their basic structure i.e., the presence of an aromatic ring with one or more hydroxyl groups, they exhibit important antioxidant properties and are also able to chelate some transition metals (Huang et al., 2005). On the other hand, the interest focused on antioxidant properties of plant-derived foods and medicinal plants has expanded for the last years, since antioxidants are involved in protection of human wellbeing. As well, plant extracts with antioxidant properties are becoming more and more prominent for the food industry, where, among other abilities, they are considered as a potential “natural” alternative to synthetic antioxidants (Lee et al., 2017; Sharifi-Rad et al., 2018). Thus, the appraisal of antioxidant properties of traditional medicinal plants, which are extensively utilized, is an essential concern in the quest for new wellsprings of natural antioxidants (Falleh et al., 2011; Giorgi et al., 2009; Mishra et al., 2018; Ramírez-Atehortúa et al., 2018; Salehi et al., 2018). Polyphenol biosynthesis and accumulation are commonly stimulated in response to biotic/abiotic stresses (Naczk and Shahidi, 2004), including Fe deficiency. Thus, deficient Fe-stressed plants might represent potential sources of polyphenols valuable for economical exploitation. However, as environmental constraints have contrasted effects on polyphenol yield, i.e., the augment polyphenol concentration in the tissues are usually followed by a restrict biomass production (Sreenivasulu et al., 2002). Optimal polyphenol yield would be obtained using stress-tolerant species (Bettaieb-Rebey et al., 2012). Tunisia is gifted with a very rich flora including a diversity of aromatic and medicinal plants. These plants constitute natural sources of active biomolecules with several nutritional and therapeutic properties. Among Tunisian flora exists A. graveolens i.e. dill (Apiaceae family, Umbelliferae) which is well cultivated in the Mediterranean regions, Europe and Central Southern Asia and frequently used for flavoring and seasoning of assorted foods such as pickles, salads, sauces and soups (Jana and Shekhawat, 2010). Its fresh or dried leaves are used for boiled or fried meats and fish, in sandwiches and fish sauces, as well as an essential ingredient of sour vinegar. Besides, this species has been used in traditional medicine for digestive disorders as carminative, as lactationstimulating, antispasmodic, antihyperlipidemic and antihypercholesterolemic (Oshaghi et al., 2017). Phenolic compounds isolated from A. graveolens are considered to be responsible for its antioxidant activity while the volatile aroma compounds make of it an excellent flavoring agent (Jana and Shekhawat, 2010). Recent reports have shown the presence of genotypic-dependent variability of plant responses to low soil Fe availability, both among forage legume (Jelali et al., 2017) and as well as other species (Jiménez et al., 2009; Kabir et al., 2015). However, little attention was paid to sweet varieties subjected to metal deficiency. In this context, dill represents an important case of study, owing to its nutritive, medicinal and therapeutic values known around the world and because this is widely consumed in Tunisia by humans and animals as well. Thus, this study aims, on one hand, to evaluate the impact of Fe lacking on growth activity, iron status and photosynthetic capacity of dill and, on other hand, to identify the responses of secondary metabolism induced by low

2. Material and methods 2.1. Solvents and reagents Phosphate buffer saline (PBS) reagents (sodium salt, sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate), iron(II)sulfate, potassium hexacyanoferrate (III), iron chloride(III), diammonium salt, 2,3-terc-butil4-hidroxianisol(BHT), trichloroacetic acid (TCA), 2,2-diphenyl-1-picrylhydrazyl and gallic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA), while thiobarbituric acid (TBA) were purchased from Acros Organics (Geel, Belgium). Ascorbic acid, formic acid, Folin–Ciocalteu reagent, sodium carbonate, sodium phosphate, potassium hydroxide and ethylenediamine tetraacetic acid (EDTA) were purchased from Panreac (Barcelona, Spain). Hydrogen peroxide and sodium hydroxide were purchased from Fisher Scientific (Hampton, USA). Desoxyribose was purchased from Alfa Aesar (Massachusetts, USA) and mannitol from Merck (New Jersey, USA). Solvents including ethanol and methanol of high-performance liquid chromatography (HPLC) purity were purchased from Lab-Scan (Lisbon, Portugal). The phenolic standard chlorogenic acid was purchased from Acros Organics (Geel, Belgium). Dicaffeoylquinic acid, caffeic acid, quercetin-3-O-rutinoside, ferulic acid, isorhamentin-3-O-rutinoside and kaempferol-3-Oglucosidewere obtained from Extrasynthese (Genay Cedex, France).

2.2. Plant material, growth conditions and harvesting Seeds of A. graveolens were collected from provenance Manouba (36°51′02″Nord 9° 56′ 10″ East, Tunisia). Seeds were disinfected with a saturated solution of calcium hypochlorite (30%) for 2 min, and then abundantly rinsed in distilled water. After a 4h-imbibition phase, they were germinated for 10 days at 19 °C in Petri dishes with filter paper regularly moistened with 0.1 mM CaSO4. Ten-day-old seedlings were then transferred to a half strength aerated nutrient solution for 20 days and then similar sized seedlings were selected and cultured as groups of 10 plants in 10 L of full strength aerated nutrient solution. The composition of nutrient solution, as determined by the methodology described by Arnon and Hoagland (1940), was: 1.25 mM Ca(NO3)2, 1.25 mM KNO3, 0.5 mM MgSO4, 0.25 mM KH2PO4 and 10 μM H3BO3, 1μM MnSO4, 0.5 μM ZnSO4, 0.05 μM (NH4)6Mo7O24 and 0.4 μM CuSO4. Two treatments were established for 14 days as follows: control (presence of iron at 30 μM: +Fe) at pH 6.0and indirect iron deficiency (30 μM Fe + 0.5 g L−1CaCO3 + 10 mM NaHCO3: +Fe + Bic) for which the pH was 8.2. Iron was supplied in the form of Fe(III)-EDTA. Hydroponic cultures were maintained in a growth chamber with a day/ night regime of 16/8 h, 24 °C/18 °C regimes, photosynthetic photon flux density (PPFD) of 200 μmol m−2 s−1 at the plant level and a relative humidity of 70%. Nutrient solutions were renewed weekly and continuously aerated with an air-in floating pump linked to capillaries introduced into each pot. At the harvest (after 14 days of treatment), shoots and roots were separated, rinsed three times with cold distilled water and blotted between two layers of filter paper.

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TPC content was assessed using an ELX800 microplate reader (Bio-Tek Instruments, nc; Winooski, VT, USA) at 750 nm. The amount of total phenolic compounds was expressed as gallic acid equivalent (mg GAE)/ g dried weight of plant material, using a calibration curve of gallic acid as standard (0.001–0.01 μgmL−1).

2.3. Chlorophyll concentration For the measurement of chlorophyll concentration, weighed fresh leaves (200 mg) were grinded using a mortar and pestle and immersed in 10 mL of 100% acetone. Samples were then homogenized with the BBraun type homogenizer (Labequip, Markam, ON, Canada) at 1000 rpm for one minute. The homogenate was filtered through two layer of cheesecloth, and was centrifuged at 2500 rpm for 10 min. The supernatant was separated and placed in quartz cuvettes and absorbance measured against a blank of 100% acetone at 2 wavelengths (662 nm and 645 nm, used as the peak absorbances of chlorophyll-a and chlorophyll-b). The total amount of chlorophyll-a and chlorophyll-b were then calculated according to the formulas of Lichtenthaler and Wellburn (1985).

2.8.2. Total flavonoid content (TFC) Total flavonoids of the hydromethanolic extracts were measured using a colorimetric assay developed by Gajula et al. (2009). An aliquot of the samples or (+)-catechin standard was added to test tubes containing 7.5 μL of NaNO2 solution (5%) and mixed for 5 min. Then, 15 μL of a freshly prepared 10% AlCl3solutionwas added. After 5 min at ambient temperature, 0.5 mL of 1 M NaOH was added. The final volume was adjusted to 2.5 mL with distilled water and thoroughly mixed. Absorbance of the mixture was determined at 510 nm against the same mixture without the sample as a blank using an ELX800 microplate reader (Bio-Tek Instruments, nc; Winooski, VT, USA). The concentrations of flavonoid compounds were calculated according to the equation that was obtained from the standard (+)-catechin graph, and were expressed as mg catechin equiv. g−1DW (mg CE g−1DW).

2.4. Evaluation of plant growth Fresh weight was immediately assessed, and the dry weight was determined after 48 h of the desiccation in an oven at 60 °C. The relative growth rate (RGR) values, based on whole plant dry weight, were determined as following: (RGR, day−1) = ln W2 - ln W1/21), whereW1andW2are the dry weights at the beginning and the end of the treatment period, and 21 is the duration of the period in days.

2.8.3. Analysis by UHPLC-ESI-DAD-MSn This analysis was performed on an Ultimate 3000 (Dionex Co., USA) apparatus equipped with an ultimate 3000 Diode Array Detector (Dionex Co., USA) and coupled to a mass spectrometer, following the general procedure previously described by Beder-Belkhiria et al. (2018). The chromatographic apparatus was composed of a quaternary pump, an autosampler, a photodiode-array detector and an automatic thermostatic column compartment. The column used was a 100 mm length, 2.1 mm i.d., 1.9 μm particle diameter, end-capped Hypersil Gold C18 column (Thermo Scientific, USA) and its temperature was maintained at 30 °C. Gradient elution was carried out with a mixture of 0.1% (v/v) of formic acid in water (solvent A) and acetonitrile (solvent B), which was degassed and filtered before use. The solvent gradient consisted of a series of linear gradients, starting with 15–28% of solvent B over 5.6 min, increasing to 29% at 8.8 min, 100% of solvent B at 13.1 min and keeping up to 17 min, followed by the return to the initial conditions, with total running of 20 min. The flow rate used was 0.2 mL min–1 and UV–vis spectral data for all peaks were accumulated in the range 200–600 nm. The mass spectrometer used was a Thermo LTQ XL (Thermo Scientific, USA) ion trap MS equipped with an ESI source. Control and data acquisition were carried out with the Thermo X calibur Qual Browser data system (Thermo Scientific, USA). Nitrogen above 99% purity was used and the gas pressure was 520 kPa (75 psi). The instrument was operated in the negative-ion mode with ESI needle voltage set at 5.00 kV and an ESI capillary temperature of 275 °C. The full scan covered the mass range from m/z 100 to 2000. CID–MS/MS and MSn experiments were simultaneously acquired for precursor ions using helium as the collision gas with collision energy of 25–35 arbitrary units.

2.5. HCl-extractible iron HCl-extractible iron (Fe2+) was determined according to the method of M’sehli et al (2014). After desiccation with the drying oven (at 60 °C for 72 h), vegetable matter was finely crushed by standard agate crusher “FRITSH”, in order to avoid powder contamination by iron traces. Then 2 g of dry samples were digested with 15 mL HCl (1 N) and rigorously stirred, the digests kept for 4 h in the vessels, filtered and the volume adjusted to 25 mL with distilled water. Active iron (Fe2+) was quantified by atomic absorption spectrophotometer (VARIAN 220 FS). 2.6. Fe-use efficiency Shoot Fe-use efficiency was calculated based on the ratio of shoot biomass (mg) to shoot Fe concentration (μg Fe). 2.7. Preparation of extracts The A. graveolens samples were extracted according to the extraction protocol reported by Abu-Reidah et al. (2015), with slight changes. Leaves and roots of dill plants submitted to two regimes of treatments: +Fe (30 μM) and + Fe + Bic (30 μM Fe + 0.5 g L−1CaCO3 + 10 mM NaHCO3) were screened for variations of phenolic compounds and antioxidant abilities. For that, the ground leaves/roots of A. graveolens (0.5 g) were extracted with methanol (80%, v/v) and sonicated for 30 min at room temperature. The mixture was then centrifuged for 15 min at 3,750g and the supernatant was poured into a round-bottom flask. The extraction procedure was repeated three times. The extraction mixtures were filtered through a G4 sintered plate filter and the filtrated solutions were combined and concentrated until dryness under reduced pressure in a rotary evaporator at 40 °C. The dried material was then solubilized in 10 mL of methanol/water (80/20, v/v).

2.8.4. Quantitative analysis of phenolic compounds For quantitative analysis, the limits of detection and quantification were calculated from the parameters of the calibration curves obtained by injection of known concentrations of different standard compounds namely caffeoylquinic acid (y = 20,203.919x + 411.856; R2 = 0.999), dicaffeoylquinic acid (y = 18,756.050x + 2742.301 R2 = 0.999); caffeic acid (y = 304.25x− 248.06; R2 = 1.000); quercetin-3-O-glucoside (y = 16,529.665x – 1846.223; R2 = 0.999); ferulic acid 2 (y = 48.436,877x + 16.441,883; R = 1.000), isorhamentin-3-O-rutinoside (y = 12.019,444x-1520,449; R2 = 0.999) and kaempferol (y = 24, 120.383x+ 3618, 207; R2 = 1.000). Phenolic compounds for which a commercial standard was not available were quantified using phenolic compounds of the same group. The results were expressed in mg per g of dried extract.

2.8. Phenolic compounds 2.8.1. Total phenolic content (TPC) The TPC of the hydromethanolic extracts were quantified through Folin–Ciocalteu method (Pereira et al., 2012) with some modifications. Briefly, 15 μL of sample extract was dissolved in 60 μL of distilled water and 15 μL of Folin-Ciocalteu reagent. The mixture was shaken, before addition of 150 μl of 7% Na2CO3. After 60 min of incubation in the dark, 468

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absorbance against the concentrations, and the EC50 value was determined considering the extract concentration that provides 0.5 of absorbance. BHA was used as the positive control for comparison.

2.9. Antioxidant proprieties 2.9.1. ABTS%+ discoloration assay This method was performed according to the procedure of Catarino et al. (2017) with some modifications, as described elsewhere. The ABTS%+solution was prepared by reacting the stock solution of ABTS (7 mM) with potassium persulfate (2.45 mM) in a ratio of 1:1. The solution was stand in the dark at room temperature for 12–16 h. Before usage, the stock solution was diluted with ethanol to get an absorbance of 0.70 ± 0.020 at 734 nm. Several concentrations of sample extracts/ standard were dissolved in 250 μL of diluted ABTS%+ solution. After 6 min of incubation, the absorption at 734 nm was measured using an ELX800 microplate reader. The percentage of inhibition was calculated using the equation:

2.9.5. Assay of the oxygen radical absorbance capacity (ORAC) The procedure was modified from the method described by Ou et al. (2001), using black round bottom 96-well microplates (Costar) on a Fluoroskan Ascent FL™ plate reader (Labsystems) with an automated injector. The experiment was conducted at 37.5 °C and in pH 7.4 phosphate buffer, with a blank sample in parallel. Four concentrations of Trolox as the control standard were used in quadruplicate, and a gradient of sixteen concentrations of the extracts was set without replication. The fluorimeter was planned to register the fluorescence (λ ex.: 485 nm/em.: 530 nm) of fluorescein each minute once adding of 375 mM of 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH), for 35 min. The results were calculated using the net area under the curves of the sample concentrations for which reduction of at least 95% of fluorescence was observed at 35 min and which also presented a linear dose response pattern. ORAC values were expressed in micromoles of Trolox equivalents per g (μmol TE g_1 DW).

ABTS scavenging activity (%) = (Abscontrol - Abssample)/ Abscontrol* 100 where Abscontrol is the absorbance of ABTS radical the control without extract addition and Abssample is the absorbance of ABTS radical with extract. The results were expressed as IC50(concentration of the extract able to inhibit the 50% of the ABTS%+) of each extract. Ascorbic acid was used as a positive control for comparison.

2.10. Statistical analyses Data were analyzed using one-way analysis of variance using Graph Pad Prism, version 6. Means were compared according to Tukey’s test at P < 0.05 when significant differences were found. Correlation analyses were performed by using a two-tailed Pearson’s correlation test. All the analyses were carried out by using SPSS v22.0 software.

2.9.2. Superoxide anion-radical scavenging activity The superoxide quenching activity of samples was determined according to the method previously described by Valentão et al. (2001) with slightly modification. The method involved mixing 75 μL of diluted extract with 75 μL NBT (0.2 mM) and 100 μL β-NADH (0.3 mM) followed by 50 μL phenazine methosulfate (0.06 mM). After incubation at ambient temperature for 5 min, the absorbance was read at 560 nm against a blank. The inhibition percentage of superoxide anion generation was calculated by the formula given bellow:

3. Results and discussion 3.1. Effects of induced Fe deficiency on plant growth, photosynthetic pigment and bivalent iron content

Superoxide anion-radical scavenging activity % = [(A0 − A1/A0) * 100] Iron (Fe) is an essential element for many plant physiological and metabolic processes, including photosynthesis, oxygen transport, energy metabolism, DNA synthesis, nitrogen assimilation, and other metabolic functions involving oxido-reduction reactions closely related to the plant iron status (Ferraro et al., 2003; Pestana et al., 2005). Dill grown under Fe-deficient media exhibited classical symptoms of deficiency of this micronutrient after a two-week treatment. In fact, dill old leaves were reduced in size and had a pale yellow colour (Fig. 1), indicating decreased chlorophyll contents, which may represent a survival strategy adopted by plants exposed to Fe deficiency. This strategy consists in the mobilization of Fe from mature and senescing organs to make it available for the youngest ones (Jelali et al., 2010). Moreover, we noticed a reduction in total chlorophyll contents in bicarbonatetreated plants with respect to the control as a result of the decline of Chl a levels (-23%) and mostly those of Chlb(-37%) (Table 1). These findings corroborate those of Jelali et al. (2011) and Houmani et al. (2012) and could be explained by the primordial role of Fe2+ in the biogenesis of chlorophyll precursors such as 5-aminolevulinic acid and protochlorophyllide (Marschner, 1995). Indeed, iron is a necessary micronutrient required for chlorophyll biosynthesis as well as for thylakoid and granum formation (Briat et al., 2015;M’sehli et al., 2014; Rout and Sahoo, 2015). Accordingly, iron chlorosis is due to chlorophyll dilution when leaves continue to grow at a normal rate under iron deficiency conditions (Abadía et al., 2002) and to cell inability to produce and/or to stabilize new chlorophyll molecules in thylakoid membrane (Belkhodja et al., 1998). Fe deficiency has been generally reported to adversely affect the growth activity in many plant species (De la Guardia and Alcántara, 2002). In this work, we have noticed a clear depressive effect of induced-Fe deficiency on the shoot height, dry weight and relative growth rate as compared with those of the control reaching respectively 23, 56 and 41% (Table 1). However, it is worthy to mention that A. graveolens

in which A1 and A0 are absorbances of solvent with and without sample, respectively. The results were expressed as IC50 values (mg mL−1). Gallic acid was used as positive control for comparison. 2.9.3. HO% scavenging assay This assay was conducted following the procedure of Catarino et al. (2017). A solution composed of equal volumes of FeCl3 300 μM, EDTA 1.2 mM, phosphate buffer 100 mM, pH 7.4 and H2O2 33.6 mM (140 μL) was mixed with 210 μL of A. graveolens extract solutions at different concentrations, 35 μL of ascorbate 1.2 mM and 35 μL of deoxyribose 33.6 mM. The reaction mixture was then incubated at 37 °C over1 h to allow the generation of HO• by ferric-ascorbate-EDTA-H2O2 interactions. Afterwards, 350 μL of 1% (w/v) TBA (prepared in 50 mM of NaOH) and equal volume of 5% (w/v) TCA were added and the solutions were placed in a boiling water bath for 20 min, to allow the formation of the pink chromogen. The reactions were then interrupted in an ice bath and the absorbance was measured in the spectrophotometer at 532 nm and HO% scavenging assay was expressed as IC50 (mgmL−1). 2.9.4. Ferric reducing antioxidant power (FRAP) assay The reducing power of each standard/extract was determined in the microplate reader as mentioned above using an absorbance at 690 nm A volume of 500 μL of different concentrations of dill extracts/standard was mixed with 500 μL of 200 mM sodium phosphate buffer (pH6.6) and 500 μL of (1% w/v) potassium ferricyanide. The mixture reaction was incubated at 50 °C for 20 min. Subsequently, 500 μL of 10% TCA was added to the mixture was centrifuged at 650 g for 10 min. The upper layer fraction (75 μL) was mixed with 75 μL mL of deionised water and 15 μL of ferric chloride solution (0.1%) (Catarino et al., 2017). A linear regression analysis was carried out by plotting the mean 469

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Fig. 1. Aspect of Anethum graveolens plants cultivated in the presence of Fe (+Fe) or in the presence of Fe plus bicarbonate (+Fe + Bic) during the treatment period (14 days).

relatively tolerant to Fe chlorosis.

Table 1 Growth parameters, photosynthetic pigment amounts, bivalent Fe contents in shoots and roots and shoot use efficiencies (FeUE) of Anethum graveolens plants grown in the presence of Fe (+Fe) or in the presence of Fe plus bicarbonate (+Fe + Bic). +Fe Chl a (mg/g FW) Chl b (mg/g FW) Chl a +Chl b (mg/g FW) Shoot lengh (cm plant−1) Root elongation (cm plant−1) Shoot DW (g plant−1) Root DW (g plant−1) Shoot RGR (day−1) Root RGR (day−1) Shoot iron content (μg. g −1 DW) Root iron content (μg. g −1 DW) Fe use efficiency (FeUE) (mg DW μg_1 Fe)

3.2. Involvement of secondary metabolism in the response of A. graveolens to Fe deficiency

+Fe + Bic a

0.53 ± 0.04 0.38 ± 0.02a 0.91 ± 0.06a 31.10 ± 1.09a 23.92 ± 5.59b 0.40 ± 0.06a 0.09 ± 0.01a 0.18 ± 0.04a 0.14 ± 0.01a 299.9 ± 12.3a 1576.1 ± 35.17a 1.33 ± 0.09b

In general, plants respond to oxidative stress by including the accumulation of secondary metabolites able to face various challenges, through variation of cellular metabolism (Ksouri et al., 2007). Among secondary metabolites, phenolic compounds play an important role in the adaptive response to Fe deficiency through their antioxidant and chelating properties (Tato et al., 2013). In this context, monitoring of total phenolic content (TPC) was assessed in control and treated samples. Phenolic compound and flavonoid levels were higher in leaves than in roots for the control plants (Table 2). Notably, the contents of phenolic compounds in organs of Fe-treated plants, as well as those of

0.41 ± 0.07b 0.24 ± 0.04b 0.65 ± 0.11b 23.94 ± 1.02b 32.50 ± 4.17a 0.177 ± 0.03b 0.081 ± 0.01a 0.108 ± 0.05b 0.136 ± 0.02a 116.23 ± 2.89b 909.20 ± 0.176b 1.75 ± 0.12a

Table 2 Changes in total polyphenols and flavonoids content in leaves and roots of Anethum graveolens plants grown in the presence of Fe (+Fe) or in the presence of Fe plus bicarbonate (+Fe + Bic).

Values are the means of six replicates ± SD. In the case of significant interaction between Fe treatment, means followed by different letters are significantly different at P < 0.05 according to Tukey’s test.

Total polyphenols (mg GAEg −1 DW)

roots were able to maintain their growth. Similar behavior has already been described for other tolerant genotypes (Pestana et al., 2005), a fact often associated with an increased number of secondary and tertiary roots, aiming at a more efficient exploration of the soil (Bavaresco and Poni, 2003). Interestingly, A. graveolens plants grown under Fe deficiency conditions increased their shoot Fe use efficiency in comparison to the control (Table 1), which allows this species to maintain its growth. Therefore, according to these findings and based on root biomass production and Fe use efficiency (FeUE), A. graveolens seems to be

+Fe L +Fe + Bic L +Fe R +Fe + Bic R

5.38 9.97 2.34 3.97

± ± ± ±

0.16b 0.05a 0.08d 0.03c

Flavonoids (mg CEg −1 DW) 2.23 3.86 0.40 0.52

± ± ± ±

0.07b 0.11a 0.01d 0.02c

Values are means of three replicates ± SD. In the case of significant interaction between Fe treatment, means followed by different letters are significantly different at P < 0.05 according to Tukey’s test. 470

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flavonoids in leaves, were about 1.7–1.9 times those of control plants. The higher phenolic content suggests that such species may be more efficient in accumulating phenolic compounds in order to adapt to the bicarbonate induced stress. According to Perron and Brumaghim (2009), phenols can act as antioxidants by chelating metal or scavenging ROS for fortification against oxidative damage. It was reported that phenolic compounds released by roots of red clover play a pivotal role in the regulation of Fe absorption via increased Fe accumulation in the root apoplast, suggesting that these metabolites could enhance the use of apoplastic Fe thus enhancing Fe nutrition in the shoot (Jin et al., 2007). Such finding would confirm the fact that Fe deficiency-induced phenolics plays a key role in Fe-efficiency in strategy I plants. Another mechanism underlying the antioxidant properties of phenols is the ability of flavonoids to alter peroxidation kinetics by modification of the lipid packing order and decrease of membrane fluidity which could hinder diffusion of free radicals and restrict peroxidative reactions (M’sehli et al., 2014). The accumulation of phenolic compounds might arise due to phenylalanine ammonia-lyase (PAL, E.C.4.3.1.5) stimulation under Fe deficiency conditions. Overall, PAL catalyzes the transformation of L-phenyalanine into trans-cinnamic acid, which is the prime intermediary in phenolics biosynthesis (Giorgi et al., 2009). The action of this enzyme enhances activity in light of stress and is considered by the most instigators to be one of the fundamental plant lines for cell acclimation against stress (Kacperska, 1993; Leyva et al., 1995). According to Lin et al. (2006), improvement of the antioxidant capacity both by increasing contents and quality of phenolic compounds may assume a critical part in the resistance of plants to abiotic stresses. This implies that plants react via a versatile mechanism promoting genes involved in the biosynthesis of antioxidants in stress conditions (Oh et al., 2009). Besides, studies showing that mutants deficient in secondary metabolites or plants with blocked PAL activity are sensitive to environmental stresses obviously display the role of these antioxidants in plant acclimation (Gitz et al.,2004).

Amongst those, 5-CQA and FQA were the major hydroxycinnamic acids from leaves, with respective amounts of 2.11 ± 0.07 and 0.243 ± 0.00 mg g-1DW in control plants and accounting for 58% and 6.7% of total phenolic compounds, respectively (Table 4). In turn, flavonoids accounted for about 31% of the total phenolic constituents of leaf extract in control plants, being mostly represented by quercetin-3O-glucuronide (peak 14, UVmax at 256, 354 nm, [M−H]−at m/z 477→ 301), and containing minor amounts of quercetin-3-O-rutinoside ([M−H]−at m/z 609→301 eluted in peak in peak 11), kaempferol-3-Oglucuronide ([M−H]- at m/z 461→285 eluted in peak 17) and isorhamnetin-3-O-glucuronide ([M−H]− at m/z 491→315 eluted in peak 19) (Justesen, 2000) (Table 3). As mentioned above and conversely to the leaf phytochemical profile, quercetin-3-O-rutinoside and quercetin-3-O-glucuronide were the only flavonoids in root extracts (vestigial) and thus, caffeic and ferulic acids derivatives were the only quantified phenolic components in this organ (Tables 3 and 4). Caffeic acid and the 3 monocaffeoylquinic acids isomers were detected in roots and, similarly to leaves, 5-CQA was the most relevant, accounting for 0.313 ± 0.007 mgg−1 DW in control plants. It is worthy to notice that the most of the hydroxycinnamic acid pool in root extract differed from leaf one. The main phenolic compound in roots (eluted in peak 19), showed a UVmax at 301sh and 326 nm and a [M−H]- at m/z 527 in full MS, which in turn fragmented to ions at m/z 365 and m/z 203, due to the loss of 162 and 324 Da, being thus tentatively assigned to caffeoyl N-tryptophan hexoside (Barros et al.,2012). This compound was detected with a quantity of 0.656 ± 0.005 mgg−1 DW, thus corresponding to about 33% of root phenolic pool, while it was not detected in leaves. Besides, caffeic acid (peak 7, [M−H]- at m/z 179→135, 179), caffeoyl N-tryptophan (peak 10, [M−H]- at m/z 365 →203, 185, 179), malonyl-tri-Ocaffeoylquinic acid (peak 23, [M−H]- at m/z 763 →677, 719, 539, 557, 395) and the dicaffeoylquinic acids: 3,5-di-O-caffeoylquinic acid (peak 15, [M−H]- at m/z 515 →353, 191, 179) and 1,4-di-O-caffeoylquinic acid (peak 19, [M−H]- at m/z 515 →353, 191, 179) were also detected in relevant concentrations in roots, albeit their absence in leaves (Martins et al., 2016). In the same trend, contrarily to leaf extracts, those from root were characterized by distinct ferulic acids derivatives besides ferulic acid (peak 12, UV max at 297sh and 322 nm, [M−H]− at m/z 193→149, 178, 134) and feruloylquinic acid (peak 9, UV max at 289sh and 324 nm, [M−H]- at m/z 367→191,193). These compounds included four feruloyl tryptophan conjugates, tentatively assigned to feruloyl N-tryptophan (peak 15, [M−H]- at m/z 379→185, 193, 203, 141), two feruloyl N-tryptophan hexoside isomers (peaks 21 and 23) and a diferuloyl Ntryptophan (peak 26, [M−H]- at m/z 555→193, 361, 379), as well as a protocatechuic acid derivative of ferulic acid, which was eluted in peak 25 ([M−H]- at m/z 329→193, 135) and two unknown derivatives of ferulic acid (eluted in peaks 27 and 29 of [M−H]- at m/z 313 and 625, respectively) (Barros et al., 2012; Martins et al., 2016). Overall, ferulic acid derivatives in roots accounted for about 0.5 mgg-1 DW in control plants and, together with caffeic acid derivatives, total hydroxycinnamic acids amounts were close to 2 mg g-1 DW. Bicarbonate supply did not induce qualitative modification in the UHPLC-phytochemical profiles, as compared to those of non-treated plants. Nevertheless, phenolic levels significantly changed (Table 4). In leaves, the amounts of the main caffeic acid derivative, i.e. 5-CQA, was raised by about 69% and overall, hydroxycinnamic acid levels reached 7.6 mg g−1 DW in the treated plants. Likewise, flavonoid levels in Fetreated plants were also incremented compared to control. Indeed, excluding kaempferol-3-O-glucuronide (the less representative flavonoid in the plant), the remaining flavonoids were raised, overall doubling the total flavonoids amounts. As for root case, results indicated that excepting for 3,5-di-O-caffeoylquinic acid and tri-O-caffeoylquinic acid, a significant increase in the biosynthesis of phenolics was noticed, in particularly with respect to ferulic acid and its derivatives. Likewise, caffeic acid and its derivatives

3.3. Characterization of the leaf and root fractions by UHPLC- DAD-ESIMSn Dill hydromethanolic extracts from leaves and roots of plant grown under control or induced Fe deficiency condition were analyzed by UHPLC-DAD-ESI-MSn to better understand the variations of metabolites to Fe deficiency stress, in particular phenolic components, under Fedeficiency conditions. To our knowledge, there is no previous information regarding the phenolic composition of dill root samples, while a few studies have reported the phenolic composition of dill leaves, namely in plants originating from Netherlands (VallverdúQueralt et al., 2015), Turkey (Orhan et al., 2013) and Denmark (Justesen, 2000) origins. Fig. 2 depicts leaf and root chromatographic profile of control plants whereas Table 3 summarizes the retention time, UV–vis and MSn spectral data of the identified compounds and also indicates their presence (or absence) in the two organs, for the experimental conditions + Fe and + Fe + Bic. The detected metabolites in both organs varied, which might be associated to their specific roles. In this context, phaeophytins a and b, as well as free amino acid N-tryptophan were only detected in leaf hydromethanolic extracts while the fatty acids hydroxy-octadecenoic acid and hydroxy-octadecatrienoic acid were exclusively found in root. Notably, the phenolic composition of the two organs was also very distinct. Indeed, regardless extracts from both organs were particularly rich in caffeic and ferulic acid derivatives, clear differences were found on specific components and their respective abundances. Moreover, flavonoids were particularly abundant in leaves but only detected as vestigial components in roots extracts. In more detail, caffeic acid ([M−H]− at m/z 179, UVmax at 289sh and 323 nm) and its monoderivatives 3-CQA, 5-CQA and 4CQA ([M−H]− at m/z 353, eluted at in peaks 4, 6 and 7, respectively), along with a feruloylquinic acid (FQA, [M−H]− at m/z 367 eluted in peak 9) were found in leaves (Li et al., 2003; Martins et al., 2016). 471

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Fig. 2. Chromatographic profile at 280 nm of Anethum graveolens hydromethanolic extracts from leaves and roots. Numbers in the figure correspond to eluted UHPLC peaks for which UV and MS data is summarized in Table 3.

nutrient uptake (Donnini et al., 2011). On other hand, the accumulation of flavonoids, in particular flavonols, could possibly be related to a potential stress response which participate in the modulation of plant cell growth and differentiation, as well as the regulation of the activity of different protein kinases. These protein kinases are, in turn, responsible for mediating ROS-induced signaling cascades that are vital for cell growth and differentiation (Agati et al., 2011; Brunetti et al., 2013).This hypothesis is supported by the fact that nutrient deficiency (nitrogen and phosphorus) affected photosynthesis by decreasing available chlorophyll level and upsetting photosynthetic membranes due to starch accumulation, which ultimately stimulated the production of flavonols, aiming to protect from light-induced oxidative damage (Lillo et al., 2008). Note that quercetin and/or quercetin derivatives are flavanols widespread in fruits and vegetables, known to exert versatile biological effects, including iron-chelating and ROS-scavenging activities under Fe-deficiency grown conditions, as well as in the process of iron absorption and the regulation of iron homeostasis maintainer (Xiao et al., 2018).

(5-CQA, CA and 1,4-dCQA) were incremented by 36%, 43% and 42% respectively over control. Levels of cinnamoyl-amino acids, including caffeoyl N-tryptophan, caffeoyl N-tryptophan hexoside, and feruloyl Ntryptophan hexoside isomers were also found to be higher in bicarbonate-treated roots as compared to control. Fe deficiency, as an abiotic stress for plants, was shown to affect the expression and the activity of certain peroxidase isoenzymes and to induce secondary oxidative stress in dicotyledonous species (Ranieri et al., 2001). The increase in caffeic acid and its derivatives levels in both studied tissues (leaves and roots) could be associated to their antioxidant functions (Niggeweg et al., 2004). Indeed, this family of compounds has strong antioxidant properties displaying high cytoprotection activity against ROS, and some of them could display interesting Fe chelating activity. E.g. CQA isomers are known to be involved in responses to different biotic and abiotic stresses in several plant species, probably through its ability to quench ROS (Niggeweg et al., 2004). The increment of phenolic compounds could be however a consequence of the reduction of lignin biosynthesis. Note that phenolic compounds such as hydroxycinnamic acids (e.g caffeic, ferulic and ultimately chlorogenic acid) are lignin precursors (Hoffmann et al., 2004), while lignification decrease has been suggested to be an adaptative response to Fe deficiency stress. In fact, while stimulation of cell-wall lignification can occur as a stress response (Kim et al., 2006) in plant tolerance to salt and drought stress (Jbir et al., 2001; Lee et al., 2007). A similar tendency in Fe-deficiency conditions could further impair mineral nutrition generating higher susceptibility to this constraint. Such hypothesis was verified in tolerant genotype of pear, where the allocation of new biomass to the organs (augmenting of root elongation) through reduction of lignin synthesis could facilitate the exploration of soil and improve Fe uptake. In contracts, in quince (susceptible genotype), lignin synthesis caused reduction of root elongation and hampered

3.4. Antioxidant capacity In parallel, dill extracts were investigated for their antioxidant abilities through distinct in vitro methods, namely ABTS%+, O2%−, HO%, FRAP and ORAC assays, allowing to evaluate their scavenging ability towards distinct radicals, as well as the ability to reduce Fe3+ to Fe2+. As shown in Table 5, in control plants, with exception for O2•- assay, leaf extracts of control plants were more active than those from roots (as reflected by lower IC50and EC50values), which is probably associated to their different TPC and TFC contents. Moreover, the results confirmed an improvement of the antioxidant activity in bicarbonate-treated plants. ABTS•+ scavenging activity increased by about 34% and 45%, 472

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Table 3 Identification of UHPLC eluting fractions by UHPLC-DAD-MSn of Anethum graveolens hydromethanolic extracts. Peak

Rt (min)

λmax

M-H

ESI MS/MS fragments

Compound

Treatment +Fe

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

1.4 1.8 1.8 5.7 6.8 8.8 9.0 9.5 11.1 11.5 12.1 12.5 12.5 12.6 13.2 13.6 13.7 13.9 13.9 14.5 15.0 15.1 15.8 16.1 16.7 16.9 20.7 20.9 21.6 22.7 22.7 23.4 24.4

224 258 258 290sh, 324 279 295sh, 325 295sh, 325 289sh, 323 289sh, 324 299sh,327 253, 354 297, 322 297sh,322 256, 354 298sh,327 300sh,328 267, 336 253, 265, 351 301sh,326 298sh,328 299sh,325 300sh,325 289sh,326 288sh,325 289,325 289sh,325 290sh,324 314 288sh,325 277 277 434,610, 651 408, 608, 664

133 191 191 353 203 353 353 179 367 365 609 193 341 477 379 515 461 491 527 515 541 677 541 763 329 555 313 307 625 297 293 883 871

115, 73 127, 173, 111, 85, 93 111, 173, 67 191, 179, 136 115; MS3 [115] : 87, 88, 85 191, 179, 173 191, 179, 173 135, 179 191, 193 203, 185, 179 301 149, 178, 134 179, 161 301 185, 193, 203, 141 353, 335, 191, 179 285 315 365, 203 353, 173,185 379, 203 497, 515, 353 379, 203 677, 719, 539, 557, 395 193, 135 193, 361, 379 193, 171 – 193, 179, 135 297, 183 249,275, 113 ND ND

Malic acidA Quinic acidA Citric acidA 3-O-caffeoylquinic acidB TryptophanB 5-O-caffeoylquinic acidB 4-O-caffeoylquinic acid B Caffeic acid B Feruloylquinic acidB Caffeoyl N-tryptophanB Quercetin-3-O-rutinosideB Ferulic acidB Caffeoyl-O-hexosideB Quercetin-O-glucuronide B Feruloyl N-tryptophan 3,5-di-O-Caffeoylquinic acidB Kaempferol-3-O-glucuronide B Isorhamnetin-3-O-glucuronide B Caffeoyl N-tryptophan hexosideB 1,4-di-O-Caffeoylquinic acidB Feruloyl N-tryptophan hexoside (isomer1) B Tri-O-caffeoylquinic acidB Feruloyl N-tryptophan hexoside (isomer 2) B Malonyl-tri-O-caffeoylquinic acidB Protocatechuic derivative of ferulic acidC Di-feruloyl N-tryptophanC Feruloyl derivativeC Unknown Feruloyl derivativeC Hydroxy-octadecenoic acidB Hydroxy-octadecatrienoic acidB Phaeophytin bB Phaeophytin aB

+Fe + Bic

L

R

L

R

+ + + + + + + + + – + + – + – – + + – – – – – –

+ + + v v + v + + + v v + v + + – – + + + + + +

+ + + + + + + + + – + + – + – – + + – – – – – –

+ + + v v + v + + + v v + v + + – – + + + + + +

– –

+ +

– –

+ +

– – – + +

+ + + – –

– – – + +

+ + + – –

A, identification was based by comparison with standard; B identification was based on the interpretation of UV spectral and MS data, plus comparison to literature; C novel compound. Mean values ± SD of three independent assays; statistical analysis was performed by one-way ANOVA (Tukey’s test).

and extract phenolic content since phenolic compounds contribute directly to antioxidant activity (Bettaieb-Rebey et al., 2012). Still, no correlation between TPC (or TFC) and antioxidant activity was observed with respect to superoxide radical quenching potential, since the estimated coefficients of determination, r values, were less than 0.5 at p < 0.05. These results suggest that the antioxidant activity of some tested extracts might have a major contribution of non-phenolic extracts constituents. Still, a high correlation was observed between the results of some the remaining activity tests [radical scavenging (ABTS%+), reducing power (FRAP) and ORAC assays] and the quantities of 5-CGA, 4-CGA, CA, Q3R, Q3Glu, and I3Glu, which suggest an efficient role of caffeic acid and its derivatives, along with flavonols, in counteracting oxidative stress events in A. graveolens grown under Fe deficiency conditions.

respectively, in leaves and by 19% and 36% respectively in roots, as compared to control. A similar trend was verified for reducing power ability, with EC50 values decreasing from 1.85 ± 0.00 to 1.34 ± 0.00 mg mL-1 in leaf and 2.15 ± 0.00 to 1.79 ± 0.00 mg mL-1 in root extracts from control and treated plants, respectively. Extracts from Fe-deficiency plants also showed enhanced activity towards peroxyl radicals, as showed by their superior ORAC values (0.61 ± 0.08 and 13 ± 0.01 μmol Trolox g-1DW for leaves and roots, respectively). The impact of induced-Fe deficiency was also noticed on O2%− and % HO scavenging capacities, despite these were not completely aligned with the previous tests. In fact, the O2%- rescuing potential of bicarbonate-treated plants was raised with respect to those of control. Despite this raise, this leaf effect was twice that of roots. On the other hand, the inhibition activity against hydroxyl radicals was closely stimulated in both organs, by about 3-fold. These results suggest that, despite the overall increment of antioxidant activity in plants submitted to induced-iron deficiency, effects in leaves and roots can vary depending on the specific reaction and/or mechanism involved. Our results also allowed concluding that dill extracts were particularly active against HO% (IC50 ranging from 0.021 ± 0.00 to 0.094 ± 0.00 mg mL-1) as compared to the reference compound mannitol (IC50 = 0.190 mg mL-1). The dependence of antioxidant activity, obtained by different assays, in relation to the total phenolic content, showed linear correlation in the cases of ABTS (r = 0.857; r = 0.818), and HO% (r = 0.774; r = 0.582) scavenging activities as well as iron reducing power (r = 0.943; r = 0.861) and ORAC assay (r = 0.775; r = 0.995) in relation to the amount of phenols and flavonoids, respectively (Table 6). Indeed, a positive correlation is always established between antioxidant capacity

4. Conclusion This study was focused on the effects of induced-Fe deficiency on the growth, photosynthetic capacity and biochemical changes in a medicinal plant: A. graveolens, with the global purpose of predicting its effect on plant efficacy. Our results showed that A. graveolens shoot and whole plant biomass production were significantly decreased under Fe deficiency conditions, whereas that of roots was not affected by such constraint. Moreover, lime-induced Fe deficiency resulted in a significant reduction of chlorophyll and Fe concentration. Interestingly, although grown under Fe deficiency conditions, A. graveolens was able to increase their shoot iron use efficiency (FeUE), probably to stimulate acidification capacity and FCR activity in their roots. On the other 473

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Table 4 Contents of individual phenolic compounds (mg g−1 DW) in leaves and roots of A. graveolens plants grown in the presence of Fe (+Fe) or in the presence of Fe plus bicarbonate (+Fe + Bic). Plant organ

Leaves

Compound name

Hydroxycinnamic acids

Flavonoids

Roots

Caffeic acid derivatives

Ferulic acid derivatives

(mg/g) DW

Caffeic acid 5-O-caffeoylquinic acid 4-O-caffeoylquinic acid Ferulolyquinic acid Quercetin-3-O-rutinoside Quercetin-3-O-glucuronide Kaempferol-3-O-glucuronide Isorhamnetin-3-O- glucuronide ∑ hydroxycinnamic acids ∑ flavonoids ∑ Total Caffeic acid 5-O-caffeoylquinic acid 1,4- di-O-caffeoylquinic acid 3,5- di-O-caffeoylquinic acid Tri-O-caffeoylquinic acid Malonyl- tri-O-caffeoylquinic acid Caffeoyl N-tryptophan Caffeoyl N-tryptophan hexoside Ferulic acid Feruloylquinic acid Feruloyl derivative Feruloyl derivative Protocatechuic derivate of ferulic acid Feruloyl N-tryptophan Di-feruloyl N-tryptophan Feruloyl N-tryptophan hexoside (isomer 1) Feruloyl N-tryptophan hexoside (isomer 2) ∑ caffeic acid derivates ∑ ferulic acid derivates ∑ Total

+Fe

+Fe + Bic

0.038 ± 0.005b 2.116 ± 0.076b 0.085 ± 0.012b 0.243 ± 0.003b 0.061 ± 0.002b 0.870 ± 0.042b 0.046 ± 0.001b 0.166 ± 0.006b 2.482 ± 0.096b 1.143 ± 0.051b 3.625 ± 0.147b 0.007 ± 0.000b 0.313 ± 0.007b 0.049 ± 0.004b 0.217 ± 0.006a 0.053 ± 0.003a 0.056 ± 0.000 0.091 ± 0.002b 0.656 ± 0.005b 0.069 ± 0.001b 0.143 ± 0.004b 0.110 ± 0.002b 0.028 ± 0.003b 0.034 ± 0.002b 0.024 ± 0.002b 0.018 ± 0.001b 0.028 ± 0.007b 0.065 ± 0.001b 1.442 ± 0.027b 0.519 ± 0.023b 1.961 ± 0.05b

0.085 ± 0.0016a 6.898 ± 0.2499a 0.350 ± 0.0072a 0.272 ± 0.005a 0.125 ± 0.010a 1.643 ± 0.063a 0.014 ± 0.00a 0.691 ± 0.025a 7.605 ± 0.263a 2.473 ± 0.098a 10.078 ± 0.361a 0.012 ± 0.003a 0.491 ± 0.021a 0.084 ± 0.003a 0.217 ± 0.008a 0.058 ± 0.006a 0.072 ± 0.000 0.139 ± 0.005a 0.821 ± 0.024a 0.293 ± 0.012a 0.359 ± 0.015a 0.208 ± 0.013a 0.052 ± 0.003a 0.067 ± 0.002a 0.035 ± 0.000a 0.033 ± 0.001a 0.044 ± 0.004a 0.108 ± 0.004a 1.894 ± 0.07a 1.199 ± 0.054a 3.093 ± 0.124b

Values are the means of 6 replicates ± SD. In the case of significant interaction between Fe treatment, means followed by different letters are significantly different at P < 0.05 according to Tukey’s test.

Acknowledgments

hand, a modulation of secondary metabolite biosynthesis, namely phenolic compounds, and an enhancement of antioxidant activity, as evaluated by five different test systems (scavenging ability on ABTS%+; O2%− and HO% radical, iron reducing power and ORAC assays) suggest an adequate protection against oxidative damage, which confer a certain tolerance to Fe deficiency to this species. These results also show that such constraint could be useful to enhance the health-promoting phytochemicals in dill and through manipulation of agricultural, along with screening programs of new Fe-efficient genotypes.

This work was supported by the Tunisian Ministry of Higher Education and Scientific Research (LR15CBBC06). Thanks are due to the University of Aveiro, FCT/MEC for the financial support to the QOPNA research Unit (FCT UID/QUI/00062/2013), through national funds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement. Susana M. Cardoso thanks the research contract under the project AgroForWealth (CENTRO-01-0145-FEDER000001), funded by Centro2020, through FEDER and PT2020. Authors are also grateful to Dr. Mónica Valega from the Chemistry Department of the University of Aveiro for technical support in the LC–MS analysis.

Conflict of interest The authors have no conflict of interest.

Table 5 Antioxidant activities of methanolic extracts of Anethum graveolens plants grown in the presence of Fe (+Fe) or in the presence of Fe plus bicarbonate (+Fe + Bic). ABTS%+ (IC50, mg/mL) +Fe L +Fe + Bic L +Fe R +Fe + Bic R Ascorbic acid BHT Gallic acid Catechin Mannitol Trolox

0.100 0.054 0.151 0.096 0.019 – – – – –

± ± ± ± ±

0.02b 0.03d 0.05a 0.02c 0.00

O2%− (IC50, mg/mL)

OH% (IC50, mg/mL)

FRAP (EC50, mg/mL)

ORAC (μmol TE g−1 DW)

2.55 ± 0.23a 1.44 ± 0.12c 1.72 ± 0.04b 1.45 ± 0.01c – – 0.005 ± 0.00 – – –

0.065 ± 0.00b 0.021 ± 0.02d 0.094 ± 0.00a 0.034 ± 0.04c – – – – 0.19 ± 0.03 –

1.85 ± 0.01b 1.34 ± 0.00d 2.15 ± 0.03a 1.79 ± 0.03c – 0.0185 ± 0.00 – – – –

0.35 0.61 0.09 0.13 – – – – – 3.94

± ± ± ±

0.02b 0.08a 0.01d 0.01c

± 0.01

Values are the means of 6 replicates ± SD. In the case of significant interaction between Fe treatment, means followed by different letters are significantly different at P < 0.05 according to Tukey’s test. 474

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Table 6 Correlation coefficients between total and individual phenolic compound amounts and antioxidant activities. Variables Caffeic acid 5-O-caffeolyquinic acid 4-O-caffeoylquinic acid 1,4- di-O-caffeoylquinic acid 3,5- di-O-caffeoylquinic acid Tri-O-caffeoylquinic acid Malonyl- tri-O-caffeoylquinic acid Caffeoyl N-tryptophan Caffeoyl N-tryptophan hexoside Ferulic acid Ferulolyquinic acid Feruloyl derivative Protocatechuic derivate of ferulic acid Feruloyl N-tryptophan Di- Feruloyl N-tryptophan Feruloyl N-tryptophan hexoside Quercetin 3-O-rutinoside Quercetin-3-O-glucuronide Kaempferol-3-O-glucuronide Isorhamnetin-3-O- glucuronide TPC TFC

TPC

ABTS%+

TFC **

0.923 0.956** 0.958** −0.480 −0.572* −0.560* −0.535* −0.503* −0.540* −0.325 0.290 −0.461 −0.452 −0.512* −0.466 −0.489 0.866** 0.854** −0.028 0.959** 1.000 0.878**

**

0.994 0.978** 0.973** −0.831** −0.887** −0.885** −0.874** −0.851** −0.876** −0.668* 0.099 −0.814** −0.805** −0.858** −0.819** −0.840** 1.000** 0.998** 0.430 0.973** 0.878** 1.000

−0.855 −0.835** −0.821** 0.436 0.676* 0.639* 0.567* 0.488 0.579* 0.136 0.651* 0.395 0.376 0.509* 0.407 0.458 −0.815** −0.808** 0.255 −0.820** −0.857** −0.818** **

O2%−

HO%

−0.097 −0.206 −0.223 −0.498 −0.453 −0.465 −0.482 −0.494 −0.480 −0.479 −0.305 −0.499 −0.499 −0.491 −0.499 −0.496 0.028 0.069 0.883** −0.255 −0.472 0.002

−0.650 −0.653* −0.639* 0.086 0.371 0.322 0.235 0.144 0.249 0.214 −0.803** 0.042 0.022 0.168 0.055 0.109 ̵ 0.574* −0.559* 0.035 −0.640* −0.774** −0.582*

FRAP *

ORAC

−0.904 −0.906** −0.897** 0.452 0.646* 0.617* 0.560* 0.495 0.569* 0.197 0.544* 0.418 0.402 0.513* 0.428 0.470 −0.854* −0.842* 0.139 −0.898** −0.943** −0.861** **

0.989** 0.949** 0.936** −0.660* −0.795** −0.784** −0.750** −0.699** −0.757** −0.393 0.026 −0.626* −0.611* −0.712** −0.633* −0.676* 0.995** 0.992** 0.195 0.935** 0.775** 0.995**

Data represents Pearson Correlation Coefficient R. ; ABTS: ABTS radical scavenging activity; FRAP: FRAP reducing power potential; O2%−: superoxide quenching activity; OH: HO% quenching activity; TPC: total phenolic content; TFC: total flavonoid content). * Significant at p < 0.05. ** Significant at p < 0.01.

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