Phytohormone profile in Lactuca sativa and Brassica oleracea plants grown under Zn deficiency

Phytochemistry xxx (2016) 1e5

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Phytohormone profile in Lactuca sativa and Brassica oleracea plants grown under Zn deficiency
n a, *, Alfonso Albacete b, Alejandro de la Torre-Gonza
lez a, Juan M. Ruiz a, Eloy Navarro-Leo ~ a Blasco a Begon a b

Department of Plant Physiology, Faculty of Sciences, University of Granada, 18071, Granada, Spain Department of Plant Nutrition, CEBAS-CSIC, Campus de Espinardo, E-30100, Espinardo, Murcia, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 June 2016 Received in revised form 5 August 2016 Accepted 10 August 2016 Available online xxx

Phytohormones, structurally diverse compounds, are involved in multiple processes within plants, such as controlling plant growth and stress response. Zn is an essential micronutrient for plants and its deficiency causes large economic losses in crops. Therefore, the purpose of this study was to analyse the role of phytohormones in the Zn-deficiency response of two economically important species, i.e. Lactuca sativa and Brassica oleracea. For this, these two species were grown hydroponically with different Znapplication rates: 10 mM Zn as control and 0.1 mM Zn as deficiency treatment and phytohormone concentration was determined by U-HPLC-MS. Zn deficiency resulted in a substantial loss of biomass in L. sativa plants that was correlated with a decline in growth-promoting hormones such as indole-3-acetic acid (IAA), cytokinins (CKs), and gibberellins (GAs). However these hormones increased or stabilized their concentrations in B. oleracea and could help to maintain the biomass in this species. A lower concentration of stress-signaling hormones such as ethylene precursor aminocyclopropane-1-carboxylic acid (ACC), abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) and also CKs might be involved in Zn uptake in L. sativa while a rise in GA4, isopentenyl adenine (iP), and ACC and a fall in JA and SA might contribute to a better Zn-utilization efficiency (ZnUtE), as observed in B. oleracea plants. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Lactuca sativa Asteraceae Brassica oleracea Brassicaceae Zn metabolism Phytohormones

1. Introduction Zinc (Zn), an essential micronutrient, is necessary for normal plant growth as it is present in vital metabolic processes such as protein synthesis, maintenance of cell membrane integrity, and DNA transcription. Likewise, Zn plays important roles in starch synthesis and in ROS detoxification, as cofactor in important enzymes such as carbonic anhydrase, CueZn-superoxide dismutase (CueZn SOD) and dehydrogenases (Vallee and Auld, 1990). Being essential for plant growth, Zn deficiency can be a major threat to crops and, in fact, this is the most widespread micronutrient defi€, 1982). ciency in agriculture (Sillanp€ aa Zn deficiency causes heavy economic losses in crops due mainly to biomass loss, as for example, in tomato, lettuce, carrot, and onion (Alloway, 2008; Paradisone et al., 2015). Other symptoms are internervial chlorosis, necrotic spots, browning, rosette disposal,

* Corresponding author.
n). E-mail address: [email protected] (E. Navarro-Leo

small and deformed leaves (Alloway, 2008). Furthermore, as a result of Zn deficiency, several changes in physiological processes occur: decrease in photosynthesis rate, inhibited starch and protein synthesis, membrane destabilization, and stunted flowering and seed production (Brown et al., 1993). Considering these effects of Zn deficiency, we might expect this nutritional deficiency to affect the phytohormone profile within the plant, as observed with other micronutrients such as Fe and Cu (Gutierrez-Carbonell et al., 2015; ~ arrubia et al., 2015). Pen Plant hormones are structurally diverse compounds involved in multiple processes: the plant growth-control, development, senescence and, response to stress. The nutrient supply affects both the synthesis and effect of phytohormones and, in turn, phytohormones can alter the plant's metabolism or structure in order to improve nutrient acquisition and homeostasis within the plant (Marschner, 2012). The auxin indole-3-acetic acid (IAA) is the main auxin within plants and a plant-growth regulator controlling cell division and differentiation, root initiation, and also nutrient-stress responses (Rayle and Cleland, 1992). It has been observed that auxins mediate the P-deficiency response, modifying root

http://dx.doi.org/10.1016/j.phytochem.2016.08.003 0031-9422/© 2016 Elsevier Ltd. All rights reserved.


n, E., et al., Phytohormone profile in Lactuca sativa and Brassica oleracea plants grown under Zn Please cite this article in press as: Navarro-Leo deficiency, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.08.003


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architecture in order to absorb more P (Al-Ghazi et al., 2003). CKs such as trans-zeatine (tZ) and isopentenyl adenine (iP), control growth and developmental processes such as cell proliferation, seed germination, and nutrient mobilization. Furthermore, CKs are involved in plant responses to the availability of nutrients such as Fe, in nitrate, phosphate, and sulphate assimilation, and in abioticstress adaptation (Werner and Schmülling, 2009). GAs is another group of phytohormones that regulate developmental processes in plants, such as leaf expansion, stem elongation, and germination. Likewise, they act in stress response since these hormones are antagonistic with respect to ABA (Weiss and Ori, 2007). ABA is known as a hormone that increases their concentration in stressed plants and is key to coordinate stress responses. Under nutrient deficiency, ABA alters the shoot:root ratio in order to enhance nutrient uptake (Vysotskaya et al., 2008). Ethylene is another essential hormone for stress responses and also regulates such processes as plant growth, carbon assimilation, leaf abscission, and senescence. Ethylene, gaseous hormone synthetised from 1aminocyclopropane-1-carboxylic acid (ACC) derived from methionine, surges in production when the plant is under stress. In addition, ethylene influences nutrient uptake and it is involved in plant responses under growth-limiting conditions (Iqbal et al., 2013). Other two phytohormones relevant in stress response are salicylic (SA) and jasmonic acids (JA). It has been noted that these hormones regulate plant response to several types of abiotic stresses, including heavy-metal stress and they can participate in ion uptake and nutrient storage (Drazic et al., 2006; Wasternack and Hause, 2013). A previous study by our research group concerning Lactuca sativa cv. Phillipus and Brassica oleracea cv. Bronco plants demonstrated that these species differ in the way that nitrogen and
n et al., organic acid metabolism to Zn deficiency (Navarro-Leo 2016; unpublished data). Therefore, in this work, we analyse the role of phytohormones under Zn-deficiency conditions in order to ascertain whether these molecules are key to select and/or generate plants with Zn-deficiency tolerance. 2. Results and discussion 2.1. Biomass, Zn concentration and ZnUtE Because Zn is an essential micronutrient for plants, when its availability is limited, it may constitute a stress that can endanger plant growth and development. Thus, the main symptom of Zn deficiency is a biomass reduction, as has been reported in many crop plants such as tomato, lettuce, potato, and carrot (Alloway, 2008). In our work, we also observed lower foliar biomass in both

species under Zn deficiency; however, this decrease was two-fold higher in L. sativa than in B. oleracea (Table 1). The biomass reduction resulted from a lower Zn concentration, but, unlike biomass, the micronutrient decreased more in B. oleracea (67% lower than in control: Table 1). A reliable parameter relating biomass to Zn concentration is Zn-utilization efficiency (ZnUtE). This parameter reflects the plant's ability to manage and utilize Zn (Rengel, 2007). ZnUtE responded differently to Zn deficiency in both species: while no significant differences were observed in L. sativa plants, ZnUtE increased by a 155% in B. oleracea plants grown under Zn deficiency (Table 1). These results suggest that, under Zn deficiency, although B. oleracea is not able to keep its internal Zn concentration stable as L. sativa does, it maintains a high ability to manage Zn and so that a shortage does not severely affect plant growth. 2.2. Hormone concentration In L. sativa plants grown under Zn deficiency, we detected a general reduction in hormone concentration. This was true for all hormones analysed except for GA1, iP, and JA, with no significant changes in comparison to control plants (Table 2). By contrast, Zndeficient B. oleracea plants increased in GA4, iP, and ACC concentrations did not change with respect to control in IAA, GA3 or ABA while both JA and SA decreased in concentrations (Table 2). Previous studies on hormones regulating plant growth have shown the relationship between Zn deficiency and IAA and GAs concentrations. In Zea mays, Hordeum vulgare, and Avena sativa a decline in GA1 and IAA was observed in plants suffering from Zn deficiency and exhibiting low growth (Suge et al., 1986; Sekimoto et al., 1997). Sekimoto et al. (1997) demonstrated that this reduction in GA concentration could affect maize growth since this was partially recovered when GAs were applied to the culture medium. Furthermore, Cakmak et al. (1989) observed that IAA levels decreased in Phaseous vulgaris L. plants with Zn deficiency, and the authors postulated that this was due to greater oxidative degradation of this hormone rather than an impairment of tryptophan synthesis. Nevertheless, these authors did not find any difference regarding control plants in CK concentration. In the present study, the IAA and GA3 concentration fell sharply in Zn-deficient L. sativa plants, i.e. by 72% and 78%, respectively, than in control plants (Table 2). This plunge in IAA concentration in L. sativa plants could be because Zn is necessary for auxin precursor tryptophan synthesis (Vallee and Auld, 1990). Both the total GA and total CK concentrations dropped by 63% and 10%, respectively, from control (Fig. 1). However, in B. oleracea, the IAA and tZ concentrations were maintained and iP and GA4 concentrations significantly rose

Table 1 Root and leaf biomass and Zn concentration in L. sativa and B. oleracea plants submitted to Zn deficiency. Leaf biomass (g DW/plant) L. sativa

B. oleracea

Analysis of variance Doses (D) Species (S) DS LSD0.05

Control 0.1 mM Zn p-value LSD0.05 Control 0.1 mM Zn p-value LSD0.05

4.20 2.34 *** 0.50 3.61 2.83 ** 0.48 *** NS ** 0.30

± 0.09 ± 0.19

± 0.02 ± 0.14

Zn foliar concentration (mg g
1 PS) 44.58 26.13 *** 5.80 50.49 16.40 *** 8.85 *** NS * 4.39

± 0.18 ± 2.08

± 3.17 ± 0.31

ZnUtE 92.15 ± 1.67 83.57 ± 5.91 NS 17.05 72.05 ± 4.87 182.94 ± 4.58 *** 18.55 *** *** *** 10.46

Values are means ± S.E. (n ¼ 9) and differences between means were compared by Fisher's least-significance test (LSD; P ¼ 0.05). The levels of significance were represented by p > 0.05: NS (not significant), p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).


n, E., et al., Phytohormone profile in Lactuca sativa and Brassica oleracea plants grown under Zn Please cite this article in press as: Navarro-Leo deficiency, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.08.003


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Table 2 Phytohormones concentrations in leaves of L. sativa and B. oleracea plants submitted to Zn deficiency.

L. sativa

Control 0.1 mM Zn p-value LSD0.05 B. oleracea Control 0.1 mM Zn p-value LSD0.05 Analysis of variance Dose (D) Species (S) DS LSD0.05

IAA

GA1

GA3

6.11 ± 1.21 1.71 ± 0.57 * 3.7 67.03 ± 5.37 72.93 ± 2.21 NS 16.13

0.20 ± 0.01 0.24 ± 0.02 NS 0.07 ND ND

3.38 0.76 * 1.58 0.34 0.51 NS 0.28

NS *** NS 6.87

*** *** *** 0.27

GA4

tZ

± 0.36 ± 0.06

ND ND

± 0.03 ± 0.10

0.13 ± 0.01 0.25 ± 0.02 * 0.09

1.39 1.22 *** 0.04 2.02 1.98 NS 0.05 *** *** *** 0.03

± 0.01 ± 0.01

± 0.01 ± 0.01

iP

ACC

ABA

JA

172.78 ± 5.90 191.06 ± 3.71 NS 0.03 11.79 ± 0.61 17.68 ± 0.70 ** 2.58

264.21 ± 14.16 187.60 ± 18.45 ** 64.57 11.96 ± 1.49 27.31 ± 1.68 ** 5.09

99.86 ± 4.17 14.83 ± 0.20 *** 11.6 172.90 ± 0.84 167.33 ± 0.07 NS 6.23

1.17 1.05 NS 0.17 0.95 0.76 *** 0.03

** *** NS 8.11

*** *** *** 26.90

*** *** *** 5.47

*** *** NS 0.07

SA ± 0.06 ± 0.01

± 0.01 ± 0.01

1.19 0.41 *** 0.17 1.58 0.72 *** 0.07

± 0.06 ± 0.01

± 0.02 ± 0.01

*** *** NS 0.07

Values are means ± S.E. (n ¼ 9) and differences between means were compared by Fisher's least-significance test (LSD; P ¼ 0.05). The levels of significance were represented by p > 0.05: NS (not significant), p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). ND: Not detected.

Fig. 1. Effect of Zn deficiency on the concentrations of total gibberellins (GAs) (A) and cytokinins (CKs) (B). Values are expressed as means ± standard error of the mean. The levels of significance were represented by p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).

(Table 2), so that in this species the total GA concentration increased by 63% and the total CK concentration remained stable. These three hormones are important for plant growth and development, this perhaps explaining the significant biomass reduction occurring in L. sativa under Zn deficiency and why biomass loss was lower in B. oleracea plants (Table 1). Furthermore, these hormones could improve Zn homeostasis and utilization in this species under Zn shortage. To our knowledge, there are no studies available about hormones controlling stress response, like ethylene, in plants under Zn deficiency. In our study, the concentration of the ethylene precursor ACC showed opposite effects in the two species under Zn deficiency, since L. sativa values were 49% lower but in B. oleracea 128% higher than control (Table 2). It was observed that ACC levels are

correlated with ethylene levels and when ACC levels increase usually this is accompanied by a rise in ethylene concentration (Ghanem et al., 2008). However this warrants caution because these authors did not directly determine the ethylene levels. Kabir et al. (2012) growing Pisum sativum and Li et al. (2014) raising Medicago truncatula plants under Fe deficiency noted that Feefficient cultivars had higher levels of ethylene than did Feinefficient ones. This could result because ethylene can promote mechanisms to cope with Fe-deficiency, such as the IRT and AHA (Hþ-ATPase) genes activation, and the increase in flavin and phenolic compounds synthesis. In addition ethylene induces structural changes in root in order to facilitate Fe absorption (Lucena et al., 2015). All of these mechanisms can also promote Zn
gue
la et al., 2008) however, absorption (Marschner, 1993; Se although if they acted in L. sativa they do not appear to be triggered by the ethylene signaling pathway, since the concentration of this hormone decreased (Table 1). Another factor relating Zn to ethylene is the fact that this element is necessary for ethylene response as it is part of its receptor, and thus Zn deficiency might induce lower sensitivity to ethylene in the plant (Burg and Burg, 1967). This could have occurred in our B. oleracea plants in which the response to this hormone would be lower despite the higher levels of ethylene precursor ACC. A weaker ethylene response would slow the activation of senescence in this species, thus reducing the loss of biomass. Another hormone involved in stress signaling is ABA. The few studies linking ABA to nutrient deficiencies present diverse results. Vysotskaya et al. (2008) observed that ABA accumulated in the leaves of wheat submitted to nutrient deficiency. The authors demonstrated that ABA was key to control the plant shoot:root ratio, favouring root growth in order to face nutrient deficiency. In barley, exogenous ABA stimulates glucose 6P dehydrogenase expression and activity (Cardi et al., 2011) in order to sustain N assimilation (Esposito et al., 1998). By contrast, another study reported that ABA declined in leaves of Phaseolus vulgaris plants grown under Zn deficiency (Cakmak et al., 1989). In our study, the Zn-deficiency treatment lowered the ABA concentration in L. sativa by 85% while no changes from control were detected in B. oleracea
gue
la et al. (2008) demonstrated that ABA and also CKs (Table 2). Se inhibit Fe deficiency response by repressing IRT, which is involved in Fe absorption. In addition to Fe, it was shown that IRT1 can transport Zn inside the plant. Likewise, CKs are reportedly able to repress the deficiency response of several nutrients (Vert, 2002). Thus, in our deficient L. sativa plants the lower CK and ABA levels could stimulate the Fe-deficiency response, which in turn could promote Zn uptake, as we found in this species, which accumulated a significant Zn concentration under deficiency conditions


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(Table 1). SA and JA are two other hormones that can help plants to cope with stress conditions. Regarding nutritional stress, it has been reported that SA treatments can stimulate the uptake of nutrients such as K, Mg, and Mn in maize plants (Gunes et al., 2005) and Fe in soybean (Aly and Soliman, 1998). Moreover, in a recent study with several species grown under P deficiency noted that the JA concentration increased (Khan et al., 2016). However, in our study, Zn deficiency caused a reduction in both JA and SA concentrations (Table 2), and therefore the synthesis of these hormones could have been affected by Zn shortage and their reduction could somehow benefit plants to cope with Zn deficiency.

could negatively affect L. sativa growth under Zn deficiency, while in B. oleracea these hormones could sustain plant growth as they increase or maintain their concentration. A decline in the concentration of stress-signaling hormones such as ethylene precursor ACC, ABA, SA, and also CKs might be involved for Zn absorption in L. sativa, while an increase in GA4, iP, and ACC and a decrease in JA and SA might contribute to better Zn homeostasis in B. oleracea plants. This work points out some hormones that could be key for plant breeding under low Zn-bioavailability conditions, but further research is required to clarify the role of hormones in plant adaptation to this micronutrient deficiency.

4. Experimental

2.3. Correlation analysis In this work, we seek to identify the hormones that are mainly affected by Zn deficiency in both species assessed. For this purpose we carried out a linear regression analysis using foliar biomass, foliar Zn concentration, and ZnUtE. According the correlation coefficients in L. sativa plants, the decrease in GA3, tZ, ABA, ACC and SA correlated well with the decline in biomass and Zn concentration while no hormone correlated with ZnUtE (Table 3). In this species, the decrease in GAs and CKs appeared to affect biomass since these hormones are involved in plant growth while ACC, ABA, and SA reduction might limit the stress response to Zn deficit and thereby affect plant biomass. However, the reduction of the concentration of these three hormones could somehow favour Zn accumulation in L. sativa plants. No strong correlations with ZnUtE appeared (Table 3), suggesting that this parameter in L. sativa could not present significant changes between Zn deficient and control plants (Table 1). In B. oleracea plants the reduction of biomass and Zn concentration correlated positively with JA and SA concentrations and negatively with the ACC concentration (Table 3). A negative correlation was found between ZnUtE, JA, and SA while a positive correlation appeared with GA4, iP, and ACC (Table 3). In this species JA and SA could, in some unknown way, affect Zn accumulation in leaves or, conversely, Zn deficiency could negatively affect JA and SA synthesis given that Zn concentration and theses hormones diminished by almost the same proportion. Regarding ZnUtE, an increase in the GA4, iP, and ACC concentrations and/or a decrease in the JA and SA concentrations could in some way benefit Zn homeostasis within the plant since the results showed a good correlation between these hormones and ZnUtE (Table 3).

4.1. Plant material, growth conditions, and treatments L. sativa cv. Phillipus, and B. oleracea cv. Bronco seeds were germinated and grown for 30 days in cell flats (cell size ¼ 3 cm  3 cm  10 cm) filled with a perlite mixture, and flats were placed on benches in an experimental greenhouse in southern Spain (Granada, Motril, Saliplant S.L.). The 30-day-old seedlings were transferred to a growth chamber under controlled environmental conditions with a relative humidity of 60e80%, temperature of 22/18  C (day/night) and 12/12-h photoperiod at a photosynthetic photon flux density (PPFD) of 350 mmol m
2 s
1 (measured at the top of plants with a 190 SB quantum sensor, LI-COR Inc., Lincoln, NE, USA). Plants were grown with hydroponic culture in lightweight polypropylene trays (60 cm top diameter, 60 cm bottom diameter, and 7 cm high) with a volume of 3 l. Throughout the experiment the plants received a growth solution composed of 4 mM KNO3, 3 mM Ca(NO3)2  4 H2O, 2 mM MgSO4  7 H2O, 6 mM KH2PO4, 1 mM NaH2PO4  2 H2O, 2 mM MnCl2  4 H2O, 0.25 mM CuSO4  5 H2O, 0.1 mM Na2MoO4  2 H2O 5 ppm, Fe-chelate (Sequestrene; 138FeG100) and 10 mM H3BO3. This solution, with a pH of 5.5e6.0, was changed every three days. Treatments were started 30 days after germination and were maintained for 21 days. Plants were grown with different Zn doses: 10 mM of ZnSO4 as control and 0.1 mM of ZnSO4 as deficiency treatment. The experimental design consisted of randomized complete block with four treatments (L. sativa-control, B. oleracea-control, L. sativa-0.1 mM Zn, B. oleracea-0.1 mM Zn), eight plants per treatment and three replications each.

4.2. Plant sampling 3. Conclusions According to our results, both species assessed presented a different response when submitted to Zn deficiency. L. sativa showed a better ability to accumulate Zn in leaves, while B. oleracea registered a minor loss in biomass through a greater ZnUtE. A decline in growth-promoting hormones such as IAA, GAs, and CKs

Plants from each treatment were divided into roots and leaves, washed with distilled water, dried on filter paper, and weighed for fresh weight (FW). Half of the roots and leaves from each treatment were frozen at
30  C for later biochemical assays and the other half of the plant material was lyophilized to measure the dry weight (DW) and the Zn concentration was determined.

Table 3 Linear correlation coefficients between leaf biomass, Zn foliar concentration and ZnUtE and hormonal concentration in leaves of L. sativa and B. oleracea plants submitted to Zn deficiency.

Leaf biomass Zn foliar concentration ZnUtE

L. sativa B. oleracea L. sativa B. oleracea L. sativa B. oleracea

IAA

GA1

GA3

GA4

tZ

iP

ACC

ABA

JA

SA

0.84*
0.55 0.82*
0,43 0.59 0.49


0.50 e
0.44 e
0.43 e

0.99***
0.48 0.97**
0.51 0.57 0.58

e
0.83* e
0.91* e 0,92*

0.99*** 0.75 0.95** 0.78 0.62
0.68


0.78
0.88*
0.83*
0.96**
0.35 0.98***

0.95**
0.90* 0.96** 0.93** 0.50 0.97**

0.99*** 0.77 0.97** 0.72 0.55
0.73

0.75 0.95** 0.71 0.98*** 0.59
0.99***

0.99*** 0.96** 0.97** 0.98*** 0.54
0.99***

p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***), n ¼ 9.


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4.3. Analysis of Zn and ZnUtE The Zn concentration was determined by ICP-MS after a sample of 150 mg dry material was subjected to a process of mineralization with sulphuric acid and H2O2 (Wolf, 1982). Zn-use efficiency parameters (ZnUtE) were calculated as leaf-tissue dry weight (LDW) divided by Zn concentration (g2 LDW mg
1 Zn) (Siddiqi and Glass, 2008). 4.4. Hormone extraction and analysis IAA, GAs (GA1, GA3 and GA4), CKs (tZ and iP), ethylene precursor ACC, ABA, SA and JA were analysed as in Ghanem et al. (2008) with some modifications. Briefly, 30 mg of homogenized dry material were dropped in 0.5 ml of cold (
20  C) extraction mixture of methanol/water (80/20, v/v). Solids were separated by centrifugation (20000 g, 15 min) and re-extracted for 30 min at 4  C in additional 0.5 ml of the same extraction solution. Pooled supernatants were passed through Sep-Pak Plus yC18 cartridge (SepPak Plus, Waters, USA) to remove interfering lipids and part of plant pigments and evaporated at 40  C under vacuum either to near dryness or until the organic solvent was removed. The residue was dissolved in 1 ml methanol/water (20/80, v/v) solution using an ultrasonic bath. The dissolved samples were filtered through Millex nylon membrane filters 13 mm diameter of 0.22 mm pore size (Millipore, Bedford, MA, USA). Next, 10 ml of filtrated extract were injected in a U-HPLC-MS system consisting of an Accela Series UHPLC (ThermoFisher Scientific, Waltham, MA, USA) coupled to an Exactive mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) using a heated electrospray ionization (HESI) interface. The mass spectra were determined using the Xcalibur software version 2.2 (ThermoFisher Scientific, Waltham, MA, USA). For quantification of the plant hormones, calibration curves were constructed for each component analysed (1, 10, 50, and 100 mg l
1) and corrected for 10 mg l
1 deuterated internal standards. Recovery percentages ranged between 92 and 95%. 4.5. Statistical analysis Data were subjected to a simple ANOVA at 95% confidence, using the Statgraphics Centurion XVI program. A two-tailed ANOVA was applied to ascertain whether the doses of Zn and the species significantly affected the results and means were compared by Fisher's least significant differences (LSD). The significance levels for both analyses were expressed as * P < 0.05, **P < 0.01, ***P < 0.001, or NS (not significant). Acknowledgments This work was financed by the PAI programme (Plan Andaluz de
n, Grupo de Investigacio
n AGR161) and by a Grant from Investigacio
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n, E., et al., Phytohormone profile in Lactuca sativa and Brassica oleracea plants grown under Zn Please cite this article in press as: Navarro-Leo deficiency, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.08.003