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PLPH-51786; No. of Pages 9

Journal of Plant Physiology xxx (2013) xxx– xxx

Contents lists available at ScienceDirect

Journal of Plant Physiology

j o ur na l ho me page: www.elsev ier .com/ locate / jp lph

hysiology

ariation of antioxidants and secondary metabolites initrogen-deficient barley plants

ozef Kovácika,∗, Borivoj Klejdusa, Petr Babulab, Markéta Jarosováa

Institute of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelská 1, 613 00 Brno, Czech RepublicDepartment of Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackého 1/3, 612 42 Brno, Czech Republic

r t i c l e i n f o

rticle history:eceived 3 June 2013eceived in revised form 8 August 2013ccepted 12 August 2013vailable online xxx

eywords:luorescence microscopyineral nutritionxidative stress

a b s t r a c t

Barley (Hordeum vulgare cv. Bojos) plants cultured in low nitrogen (N) containing Hoagland solution(20 mg/l) were exposed to N deficiency (−N) over 15 days. Plants revealed relatively high tolerance tototal N deficit because shoot length was not altered and dry biomass was depleted by ca. 30% while rootlength increased by ca. 50% and dry biomass remained unaffected. Soluble proteins and free amino acidsdecreased more pronouncedly in the roots. Antioxidants (glutathione and ascorbic acid) decreased in theshoots but increased or were not affected in the roots. Ascorbate peroxidase and glutathione reductaseactivities were depleted in shoots and/or roots while guaiacol peroxidase activity was stimulated in theshoots. In accordance, fluorescence signal of reactive oxygen species (ROS) and nitric oxide was elevated inshoots but no extensive changes were observed in roots if +N and −N treatments are compared. At the level

henolic metabolismeactive oxygen species

of phenolic metabolites, slight increase in soluble phenols and some phenolic acids and strong elevationof flavonoid homoorientin was found in the shoots but not in the roots. Fluorescence microscopy in termsof detection of phenols is also discussed. We also briefly discussed accuracy of quantification of someparameters owing to discrepancies in the literature. It is concluded that N deficiency induces increasein shoot phenolics but also elevates symptoms of oxidative stress while increase in root antioxidantsprobably contributes to ROS homeostasis aimed to maintain root development.

ntroduction

Nitrogen (N) is essential plant macronutrient because it isnvolved in the biosynthesis of amino acids, proteins and enzymesScheible et al., 2004; Kovácik and Backor, 2007). Owing to N impor-ance in metabolism, their limited availability or deficiency resultsn reduced growth and lower yield of plants (Rubio-Wilhelmi et al.,011). At the same time, shift from N-based to C-based com-ounds is usually observed (Rubio-Wilhelmi et al., 2012a,b). These-based metabolites include mainly phenolics, such as phenoliccids, flavonoids as well as coumarins (Kovácik et al., 2007; Giorgit al., 2009; Rubio-Wilhelmi et al., 2012a). This effect of N deficiencyn phenolic content is typical because depletion of other macronu-rient such as potassium does not elevate phenols (Nguyen et al.,010) while phosphate deficiency symptoms are partially similaro those of N deficit (Juszczuk et al., 2004).

Phenolics (or part of phenolics) are important so-called

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on-enzymatic antioxidants and include several thousands ofompounds. They act as efficient antioxidants both in plantsnd in human diet, therefore studies of their accumulation in

∗ Corresponding author. Tel.: +420 545 133281; fax: +420 545 212044.E-mail address: jozkovacik@yahoo.com (J. Kovácik).

176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.jplph.2013.08.004

© 2013 Elsevier GmbH. All rights reserved.

crop/medicinal plants received greater attention owing to producemore healthy preparations (Giorgi et al., 2009). Owing to direct con-nection between C and N metabolism, manipulating the N level isa good tool how to increase amount of phenols in plants (Kováciket al., 2011). Besides, such studies are also important in terms ofmore complex responses of metabolites and metabolism withinplant tissue, providing information about stress tolerance.

Environmental stress typically stimulates enhanced productionof reactive oxygen species (ROS) in aerobic organisms includ-ing plants (Noctor and Foyer, 1998). Nitric oxide (NO) is anotherimportant gaseous molecule being involved in the regulation ofmetabolism (Kovácik et al., 2010). These molecules are also formedunder N deficiency (Kovácik et al., 2009) or potassium deficiency(Hernandez et al., 2012). ROS overproduction is therefore con-trolled by various non-enzymatic (phenolics, glutathione, ascorbicacid) and enzymatic (ascorbate peroxidase, guaiacol peroxidase,glutathione reductase) antioxidants (Sakihama et al., 2002; Chenet al., 2010; Gajewska and Skłodowska, 2010).

Barley (Hordeum vulgare) is an important crop species for food-stuff and beer brewing industry and involves numerous cultivars

and secondary metabolites in nitrogen-deficient barley plants. J Plant

(Dvoráková et al., 2008). Its responses to environmental stresswere tested mainly after application of heavy metals (Chen et al.,2010). Known metabolic profile involves phenolics in caryopsis(Dvoráková et al., 2008) or excellently identified flavones in leaf

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Fig. 1. Representative photo of Hordeum vulgare cultivation in hydroponics. Noteslight chlorosis of N-deficient (−N) plants in comparison with control (+N) after 15d

baaie

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P

wwpsi1E0apstcwfl

ays of N deficiency.

iomass (Ferreres et al., 2008). Surprisingly, basic antioxidants suchs ascorbate and glutathione involve such variable values in leafnd/or root tissue that they could only hardly be ascribed to var-ous cultivars (Palatnik et al., 2002; Finkemeier et al., 2003; Chent al., 2010).

Although manipulation of nitrate availability was intensivelytudied in various plants (see above), alteration of metabolism inerms of N nutrition in barley is only poorly known (Finkemeiert al., 2003). Quantitative data are completely missing in termsf secondary metabolites in vegetative organs. We therefore com-lexly studied response of known Czech barley cultivar (cv. Bojos)o nitrate deficiency over sufficiently long exposure period untilppearance of visible symptoms. Growth parameters, amino acids,henolics and other antioxidants including selected enzymaticctivities were monitored and are complexly discussed in rela-ion to growth changes. Extensive fluorescence microscopy waslso performed to allow visualization of some parameters. Manyarameters in barley plants are presented here for the first time andesponses to N deficiency are therefore compared with other plantpecies or other stress impacts. We also briefly discussed accuracyf quantification of some parameters measured in barley owing toiscrepancies in the literature.

aterials and methods

lant culture, experimental design and statistics

Caryopses (grains) of barley (Hordeum vulgare cv. Bojos)ere surface-sterilized with 70% ethanol for 1 min, rinsedith deionised water and placed on Petri dishes with filteraper and deionised water. After 48 h, homogenous germinatedeedlings were placed to 1/10-strength Hoagland solution (contain-ng 403 �M Ca(NO3)2·4H2O, 52.2 �M NH4H2PO4, 604 �M KNO3,99 �M MgSO4·7H2O, 35.6 �M NaOH, 28.8 �M KOH, 8.92 �MDTA, 8.96 �M FeSO4·7H2O, 9.68 �M H3BO3, 2.03 �M MnCl2·4H2O,.314 �M ZnSO4·7H2O, 0.210 �M CuSO4·5H2O, 0.139 �M Na2MoO4nd 0.0859 �M CoCl2·6H2O, pH maintained at 6.0) in 7-L brownlastic pots with continual aeration (Fig. 1). One pot contained 25eedlings and solutions were changes every 5 days. Whole cul-

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Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.08.004

ivation was performed in a growth chamber under controlledonditions: 12 h day (6.00 am to 6.00 pm); photon flux densityas ∼250 �mol m−2 s−1 PAR at leaf level supplied by cool whiteuorescent tubes L36W/840 (Lumilux, Osram, Germany); 25/20 ◦C

PRESSysiology xxx (2013) xxx– xxx

day/night temperature; and relative humidity ∼60%. After 15 daysof cultivation, part of plants was subjected to nitrogen (N) defi-ciency by substituting N-containing salts with equimolar saltswithout N (Kovácik and Backor, 2007; Kovácik et al., 2011). Con-trol plants were further cultured in N-containing medium. Becausefirst symptoms of N deficiency on shoots appeared after 10–11 days,experiment was closed after 15 days of N-deficient conditions. Thenplants were harvested and separated to shoots and roots and length,fresh and dry mass were measured. For parameters measured infresh samples, whole shoots or roots were powdered using liq-uid N2 and assayed as described below. Spectrophotometry wascarried out with Agilent/HP DAD UV/Vis 8453 Spectrophotometer.Fluorescence microscopy was done with Axioscop 40 microscope(Carl Zeiss, Germany) equipped with appropriate set of excita-tion/emission filters.

Data were evaluated using Student’s t-test by comparison con-trol (+N) and nitrogen-deficient (−N) variant for each parameter.Number of replications (n) in tables/figures denotes individualplants measured for each parameter. Two independent repetitionsof the whole experiment were performed in order to check repro-ducibility.

Measurement of growth, tissue water content and nitrogenousmetabolites

Fresh and dry matters were measured in order to determine theplant water content [100 − (dry mass × 100/fresh mass)] allowingrecalculation of parameters measured in fresh samples. These driedsamples were ground to a fine powder and analyzed for free aminoacids and phenolics.

Soluble proteins were quantified according to Bradford method(1976) in homogenates prepared using 50 mM potassium phos-phate buffer containing 5 mM insoluble PVPP (pH 7.0, 1 g FW/5 ml)and bovine serum albumin as standard. Free amino acids wereextracted with 80% aqueous ethanol using computer controlledIKA Werke 50 device related to Soxhlet apparatus and analyseswere performed on an HP 1100 liquid chromatograph (HewlettPackard, Waldbronn, Germany) with fluorometric detector FLD HP1100 and using precolumn derivatization with o-phtalaldehydeand 9-fluorenylmethyl chloroformate (Kovácik et al.,2011).

Quantification of glutathione, ascorbic acid and antioxidativeenzymes

Reduced (GSH) and oxidized glutathione (GSSG) and ascorbicacid (AsA) were extracted with 35 mM HCl (0.2 g FW/2 ml) andquantified using LC–MS/MS (Agilent 1200 Series Rapid Resolu-tion LC system coupled on-line to a detector) Agilent 6460 Triplequadrupole with Agilent Jet Stream Technologies (Kovácik et al.,2012) at m/z values 308/76, 613/231 (Airaki et al., 2011) and 177/95in positive MRM mode, respectively. Separation was done using col-umn Zorbax SB-C18 50 × 2.1 mm, 1.8 �m particle size and mobilephase consisting of 0.2% acetic acid and methanol (95:5). The flow-rate was 0.6 ml/min and column temperature was set at 25 ◦C.Freshly prepared standards were used for calibration and quan-tification.

Activities of antioxidative enzymes were measured in potas-sium phosphate buffer homogenates prepared as mentioned above.Ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glu-

and secondary metabolites in nitrogen-deficient barley plants. J Plant

tathione reductase (GR) activities were measured as the oxidationof ascorbate (290 nm) and guaiacol (470 nm) and the reduction ofGSSG (412 nm), respectively (Kovácik and Backor, 2007; Kováciket al., 2009).

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Table 1Growth and physiological responses in Hordeum vulgare plants cultured either in nitrogen-sufficient (+N) or nitrogen-deficient (−N) solution over 15 days.

Shoot Root

+N −N +N −N

Length (cm plant−1) 30.8 ± 2.21 27.1 ± 3.08 20.6 ± 2.37 32.6 ± 2.84***

Dry weight (mg plant−1) 119.1 ± 18.3 75.7 ± 13.4*** 41.9 ± 7.05 44.7 ± 8.25Water content (%) 85.57 ± 0.62 85.37 ± 0.86 91.16 ± 0.39 89.34 ± 0.42***

Soluble proteins (mg g−1 DW) 45.6 ± 1.12 38.7 ± 2.24** 44.5 ± 2.75 27.9 ± 1.80***

Data are means ± SDs (n = 5 for proteins and n = 10 for other parameters).* Significant difference at 0.05 level of Student’s t-test between control and −N.

** Significant difference at 0.01 level of Student’s t-test between control and −N.*** Significant difference at 0.001 level of Student’s t-test between control and −N.

A

as

iunbcTTgas

wqcimf

F

aprR2cwR(rdPAaeasbtUt3a

(Table 2).

Table 2Accumulation of free amino acids (�mol g−1 DW) in Hordeum vulgare plants culturedeither in nitrogen-sufficient (+N) or nitrogen-deficient (−N) solution over 15 days.

Shoot Root

+N –N +N –N

Aspartic acid 0.93 ± 0.08 0.52 ± 0.02** 2.83 ± 0.30 0.86 ± 0.03***

Glutamic acid 0.49 ± 0.02 0.40 ± 0.04* 3.71 ± 0.18 1.47 ± 0.20***

Serine 3.19 ± 0.12 3.16 ± 0.17 5.44 ± 0.35 2.91 ± 0.32***

Histidine 0.27 ± 0.03 0.26 ± 0.02 0.65 ± 0.04 0.40 ± 0.05**

Glycine 3.77 ± 0.27 2.35 ± 0.21** 4.81 ± 0.34 2.13 ± 0.26***

Threonine 0.28 ± 0.02 0.73 ± 0.05*** 0.47 ± 0.01 0.36 ± 0.04**

Arginine 0.75 ± 0.04 1.09 ± 0.11** 0.90 ± 0.08 0.45 ± 0.03***

Alanine 13.57 ± 1.11 6.23 ± 0.22*** 8.61 ± 0.31 3.43 ± 0.32***

Tyrosine 1.20 ± 0.03 1.11 ± 0.08 0.77 ± 0.03 0.41 ± 0.03***

Cysteine 0.87 ± 0.06 0.80 ± 0.02 1.12 ± 0.09 0.56 ± 0.02***

Valine 0.79 ± 0.01 1.01 ± 0.06** 1.52 ± 0.22 0.94 ± 0.07*

Methionine 2.57 ± 0.11 2.48 ± 0.21 3.35 ± 0.25 1.13 ± 0.20***

Phenylalanine 0.49 ± 0.01 0.51 ± 0.04 1.03 ± 0.02 1.02 ± 0.17Isoleucine 1.37 ± 0.04 1.33 ± 0.07 0.63 ± 0.03 0.39 ± 0.03***

Leucine 2.77 ± 0.08 3.01 ± 0.20 2.46 ± 0.28 1.59 ± 0.20*

ssay of phenolic metabolites

Total soluble phenols were extracted with 80% methanolnd quantified using Folin–Ciocalteu method with gallic acid astandard with detection at 750 nm (Kovácik and Backor, 2007).

Selected cinnamic and benzoic acid derivatives were measuredn 80% methanol extracts (free acids). Quantification was donesing Agilent 1200 Series Rapid Resolution LC system (Agilent Tech-ologies, Waldbronn, Germany) consisted of on-line degasser, ainary pump, a high performance SL autosampler, a thermostatedolumn compartment, and a photodiode array UV–vis detector.he system was coupled on-line to an MS detector Agilent 6460riple quadrupole LC–MS/MS with Agilent Jet Stream Technolo-ies. Compounds were identified based on the specific m/z valuesnd retention time and quantified using commercially availabletandards (Klejdus et al., 2013).

Flavonoid homoorientin (=isoorientin, luteolin-6-C-glucoside)as extracted with 80% ethanol. Quantification was done by triple

uadrupole LC–MS/MS device mentioned above using standardompound (Extrasynthese, France). Identity was verified by mon-toring MRM at m/z value 447/327 in negative ESI mode for this

etabolite (Ferreres et al., 2008); collision energy was 16 eV andragmentor value 170 V.

luorescence microscopy

Sections from shoots were taken ca. 3 cm above stem basend were immediately stained and observed. In roots, variousarts were excised and stained (seminal root, lateral root andoot tip). ROS and RNS/NO were stained using CellROX® Deeped Reagent (644ex/665em, Life Technologies Corporation) and,3-diaminonaphthalene (Sigma–Aldrich) forming highly fluores-ent 1H-naphthotriazole product (365ex/415em) in accordanceith manufacturer’s instructions. Stock solution of CellROX® Deeped Reagent in DMSO was diluted by Phosphate Buffered SalinePBS) buffer (0.05 M, pH 6.8) to final concentration of 5 �M,oots were stained for 60 min at 37 ◦C. Stock solution of 2,3-iaminonaphthalene (DAN) in 0.62 M HCl was used diluted byBS buffer (0.05 M, pH 6.8) to the final concentration of 250 �M.fter incubation, roots were washed three times by PBS buffernd observed. For NO microscopy, 4,5-diaminofluorescein diac-tate (495ex/515em, Life Technologies Corporation) was also testednd samples revealed trend identical to that of DAN (data nothown). Staining was carried out in the dark to avoid possi-le light-accelerated oxidation. Phenolics were visualized usinghe NPR reagent (2-aminoethyl diphenylborinate, Sigma–Aldrich,

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Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.08.004

SA). Due to its low solubility in water, a 1% (w/v) ethanolic solu-ion was used (Gitz et al., 2004). Plant material was incubated for0 min at room temperature, then washed three times in PBS buffernd observed.

Results

Growth and physiological responses of barley to N deficiency

After 15 days of N deficiency, barley plants showed slight chloro-sis and yellowish apex of the oldest leaves (Fig. 1). Shoot lengthwas not affected while root length increased by ca. 50% (Table 1).On the other hand, shoot dry weight decrease by ca. 30% but rootdry weight was not significantly altered though low stimulationappeared. Tissue water content was depleted only in the roots(Table 1).

Changes of nitrogenous metabolites

Soluble proteins decreased in whole plants being depressedmore in the roots (Table 1). Accumulation of free amino acidsrevealed trend similar to that of soluble proteins. In shoots, manycompounds showed no response to N deficiency while Asp, Glu,Gly and Ala decreased but Thr, Arg, Val and Lys increased; how-ever, sum of amino acids decreased by ca. 22% (Table 2). Almostall quantified amino acids in roots were depleted, except forPhe and Pro: this led to lower sum of amino acids by ca. 50%

and secondary metabolites in nitrogen-deficient barley plants. J Plant

Lysine 0.94 ± 0.06 1.37 ± 0.06** 0.57 ± 0.06 0.34 ± 0.05**

Proline 1.78 ± 0.11 1.80 ± 0.22 0.97 ± 0.10 0.85 ± 0.08Sum 36.02 ± 2.33 28.17 ± 1.66* 39.83 ± 2.04 19.24 ± 1.67***

Data are means ± SDs (n = 5).

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Shoot

***

0

25

50

75

100

GS

H (

µg

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DW

)

Root

**

0

15

30

45

60

***

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2

4

6

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SG

(µ

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-1 D

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*

0

1

2

3

*

0

40

80

120

160

As

A (

µg

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0.2

0.4

0.6

0.8

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0

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10

15

N deficitcontrol

So

lub

le p

hen

ols

(m

g g

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0

2

4

6

8

10

N deficitcontrol

Fig. 2. Quantitative changes of the main antioxidants in Hordeum vulgare plants cultured either in nitrogen-sufficient (control, +N) or nitrogen-deficient (−N) solution over15 days. Data are means ± SDs (n = 5). *, **, *** Indicate significance at 0.05, 0.01 and 0.001 level of Student’s t-test between control and −N. GSH – reduced glutathione, GSSG– . Note

R

iii(

ib(a

oxidized glutathione, AsA – ascorbic acid, soluble phenols – total soluble phenols

esponses of antioxidants and antioxidative enzymes to N deficit

Reduced (GSH) and oxidized (GSSG) glutathione was depletedn N-deficient shoots (−43% and −72%) but increase significantlyn the roots (+104% and +56%, Fig. 2). Ascorbic acid also decreasedn shoots (−34%) but no significant alteration was found in rootsFig. 2).

Activity of ascorbate peroxidase decreased (−43%) while gua-

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acol peroxidase activity increase (+102%) in shoots of N-deficientarley plants but no significant changes were observed in the rootsFig. 3). Glutathione reductase activity was depleted in both shootsnd roots (−21% and −45%).

strong quantitative difference of AsA between shoot and root.

Fluorescence microscopy of ROS and NO under N deficiency

Staining of reactive oxygen species and nitric oxide revealedintensification of fluorescence signal in stem cross sections underN deficiency (Fig. 4). On the contrary, seminal root, lateral roots androot tips of barley did not show any intensive changes (Fig. 5).

Alteration of phenolic metabolism

and secondary metabolites in nitrogen-deficient barley plants. J Plant

Total soluble phenols slightly increased in shoots (+27%) butwere unaltered in roots (Fig. 2). Among derivatives of benzoicand cinnamic acids, so-called phenolic acids, accumulation of

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***

0

300

600

900

AP

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N deficit

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2

4

6

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X a

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y

(µm

ol m

in-1

mg

-1 p

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**

**

0

30

60

90

toortoohs

GR

acti

vit

y

(nm

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in-1

mg

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rote

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Fig. 3. Changes of ascorbate peroxidase (APX), guiacol peroxidase (GPX) and glu-tnO

psasof(

D

iessi

athione reductase (GR) activities in Hordeum vulgare plants cultured either initrogen-sufficient (control, +N) or nitrogen-deficient (−N) solution over 15 days.ther details are the same as in Fig. 2.

-hydroxybenzoic acid and its aldehyde, vanillic and salicylic acidignificantly increased in shoots. In the roots, gallic acid, vanilliccid and vanillin were elevated (Table 3). Notwithstanding this,um of acids was not enhanced by N deficiency either in shootsr roots because quantitatively dominant derivatives (p-coumaric,erulic and sinapic acid in shoot or root) remained unalteredTable 3).

iscussion

Barley cultivar (cv. Bojos) is one the most cultured cultivarsn the Czech Republic though it was not subjected to extensive

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xperimental investigation (Dvoráková et al., 2008). Plants revealedimilar growth intensity if compared to cv. Gerbel (30 cm perhoot/30 days in our study, 15 cm per shoot/10 days of culturen a study by Finkemeier et al., 2003) but our cultivar contained

PRESSysiology xxx (2013) xxx– xxx 5

higher amount of proteins despite low nitrogen content in theculture solution. Although inter-experiment comparison could beaffected by various other conditions, this still indicates that cv.Bojos is robust enough for both field and laboratory studies. Wealso observed considerable tolerance to N deficiency as plants didnot reveal growth depression after 7 days under total N deficitand even after 15 days shoot length was not affected and drybiomass decreased by about 30% (including slight chlorosis, Fig. 1).Besides, plants had two leaves after 7 days of N deficiency (photonot shown) and even third leaf appeared during next deficiency(containing three leaves per plants at the end of the experiment).This is another indication about good adaptability of this cultivar,considering the fact that older plants such as chamomile culturedin solution with 10-times higher N content (expecting they containhigher endogenous sources of nitrogen) exposed to N deficiencyshowed more pronounced decrease of shoot biomass and pro-tein content after 12 days of N-deficient conditions (Kovácik andBackor, 2007). Besides, barley cv. Gerbel revealed protein deple-tion by about 50% (Finkemeier et al., 2003) while our cv. Bojosshowed less-expressive decrease (Table 1). Another interestingobservation is high sum of amino acids: this value is similar tothat observed in chamomile plants cultured with higher N contentin solution (Kovácik et al., 2011). Slightly affected accumulationof individual amino acids in shoots (Table 2) could be influencedby at least two reasons: (i) protein catabolism (as confirmed bydecrease of proteins) and (ii) amino acid biosynthesis (as confirmedby even increased accumulation of some acids). In accordance withour study, even discontinuous changes of some individual aminoacids were observed in tobacco plants at various levels of N star-vation (Rubio-Wilhelmi et al., 2012b). One possible explanationcould be various regulations of genes involved in the metabolism ofamino acids as observed in Arabidopsis N-deprived plants: nitratere-addition led to increase in amino acids involved in centralmetabolism while minor amino acids decreased (Scheible et al.,2004). Root growth response was more expressive than those ofshoots in response to N starvation and increase by ca. 50% wasobserved despite unaltered root biomass (Table 1). This enlarge-ment of root system aimed to “search” nitrogen source in orderto intensify its uptake was also observed in other barley cultivar(Finkemeier et al., 2003) and in other plant species (Kovácik andBackor, 2007). Such responses were also observed in Arabidop-sis thaliana where only 2-days long N starvation induced growthof roots though they were thin (Scheible et al., 2004). On barleyroots also numerous root hairs appeared after 15 days of N defi-ciency (Fig. 5). More pronounced depletion of amino acids in theroots could be related just to root development and various planthormones and genes were reported to participate in this process(Scheible et al., 2004; Rubio-Wilhelmi et al., 2011).

Glutathione is one of important antioxidants of living cellsbeing involved also in responses of plants to environmental stress(Kovácik et al., 2012) including barley (Chen et al., 2010). Ele-vated amount of GSH but also of GSSG in roots indicates thatdespite depleted root GR activity, enhanced biosynthesis of GSHoccurs. This is in contradiction to data observed in barley cv. Ger-bel, where unaltered GR activity but depletion of total glutathionewas observed (Finkemeier et al., 2003). Enhanced synthesis of GSHin N-deficient barley roots (Fig. 2) could also be inferred from deple-tion of its precursors (Glu, Gly, Cys) mainly in the roots (Table 2).Our cultivar seems therefore be more tolerant in terms of GSHsynthesis. Alternatively, less accurate quantification as “total glu-tathione” (Finkemeier et al., 2003) could be a reason for differentresponses because we quantified GSH and GSSG separately and

and secondary metabolites in nitrogen-deficient barley plants. J Plant

using sensitive LC–MS/MS method that is more precise comparedto spectrophotometry (Airaki et al., 2011). Increase in GSH amounthas also been observed in the barley plants exposed to cadmiumthough with substantially lower intensity (Chen et al., 2010). In

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F ecies

m (+N) ot

titTfFF6GO(mfaecm

TAv

D

ig. 4. Fluorescence staining of reactive oxygen species (ROS), reactive nitrogen spade cross sections of Hordeum vulgare stems cultured either in nitrogen-sufficient

he stem base. Bar indicates 100 �m.

erms of quantitative level, various amounts of GSH were reportedn barley, probably owing to use of various methods for quantifica-ion (Palatnik et al., 2002; Finkemeier et al., 2003; Chen et al., 2010).he values reported are not uniform, e.g. “total glutathione” wasound to be ca. 12 mg g−1 FW (Palatnik et al., 2002) or 105 �g g−1

W (Finkemeier et al., 2003). Our data are ca. 14 and 2 �g GSH g−1

W in shoots and roots, respectively. Our shoot value is similar to ca.0 �g g−1 DW found by Chen et al. (2010). Besides, exact GSH andSSG values have only rarely been reported as mentioned above.ur GSH/GSSG ratio is similar to that observed in Capsicum plants

Airaki et al., 2011) and it is undoubted that LC–MS/MS allows theost precise quantification currently available. The same is true

or ascorbic acid content, because very different amounts were

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lso reported in barley leaf tissue, e.g. 180–270 �g g−1 FW (Chent al., 2010) and 640 �g g−1 FW (Palatnik et al., 2002). Our valuea. 20 �g g−1 FW (=114 nmol g−1 FW) is substantially lower thanentioned values and there are not any data measured using

able 3ccumulation of free benzoic (gallic – salicylic) and cinnamic acid (chlorogenic – sinapulgare plants cultured either in nitrogen-sufficient (+N) or nitrogen-deficient (−N) solut

Shoot

+N –N

Gallic a. 0.94 ± 0.15 0.92

Protocatechuic a. 10.75 ± 0.73 13.75

Protocatechuic ald. 2.48 ± 0.36 2.53

pOHbenzoic a. 7.19 ± 0.11 4.27

pOHbenzoic ald. 6.89 ± 0.29 3.95

Vanillic a. 15.66 ± 0.50 19.37

Vanillin 6.97 ± 0.22 7.26

Syringic a. 3.81 ± 0.09 4.18

Salicylic a. 5.75 ± 0.20 7.73

Chlorogenic 0.25 ± 0.04 0.27

Caffeic a. 1.07 ± 0.20 1.03

p-coumaric a. 10.90 ± 1.27 9.89

Ferulic a. 31.96 ± 3.70 33.68

Sinapic a. 127.2 ± 9.21 114.5

sum 231.8 ± 13.6 223.4

Homoorientin 12.69 ± 2.48 59.71

ata are means ± SDs (n = 5). a. – acid, ald. – aldehyde, pOHbenzoic – p-hydroxybenzoic a

(RNS, preferentially NO) and phenolic metabolites (NPR) in freshly-prepared handr nitrogen-deficient (−N) solution over 15 days. Sections were taken ca. 3 cm above

LC–MS/MS in leaf tissue. However, recent report using anotherhighly precise detection with HPLC-ESI-TOF mass spectrometryrevealed 297 nmol g−1 FW in sugar beet leaf tissue (Rellán-Álvarezet al., 2011), providing good quantitative correlation with our valuedespite various plant species. Considering simple fact that bothbarley and sugar beet are not cultured for vitamin C production,these values are much more realistic in leaf tissue of commoncrop plants. AsA depletion in leaf tissue and unaltered amount inroots is in line with glutathione changes, indicating depletion ofascorbate–glutathione cycle intensity in shoots leading to elevatedoxidative stress. In agreement with our data, increase in GSH, GSSGand AsA was also observed in potassium-starved roots of tomato,probably contributing to ROS homeostasis connected with changes

and secondary metabolites in nitrogen-deficient barley plants. J Plant

in root growth (Hernandez et al., 2012).Reactive oxygen species formed under stress conditions are

scavenged by various non-enzymatic (such as GSH and AsAmentioned above) as well as enzymatic antioxidants. GR and APX

ic) derivatives (�g g−1 DW) and flavonoid homoorientin (�g g−1 DW) in Hordeumion over 15 days.

Root

+N –N

± 0.17 0.24 ± 0.02 0.39 ± 0.04**

± 2.06 1.38 ± 0.25 1.28 ± 0.27± 0.18 1.62 ± 0.11 1.57 ± 0.12± 0.32*** 8.20 ± 0.64 8.52 ± 0.14± 0.24*** 27.24 ± 2.16 29.25 ± 1.50± 1.59* 4.75 ± 0.17 5.51 ± 0.19**

± 0.19 13.87 ± 0.30 18.17 ± 1.08**

± 0.21 2.23 ± 0.11 2.21 ± 0.19± 0.24*** 2.21 ± 0.19 2.16 ± 0.09± 0.02 0.16 ± 0.03 0.15 ± 0.02± 0.14 0.29 ± 0.02 0.31 ± 0.04± 0.72 42.58 ± 4.35 42.71 ± 3.49± 2.30 10.23 ± 1.06 11.74 ± 1.62± 11.8 0.94 ± 0.14 1.06 ± 0.14± 14.9 116.0 ± 7.37 125.1 ± 5.39± 5.30*** nd nd

cid, nd – not detected.

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F peciesc 15 day

eaFr(

ig. 5. Fluorescence staining of reactive oxygen species (ROS), reactive nitrogen sultured either in nitrogen-sufficient (+N) or nitrogen-deficient (−N) solution over

nzymes contribute to ROS homeostasis by regeneration of GSH

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Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.08.004

nd reduction of H2O2 using ascorbate, respectively (Noctor andoyer, 1998). At least maintenance of APX activity in N-deficientoots (Fig. 3) fits well with unaltered amount of its substrateFig. 2). On the other hand, depletion of both APX and GR activities

(RNS) and phenolic metabolites (NPR) in various parts of Hordeum vulgare rootss. Bar indicates 100 �m. Note numerous root hairs on −N roots.

found in shoots is in accordance with visualized elevation of

and secondary metabolites in nitrogen-deficient barley plants. J Plant

ROS formation (Fig. 4) though the same was not observed invarious parts of roots (Fig. 5). These root-specific responses couldbe ascribed just to ROS regulation owing to intensive growth.K-deprived tomato roots also revealed rather increase in APX

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ut decrease in GR activity (Hernandez et al., 2012) as we par-ially observed (Fig. 3). Antioxidative enzymes showed rathertimulation in relation to prolonged N deficiency in chamomileKovácik and Backor, 2007) or in barley under Cd excess (Chent al., 2010). Nitrogen-deprived barley seedlings revealed unal-ered GR and dehydroascorbate reductase activity in comparisonith +N ones (based on per mg protein calculation; Finkemeier

t al., 2003) indicating maintenance as mentioned above. Thoughynamic changes of antioxidative enzymes are expected andherefore comparison of various studies is complicated, there istill good evidence that antioxidative enzymes participate in ROSomeostasis in barley. We also note extensively different unitsf enzymatic activities reported in barley shoot and root tissue∼0.6–2 mmol min−1 g−1 FW; Chen et al., 2010) compared withur values that would be ∼0.3–2 �mol min−1 g−1 FW. On the otherand, e.g. GPX activity (Fig. 3) that typically shows the highestalue among various antioxidative enzymes, fits well with shootnd root values reported in Si-treated barley (Balakhnina et al.,012).

Fluorescence microscopy is a modern and effective tool allowingo see changes of target molecules within tissue that are unable toe obtained by quantitative methods such as spectrophotometry.o our knowledge, this is first such extensive study using barley.lear enhancement of ROS signal in shoots and unaltered signal inoots in response to N deficiency is well correlated with changesf antioxidants and enzymes mentioned above. Similarly to theseesults, amount of ROS (H2O2) was elevated in shoots but depletedn roots of N-deficient chamomile related to prolonged N defi-iency (Kovácik and Backor, 2007). This is also in agreement withncrease in H2O2 content in K-starved tomato considering only 24-

exposure to K deprivation (Hernandez et al., 2012). Analyses ofarious parts of barley roots revealed excellent homogeneity ando changes to ROS or NO signal were observed (Fig. 5). No attenu-tion of ROS observed after prolonged exposure to N deficiency inoots in spite of increase in antioxidants confirms essentiality forrowth responses as also visible by appearance of root hairs (Fig. 5).t is concluded that both ROS and NO participate in sensing of min-ral nutrients deficiency through root tissue and related growthlterations (Hernandez et al., 2012; Kovácik et al., 2009 and theeferences therein).

Phenolic metabolites are widespread secondary plant productseing involved in growth processes and antioxidative protectionFinger-Teixeira et al., 2010; Kovácik et al., 2010). In terms of N defi-iency, various phenols typically show elevation in plants, beingvoked by “shift” from N-based to C-based metabolites aimed toobilize N sources within plant (Kovácik et al., 2007; Giorgi et al.,

009; Rubio-Wilhelmi et al., 2012a). It was therefore surprisingo find relatively slight increase in total soluble phenols in shootsnd no response in barley roots (Fig. 3). We therefore performeduorescence staining with universal NPR reagent: bright yellowuorescence in response to this reagent is considered to be a signal

or the presence of flavonoids (Gitz et al., 2004 and the referencesherein) but so far, no study that presents specificity of this reagentsowards individual groups/derivatives in plant tissues has beenublished. This yellow signal was also observed on cross sectionf barley stem without any extensive change if +N and −N variantsre compared (Fig. 4) but root tips revealed strong signal under-deficient conditions (Fig. 5). Another aspect is green emission

hat could indicate shift in fluorescence after NPR reaction withther groups of phenols: in fact, lateral roots and root tips (formingain portion of root system) revealed clear elevation of green flu-

rescence in N-deficient conditions. On the stem sections, rather

Please cite this article in press as: Kovácik J, et al. Variation of antioxidants

Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.08.004

ecrease was visible (we note that stem section was taken close totem base and is equivalent to part of seminal roots in Fig. 5). Takenogether, we assume that these observations, the most probably,ndicate shoot-to-root translocation of phenols and/or exudation

PRESSysiology xxx (2013) xxx– xxx

of phenols and further detailed profile of flavonoids is needed.However, it is well known that phenols (Juszczuk et al., 2004) orspecifically flavonoids and isoflavonoids (Dixon and Paiva, 1995)are exuded under low nitrogen conditions. Numerous flavone gly-cosides were reported in barley leaves, including luteolin derivativehomoorientin (isoorientin, Ferreres et al., 2008). We found increaseof this metabolite in shoot tissue but absence in roots indicatingtissue specificity of biosynthesis and/or storage form. Analyses ofphenolic acids also confirmed slight impact of N deficiency andexudation of phenolics or involvement in root growth (e.g. ligninprecursors) is therefore expected. We found no literature relatedto leaf assay of phenols in barley and therefore we are unable todiscuss this aspect deeply. However, quantitative comparison ofphenols (total soluble phenols and individual phenolic acids) withbarley grains (Dvoráková et al., 2008) shows higher amount invegetative parts as naturally expected. Enhancement of guaiacolperoxidase (GPX) activity in N-deficient shoots could also con-tribute to less visible increase of phenols: soluble GPX serves asan H2O2 scavenger in connection with phenols (Sakihama et al.,2002). In this cycle, phenolics are oxidized to phenoxyl radicalsthat can be reduced by ascorbate. Reduction of phenoxyl radical isessential part of this cycle, because it possesses prooxidant activity(Sakihama et al., 2002). These facts reinforce complexity and tightlybalanced pro/anti-oxidative properties of antioxidants. Depletionof GSH and AsA content and elevated GPX activity in shoots couldcontribute to shift towards oxidative stress, as visualized using flu-orescence microscopy of ROS.

Conclusions

Considering the fact that we exposed barley plants to total Ndeficiency over 15 days which is improbable in the field conditions,they revealed relatively considerable tolerance. This was mainlyvisible by unaltered shoot length and relatively slight growthdepression and chlorosis. On the other hand, enhanced root growthindicates ability of plants to cope with N deficiency by expansion tospace aimed to “search” for additional N sources. Though nitroge-nous metabolites (proteins and amino acids) were depressed, thisdecrease was relatively low considering severity of total N defi-ciency. On the other hand, N deficit stimulated increase in selectedphenolic metabolites including flavonoid homoorientin in barleyleaves, suggesting improved nutritional value owing to elevatedcontent of antioxidants. Elevated (GSH) or unaltered (AsA, phe-nols) level of antioxidants in the roots could provide protectionagainst oxidative stress, as visualized by no impact on ROS accu-mulation. This implies important role of antioxidative metabolitesin barley tolerance to stress impacts and further studies using var-ious cultivars will be conducted. We also urge to quantify sensitivemetabolites such as GSH and AsA using precise and fast methodsallowing comparison in the same species.

Disclosure statement

The authors declare that there are no conflicts of interest.

Role of the funding source

Sponsors had no involvement in the present study.

Acknowledgement

and secondary metabolites in nitrogen-deficient barley plants. J Plant

The work was supported by OP Education for Competitive-ness (European Social Fund and the state budget of the CzechRepublic) CZ.1.07/2.3.00/30.0017 Postdocs in Biological Sciences atMENDELU.

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