J. Plant Nutr. Soil Sci. 2011, 000, 1–8 DOI: 10.1002/jpln.

201000075 1

Phenol-containing organic substances stimulate phenylpropanoid

metabolism in Zea mays
Andrea Ertani1, Michela Schiavon1, Adriano Altissimo2, Clizia Franceschi3, and Serenella Nardi1*
1 Department of Agricultural Biotechnology, University of Padova, Agripolis, viale dell’Università 16, 35020 Legnaro, Italy
2 Landlab Associated Office, via Quintarello 12/A, 36050 Quinto Vicentino, Vicenza, Italy
3 R&S ILSA S.p.A., via Quinta Strada 28, 36071 Arzignano, Vicenza, Italy

Abstract
Intensive land use may affect soil properties (e.g., decreased soil organic matter [SOM] content)
and, consequently, reduce crop yields considerably. One way of counteracting the loss of SOM
and stimulating plant productivity could be the use of organic residues from agro-industrial pro-
cesses as bioactive products. The present study was focused on the possible effects of phenol-
containing organic substances derived from agro-industrial by-products on maize (Zea mays L.)
metabolism in a pot experiment. Plants were grown for 12 d in a nutrient solution in the absence
(control) or in the presence of either a cellulosolitic dry apple hydrolyzate (AP) or a dry blueberry
cool extract (BB) applied at two rates (0.1 and 1 mL L–1). Both products increased root and leaf
biomass and led to higher concentrations of macronutrients in the plant tissue. AP and BB also
had a positive impact on nitrogen (N) metabolism stimulating the activity and gene expression of
phenylalanine ammonia-lyase, a key enzyme of the phenylpropanoid pathway. Furthermore,
both products increased leaf concentrations of phenols (+ 28% and 49% for AP and BB, respec-
tively) and flavonoids (+ 22% and 25% for AP and BB, respectively). From our results it can be
assumed that residues from agro-industry may be successfully used as bioactive products in
agriculture to increase plant yield and resistance to stress conditions.

Key words: bioactive substances / flavonoids / gene expression / phenols / phenylalanine ammonia-lyase
(PAL)

Accepted March 21, 2011

1 Introduction
The intensive cultivation of land (e.g., monocropping, soil til- three major groups on the basis of their original matrix: humic
lage) might affect soil quality due to a reduced content of soil substances, marine bioactive substances, and amino acid–
organic matter (SOM; Francioso et al., 2000). To counteract containing products (Kauffman III et al., 2007).
SOM losses and improve crop yields, the application of man-
ure or other organic amendments to soils has been progres-
Although several authors have reported the beneficial effects
sively introduced in agricultural practices (Glaser et al.,
of biostimulants on plants (Chen et al., 2003; Parrado et al.,
2002). The addition of selected organic residues from agro-
2008; Ertani et al., 2009; Rayorath et al., 2008; Schiavon
industry to soil is a further strategy to increase SOM and, at
et al., 2008, 2010), the physiological and molecular mechan-
the same time, presents a solution for reducing organic-
isms through which such compounds act are to a certain
waste disposal (Akavia et al., 2009). Agro-industrial residues
degree unknown. Schiavon et al. (2008) showed that an
often contain bioactive molecules, including antioxidants
alfalfa-based biostimulant promoted plant growth and nitro-
(Schieber et al., 2001; Balasundram et al., 2005), which could
gen assimilation by inducing a tightly coordinated regulation
be exploited by farmers to improve crop productivity (da Silva
of the carbon and nitrogen metabolic pathways at the level of
et al., 2008). In such a case, these components can be
both gene expression and enzyme activity. Another recent
referred to as biostimulant compounds. Basically, biostimu-
study suggests that active molecules in biostimulants could
lants function as positive growth regulators at low doses by
enhance plant defense against stress through the induction
enhancing plant nutrition and metabolism (Chen et al., 2003;
of secondary metabolism, leading to the synthesis of pheno-
Schiavon et al., 2008, 2010; Ertani et al., 2009; Khan et al.,
lics (Schiavon et al., 2010).
2009). Furthermore, the use of such compounds might lead
to reduced nitrogen-application levels (Russo and Berlyn,
1991) and increase the resistance of plants to multiple abio- Therefore, a pot trial with maize was conducted to test the
tic-stress factors such as heat, drought, and salinity (de Vas- performance of a cellulosolitic dry apple hydrolysate (AP) and
concelos et al., 2009). Biostimulants are commercially avail- a dry blueberry cool extract (BB) on plant growth and on nitro-
able in different formulations and are generally classified into gen and phenylpropanoid metabolic pathways.

* Correspondence: Dr. S. Nardi; e-mail: serenella.nardi@unipd.it

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2 Ertani, Schiavon, Altissimo, Franceschi, Nardi J. Plant Nutr. Soil Sci. 2011, 000, 1–8

Table 1: Chemical properties and phenol concentrations of AP and To determine the phenolic acid composition, 1 mL of either
BB products. AP or BB extract was added with deionized water (1:2) and
shaken for 1 h at 25°C. The extracts were then centrifuged at
AP BB
13 000 g for 40 min and filtered (0.22 lm; Membra-Fil® What-
pH 4.1 3.2 man Brand, Whatman, Milano, Italy). Analyses were per-
EC§ / dS m–1 0.61 0.10 formed using an HPLC 2700 coupled with a 1806 UV/Vis
detector (Thermo Finnigan Italia SpA, Rodano, Italy). The
TOC / % 21.9 0.01
stationary phase was constituted by the column (Supelcosil
S/% 24 0.8 TM-LC 18) and precolumn (Pelliguard TM109 LC 18) of
N/% 0.20 0.01 Supelco. The mobile phase (1 L) was formed by n-butanol
(18 mL) and acetic acid 50% (1.5 mL). Phenolic compounds
Total phenols / lM 530 710
were separated (loop 20 lL) with a flux of 1.2 mL min–1 at
Gallic acid / mg L–1 n.d. 80.18 room temperature and analyzed using a UV detector at
Protocatechuic acid / mg L–1 170.56 133.67 275 nm. Each run lasted 30 min. Nine standards, including
Chlorogenic acid / mg L–1 n.d. 71.20 gallic, ferulic, vanillic, protocatetic, caffeic, p-Coumaric,
p-Hydroxibenzoic, syringic, and chlorogenic acids were used
Caffeic acid / mg L–1 n.d. n.d.
for the calibration curves.
Vanillic acid / mg L–1 n.d. 22.01
Syringic acid / mg L–1 n.d. 20.42 AP and BB showed different chemical characteristics (Tab. 1):
p-Coumaric acid / mg L–1 59.77 164.87 The pH was more acidic for BB (3.2) than for AP (4.1), and
the value of electrical conductivity (EC) was 6 times higher
Ferulic acid / mg L–1 n.d. 19.24
for AP than for BB. AP contained a significant amount of total
p-Hydroxibenzoic acid / mg L–1 n.d. n.d. organic carbon (TOC), sulfur (S), and nitrogen (N), while
§
these three elements were present only in traces in the BB
EC: Electrical conductivity; TOC: total organic carbon; S: sulfur; N:
product. The level of total phenols was appreciable in both
nitrogen. n. d. = not detectable
products. Interestingly, most phenolic compounds were
detectable only in BB.

2 Material and methods

2.1 Characterization of the test products
The two products used in this study (AP and BB) were manu-
factured by ILSA (Arzignano, Vicenza, Italy). AP was pro-
duced from dry apple material through a fully controlled enzy-
matic hydrolysis using a mixture of cellulotic enzymes (cellu-
lose, b-glucanase, xylanase, and emicellulase) at pH 6.5–7.5
and temperature ranging within 50°C–60°C for 320 min. The
ratio apple weight to enzyme mixture was 1:4 (w/v). BB was
obtained from dry blueberry material via cool extraction
according to the method of Machado (2007). BB aqueous
extracts were prepared by extracting 10 g of dried, ground
plant material with 50 mL deionized water in a shaker for 2 h
at room temperature. Both AP and BB extracts were filtered
with cellulosic membrane filters at 0.8 lm (Membra-Fil®
Whatman Brand, Whatman, Milano, Italy) before use. The pH
was determined in water (3:50 w/v), as well as the electrical
conductivity (EC) (1:10 w/v). The C, N, S concentrations
were measured in AP and BB extracts using an element ana-
lyzer (vario MACRO CNS, Hanau, Germany).
Total phenols were determined according to Arnaldos et al.
(2001). Extracts of AP and BB (1 mL) were maintained on ice
with pure methanol (1:1, v/v) for 30 min and centrifuged at
5000 g for 30 min at 4°C. One milliliter of 2% Na2CO3 and
75 lL of Folin-Ciocalteau reagent (Sigma-Aldrich) were
added to 100 lL of phenolic extract. After 15 min of incuba-
tion at 25°C in the dark, the absorbance at k = 725 nm was
measured. Gallic acid was used as standard according to
Meenakshi et al. (2009).
2.2 Plant material
Seeds of Zea mays L. (cv. DKc 5783, DeKalb, Italia) were
soaked in distilled water for one night and then surface-steri-
lized in 5% (v/v) sodium hypochlorite for 10 min while shak-
ing. Seeds were left to germinate for 60 h in the dark at 25°C
on a filter paper wetted with 1 mM CaSO4 (Nardi et al., 2002).
Germinated seedlings were transplanted into 3 L pots con-
taining an aerated Hoagland modified solution (Hoagland and
Arnon, 1950), with a density of 24 plants pot–1. The nutrient
solution was renewed every 48 h and had the following com-
position (lM): KH2PO4 (40), Ca(NO3)2 (200), KNO3 (200),
MgSO4 (200), Fe,Na-EDTA (10), H3BO3 (4.6), CuCl2 (0.036),
MnCl2 (0.9), ZnCl2 (0.09), NaMoO (0.01). Plants were culti-
vated at 14 h light/10 h dark cycle, air temperature of 27°C/
21°C, relative humidity of 70%/85%, and at a photon-flux
density of 280 mol m–2 s–1 in a climate chamber. Twelve days
after transplanting, AP or BB was added to the nutrient solu-
tion in the pots at different concentrations: 0 (control), 0.1,
and 1 mL L–1. The addition of the two products to the nutrient
solution was performed only once. After 48 h, plants were
randomly harvested from three pots of each treatment, and
then fresh samples of roots and leaves were carefully
washed and dried with blotting paper. A subsample of the
plant material was immediately frozen with liquid nitrogen
and kept at –80°C for molecular and physiological analyses.
For dry-weight measurement, thirty plants were used (ten per
treatment from each pot). Plants were divided in roots and
leaves and weighed separately. The samples were placed in
an oven for 2 d at 70°C and allowed to cool for 2 h inside a
closed bell jar. The dry weight was measured per plant.

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J. Plant Nutr. Soil Sci. 2011, 000, 1–8
2.3 Mineral-nutrient determination
The determination of mineral nutrients in leaves and roots of
maize plants was performed after an acid-digestion proce-
dure using a microwave (Milestone Ethos model 1600, Mile-
stone, Shelton, CT). All digestion reactions were carried out
in closed Teflon vessels of 120 mL volume using 500 mg plant
material and 10 mL of 30% (v/v) HCl as a solvent. Digested
samples were then diluted in 10 mL ultrapure water and
assayed via inductively coupled plasma–atomic emission
spectroscopy (Spectrum CirosCCD, Kleve, Germany).
For nitrate determinations, root and leaf tissues (1 g) of three
representative plants per pot were frozen in liquid nitrogen
and homogenized (1:5 w/v) in 10 mM HCl. The extract was fil-
tered through two layers of muslin and clarified by centrifuga-
tion at 35 000 g for 15 min. All steps were performed at 4°C.
The supernatant was filtered (0.22 lm; Membra-Fil® What-
man Brand, Whatman, Milano, Italy). The quantification of
NO3 was performed via HPLC using an AS 4S-SC anionic
exchange column (Dionex, Sunnyvale, CA, USA), equipped
with a Dionex suppressor and a 431 conductivity detector
(Waters-Millipore, Milford, MA, USA). A solution of sodium
bicarbonate and sodium carbonate (1.7 mM NaHCO3/1.8 mM
Na2CO3) was used as eluent at 2 mL min–1 flow rate. As a
reference standard, sodium nitrate was used (Fluka, Buchs,
Switzerland). Nitrate concentration is expressed as NO3
lmol (g fresh weight)–1.
2.4 Analysis of soluble proteins and sugars
For the extraction of soluble proteins, frozen foliar and root
tissues (500 mg) were ground in liquid nitrogen, vortexed with
5 mL extraction buffer (100 mM Tris HCl pH 7.5, 1 mM
Na2EDTA, 5 mM DTT), and centrifuged at 14 000 g. The
supernatants were mixed with 10% (w/v) trichloroacetic acid
(TCA) and centrifuged. The pellets obtained were resus-
pended in 0.1 N NaOH. The protein concentration was ana-
lyzed according to Bradford (1976) using a UV/VIS spectro-
photometer (Lambda 1, Perkin-Elmer, Monza, Italy) at k =
595 nm. The protein concentration is expressed in mg of pro-
tein (g fresh weight)–1.
Foliar tissues (100 mg) of five representative plants per pot
were dried for 48 h at 80°C, ground in liquid nitrogen, and
then extracted with 2.5 mL 0.1 N H2SO4. Samples were incu-
bated in a heating block for 40 min at 60°C and then centri-
fuged at 6000 g for 10 min at 4°C. After filtration (0.2 lm,
Membra-Fil® Whatman Brand, Whatman, Milano, Italy), the
supernatants were analyzed with HPLC (Perkin Elmer 410).
The soluble sugars were separated through a Biorad Aminex
87 C column (300 × 7.8 mm) using H2O as eluent at a flow
rate of 0.6 mL min–1. Sugar concentration is expressed in
g (kg dry weight)–1.
2.5 Extraction and measurement of soluble
phenols and flavonoids
Soluble phenolic acids were extracted by crushing leaves
(1 g) in a mortar in the presence of 3 mL pure methanol. The
extracts were maintained in an ice bath for 30 min and centri-
 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Phenol-containing substances 3
fuged at 5000 g for 30 min at 4°C. The supernatants were
stored at –20°C until analysis. Total phenols were measured
according to Arnaldos et al. (2001) as described above. Fla-
vonoids were extracted from leaf tissues (1 g) using 50 mL of
acidified methanol solution. The extracts were kept at 4°C for
16 h before measuring the absorbance at k = 300 nm. Flavo-
noids are expressed as gallic acid equivalents.
For phenolic acid–composition determination, fresh tissue
(1 g) from three plants per treatment was used. The protocol
was the same reported for the quantification of different types
of phenolics in AP and BB.
2.6 Phenylalanine ammonia-lyase assay
Fresh leaf tissues (1 g) were homogenized for 15 min on ice
with a glass homogenizer using 0.10 g polyvinylpyrrolidone
(PVP) and 5 mL of 100 mM potassium phosphate buffer
(pH 8.0) containing 1.4 mM 2-mercaptoethanol. The homoge-
nate was centrifuged at 15 000 g for 15 min at 4°C. The
supernatant was chromatographed using a Sephadex® G-25
column (GE Healthcare UK, Buckinghamshire, England)
equilibrated with the buffer described above. The eluate was
the enzyme extract used for the assay. The concentration of
protein in the enzyme extract was measured according to the
method of Bradford (1976). Phenylalanine ammonia-lyase
(PAL, EC 4.3.1.25) activity was assayed using the modified
method of Mori et al. (2001). The extract (0.2 mL) was mixed
with 0.4 mL of 100 mM Tris HCl buffer (pH 8.8) and 0.2 mL of
40 mM phenyl-alanine to 0.2 mL of enzyme extract. The mix-
ture was incubated for 30 min at 37°C and the reaction termi-
nated with 0.2 mL 25% TCA. Phenylalanine was added to the
controls after incubation and the addition of TCA. The assay
mixture was centrifuged at 10 000 g for 15 min at 4°C, and
absorbance of the supernatant was measured at k = 280 nm
relative to the control.
2.7 Semiquantitative RT-PCR
Isolation of RNA was performed in leaves of Z. mays plants
using the Nucleon PhytoPure kit (GE Healthcare UK, Bucking-
hamshire, England) according to the protocol provided by the
manufacturer. First-strand cDNA was synthesized from 5 lg
of RNA, after Dnase treatment with Dnase RQ1 (Promega,
Milano, Italy), using 200 U of ImProm-II™ Reverse Transcrip-
tase (Promega, Milano, Italy) and oligodT as primers in 20 lL
reactions (Schiavon et al., 2008). RT-PCR experiments with
specific primers (forward: 5′-CGCATCAACACCCTCCTC-3′;
reverse: 5′-GATGTAGGAGAGCGGGACCA-3′) were carried
out to evaluate the expression level of the Z. mays gene
encoding for PAL (ZmPAL: accession No. L77912). PCR
reactions were performed as described by Schiavon et al.
(2010). The constitutively expressed Z. mays actin gene
(J0128) was used as the internal control to normalize the
obtained gene expression results (primers: forward,
5′-TGTTTCGCCTGAAGATCACCCTGTG-3′; reverse,
5′-TGAACCTTTCTGACCCAATGGTGATGA-3′. An RT-PCR
analysis was performed using the Gen Amp PCR system
9700 (PE Biosystems, Branchburg, NJ, USA). The DNA frag-
ments were quantified through the ImageJ program (ImageJ
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4 Ertani, Schiavon, Altissimo, Franceschi, Nardi J. Plant Nutr. Soil Sci. 2011, 000, 1–8

1.23J, Wayne Rasband, National Institute of Health,
Bethesda, MD, USA). Furthermore, to confirm the expres-
sion-analysis results, PCR reactions were carried out on
cDNAs obtained from two different RNA extractions from
roots of seedlings of two independent experiments, and were
repeated at least 4 times for each cDNA.
Table 2: Root and leaf dry weight of Z. mays plants grown for 12 d in
a modified Hoagland nutrient solution and treated for 2 d with either
AP or BB at 0.1 or 1 mL L–1. Values in percentage refer to the root
and leaf growth of plants treated with AP or BB compared to the con-
trol (100%). Data are expressed in g per plant (n = 30, ± s). Values in
the same column followed by the same letter are not statistically dif-
ferent at p < 5% according to Student-Newman-Keuls test.

Treatment Root dry weight Leave dry weight

2.8 Statistical analysis
/g /% /g /%
Data of protein, sugar, element, nitrate, phenolic-compound Control 1.98 ± 0.09 b (100) 4.66 ± 0.13 c (100)
concentrations, and PAL activity represent the means of
AP 0.1 2.11 ± 0.12 ab (107) 4.28 ± 0.15 c (92)
measurements from three different pots per treatment. For
each measurement, three plants were used (± s). Analysis of AP 1.0 2.02 ± 0.07 b (102) 5.21 ± 0.09 c (112)
variance (ANOVA) was performed using the SPSS software BB 0.1 2.27 ± 0.14 a (115) 8.38 ± 0.04 a (180)
and was followed by pairwise post-hoc analyses (Student-
BB 1.0 2.28 ± 0.12 a (115) 6.43 ± 0.07 b (138)
Newman-Keuls test) to determine which means differed sig-
nificantly at p ≤ 5% (Sokal and Rohlf, 1969).
creased the foliar concentrations of N, P, K, Ca, and Mg
3 Results compared to the control (except P concentration for AP 0.1/
AP 1.0 and Ca concentration for AP 0.1; Tab. 4). These
For the apple hydrolysate a nonsignificant influence on root effects were more pronounced for N, K, Ca, and Mg when AP
and leaf biomass of plants was found, while the blueberry or BB were given to plants at the higher dose (1 mL L–1). The
cool extract significantly increased the root as well as the leaf concentration of nitrate in leaves and roots of plants treated
dry weight at both test doses compared to the control and the with either AP or BB was lower than in nontreated plants
AP treatments (Tab. 2). While no significant differences ap- (Fig. 1). The maximum decrease was observed in plants
peared between the two BB treatments on root growth, the grown in the presence of BB 1 mL L–1.
lower BB concentration led to a significantly higher leaf dry
weight. The synthesis of phenols and flavonoids was stimulated in
leaves of maize plants at both test doses of AP or BB,
To evaluate the effect of AP and BB on the N and C metabo- although not significant for AP 0.1 (Tab. 5). The blueberry
lism of maize plants, the concentrations of proteins and solu- cool extract was more effective in promoting an accumulation
ble sugars were measured (Tab. 3). Both products signifi- of phenolics, as the maximum levels of such compounds was
cantly increased the protein concentration in roots as well as observed in plants with BB at 1 mL L–1 (+49%).
in leaves, especially at the 1 mL L–1 rate. Sucrose and glu-
cose concentrations drastically decreased in foliar tissues of Concentrations of phenol compounds varied in plants in re-
plants treated with either AP or BB. The effect was more pro- sponse to AP or BB treatment (Tab. 5).
nounced for both products at the higher application rate. On
the other hand, fructose accumulated to a greater extent, Five derivatives of benzoic acid (gallic, protocatechuic, syrin-
with higher values recorded for plants grown with AP at both gic, vanillic, and p-Hydrossibenzoic acid) and three of cin-
test doses. namic acid (caffeic, p-Coumaric, and ferulic) were detected.
The concentrations of gallic, protocatechuic, syringic, and
The concentrations of several mineral nutrients were deter- p-Hydrossibenzoic acid were significantly higher in plants
mined in maize plants to evaluate whether AP and BB pro- treated with either AP or BB than the controls, while vanillic
mote plant nutrient uptake. Both products significantly in- acid was enhanced only in AP-supplied plants, in particular at

Table 3: Protein concentration in roots and leaves and leaf sugar concentrations of Z. mays plants grown for 12 d in a modified Hoagland
nutrient solution and treated for 2 d with either AP and BB at 0.1 or 1 mL L–1 . Data are the means of three replicates with three plants in each
(± s). Values in the same column followed by the same letter are not statistically different at p < 5% according to Student-Newman-Keuls test.

Treatment Proteins Sucrose Glucose Fructose

/ mg (g fresh weight)–1 / mg (g dry weight)–1
roots leaves
Control 1.01 ± 0.41 d 2.90 ± 0.37 c 116.73 ± 12.34 a 27.42 ± 1.33 a 7.43 ± 0.57 d
AP 0.1 1.36 ± 0.34 c 3.19 ± 0.17 b 77.39 ± 10.02 b 10.96 ± 1.14 c 26.66 ± 1.21 a
AP 1.0 1.94 ± 0.16 a 3.91 ± 0.10 a 3.55 ± 1.48 d 4.05 ± 1.26 d 23.18 ± 0.68 b
BB 0.1 1.85 ± 0.10 b 3.57 ± 0.16 b 22.47 ± 4.42 c 22.76 ± 1.18 b 9.79 ± 0.61 c
BB 1.0 1.99 ± 0.32 a 3.83 ± 0.09 a 8.83 ± 3.27 d 6.83 ± 2.29 d 10.12 ± 0.44 c

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J. Plant Nutr. Soil Sci. 2011, 000, 1–8 Phenol-containing substances 5

Table 4: Effect of AP and BB treatment on N, P, K, Ca, and Mg content of Z. mays plants grown for 12 d in a modified Hoagland nutrient solution
and treated for 2 d with either AP and BB at 0.1 or 1 mL L–1. Data represent the means of three replicates with three plants in each (± s). Values
in the same column followed by the same letter are not statistically different at p < 5% according to Student-Newman-Keuls test.

Treatment N P K Ca Mg
/%
Control 4.02 ± 0.20 c 0.130 ± 0.064 c 2.161 ± 0.120 d 0.910 ± 0.033 c 0.224 ± 0.048 d
AP 0.1 4.13 ± 0.3 b 0.148 ± 0.047 c 2.702 ± 0.170 c 0.986 ± 0.163 bc 0.239 ± 0.035 c
AP 1.0 4.25 ± 0.4 ab 0.110 ± 0.018 d 2.923 ± 0.112 b 1.334 ± 0.032 a 0.320 ± 0.012 a
BB 0.1 4.16 ± 0.2 b 0.220 ± 0.153 b 2.624 ± 0.180c 1.024 ± 0.010 b 0.266 ± 0.062 b
BB 1.0 4.26 ± 0.3 a 0.310 ± 0.020 a 3.186 ± 0.140 a 1.396 ± 0.0433 a 0.304 ± 0.011 a

0.1 mL L–1. Interestingly, the concentration of p-coumaric acid

a A was decreased by AP and BB at the lower dose compared to
the control, but showed a remarkable increase at 1 mL L–1. A
significant increase of caffeic acid concentration was ob-
served in plants grown with BB at both application rates and
/µmol (g f.wt.) –1

b b with AP at 0.1 mL L–1, while the concentration of ferulic acid

b
c only slightly increased.

a
B Application of AP and BB at both tested rates significantly
NO3– /µ

enhanced the activity of the PAL enzyme in leaves of maize

b plants in comparison to the control (Tab. 5). The maximum
c b increase of PAL activity (≈ +200%) was recorded when plants
d were grown in the presence of 1 mL L–1 BB.

The analysis of gene expression performed on leaves of

Figure 1: Effect of AP and BB treatment on nitrate concentration in
leaves (A) and in roots (B) of Z. mays plants grown for 12 d in a
modified Hoagland nutrient solution and treated for 2 d with either AP
and BB at 0.1 or 1 mL L–1. Different letters on bars indicate significant
differences between treatments (p < 5%).
maize showed high ZmPAL transcript accumulation only in
plants supplied with 1 mL L–1 AP (≈ +330%) or BB (≈ +450%)
(Fig. 2). No significant differences were observed between
plants treated with the lower concentration of AP or BB
(0.1 mL L–1) and the control.

Table 5: Concentrations of phenols and flavonoids, selected phenolic compounds, and PAL activity in leaves of Z. mays grown for 12 d in a
modified Hoagland nutrient solution and treated for 2 d with either AP or BB at 0.1 or 1 mL L–1. Data are the means of three measurements with
three plants in each (± s). Values in the same row followed by the same letter are not statistically different at p < 5% according to Student-
Newman-Keuls test.

Control AP 0.1 AP 1.0 BB 0.1 BB 1.0

Phenols / mg gallic acid (g fresh weight)–1 1.02 ± 0.04 c 1.13 ± 0.05 c 1.30 ± 0.05 b 1.48 ± 0.04 a 1.52 ± 0.09 a
Flavonoids / mg gallic acid (g fresh weight)–1 0.092 ± 0.005 d 0.123 ± 0.017 c 0.111 ± 0.007 c 0.204 ± 0.010 b 0.232 ± 0.013 a

Benzoic acid derivatives / lg (g dry weight)–1

Gallic acid 2.37 ± 0.62 c 13.73 ± 2.84 b 22.81 ± 1.47 a 14.52 ± 2.73 b 24.42 ± 1.12 a
Protocatechuic acid 12.8 ± 1.73 d 30.82 ± 3.23c 42.38 ± 1.99 b 50.52 ± 2.85 a 49.93 ± 2.16 a
Syringic acid n.d.§ 26.86 ± 2.44 a 28.32 ± 2.17 a 26.02 ± 2.81 a 25.92 ± 2.10 a
Vanillic acid n.d. 41.27 ± 5.61 a 20.08 ± 3.72 b n.d. n.d.
p-Hydroxibenzoic acid n.d. 5.89 ± 0.98 a 2.68 ± 0.99 b 6.15 ± 0.43 a 6.72 ± 0.35 a

Cinnamic acid derivatives / lg (g dry weight)–1

Caffeic acid n.d. n.d. 2.47 ± 0.63 b 12.13 ± 1.97 a 10.86 ± 2.09 a
Chlorogenic acid n.d. n.d. n.d. n.d. n.d.
p-Coumaric acid 55.01 ± 4.03 c 15.61 ± 2.28 e 149.70 ± 4.34 b 34.71 ± 2.04 d 172.46 ± 4.12 a
Ferulic acid 75.33 ± 5.62 c 83.41 ± 3.84 bc 87.18 ± 3.01 ab 84.65 ± 4.21 bc 91.32 ± 4.16 a
PAL activity / nmol cinnamic acid (mg protein)–1 min–1 9.481 ± 0.012 c 23.845 ± 0.008 b 21.425 ± 0.007 b 23.855 ± 0.009 b 30.399 ± 0.017 a

§ n. d. = not detectable.

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6 Ertani, Schiavon, Altissimo, Franceschi, Nardi
Figure 2: Gene expression (mRNA level) and relative transcript
accumulation of the gene-encoding Phenylalanine ammonia-lyase
(ZmPAL) in leaves of Z. mays grown for 12 d in a modified Hoagland
nutrient solution and treated for 2 d with either AP and BB at 0.1 or
1.0 mL L–1. Zmact (actin) was used as internal control. The
accumulation of the gene transcript was first normalized relatively
to the Zmact transcript and then expressed relatively to the control
(= 100%). Analyses were repeated at least three times for each cDNA
obtained from two different RNA extractions. Different letters indicate
significant differences between treatments (p < 5%) according to
Student-Newman-Keuls test.
4 Discussion
The addition of recycled residues from agro-industry to soils
during agricultural practices may offer an environmentally
friendly strategy to increase the content of soil organic matter
and improve crop yields (Mustin, 1987; da Silva et al., 2008).
Since most residues are good sources of bioactive molecules
such as phenolics (Moure et al., 2001; Balasundram et al.,
2005), two phenol-containing products were tested in this
study for their capacity to improve plant growth and enhance
the nitrogen and phenylpropanoid metabolic pathways in
maize. It is worth noting that test doses of AP and BB as low
as 0.1 and 1 mL L–1 were in the typical range of biostimulant
concentrations able to enhance plant nutrient-use efficiency
(Schmidt et al., 2003; Chen et al., 2003; Schiavon et al.,
2008, 2010).
The two products, especially the blueberry cool extract at the
lower concentration (0.1 mL L–1), increased the leaf and root
biomass of maize plants (Tab. 2). Phenolics contained in AP
and BB (Tab. 1) may act as positive growth regulators by
increasing the uptake of a number of macronutrients (N, P, K,
Ca, Mg; Tab. 4). In a study by Mugnai et al. (2007), the
enhancement of K influx observed for Vitis vinifera roots after
applying marine bioactive substances improved plant growth.
In the case of P, there is further evidence that some plants
may release phenolics (syringic, caffeic, and protocatechuic
acid) in the rhizosphere to solubilize and to take up inorganic
P (Morel and Hinsinger, 1999). Thus, it can be hypothesized
 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Plant Nutr. Soil Sci. 2011, 000, 1–8
that the protocatechuic and syringic acids in AP and BB may
induce plant P uptake by increasing the solubility of P com-
pounds similarly to the phenolics secreted by plants. The
increase of nitrogen concentration in plants could be ascribed
to the stimulation of nitrate uptake by AP and BB, as reported
for other biostimulants (Chen et al., 2003; Quaggiotti et al.,
2004).
The concentration of proteins was significantly higher in
plants treated with AP or BB (Tab. 3), probably as a result of
the increased N uptake. The enhancement of N assimilation
was concomitant with the stimulation of the secondary meta-
bolism associated with the synthesis of phenolic compounds,
as confirmed by the increase of phenylalanine ammonia-
lyase (PAL) enzyme activity Tab. 5) and gene expression in
plants supplied with either AP or BB at 1 mL L–1 (Fig. 2). High-
er activity of PAL results in a greater production of NH‡ 4 ions
that could be recycled in the glutamine synthetase–glutamate
synthase (GS/GOGAT) cycle to synthesize new amino acids
(Rösler et al., 1997; Andersen et al., 2007). The lack of a per-
fect relationship between PAL enzyme activity and transcript
accumulation suggests the existence of multiple posttransla-
tional mechanisms of PAL regulation in plants, which may
comprise PAL allosteric regulation, inactivation of specific
proteases and inhibition by end-products (cinnamic acid de-
rivatives and related compounds), as suggested by Tena et al.
(1984). The repression of PAL by end-products did not occur
in the present study, although it might arise at higher concen-
trations of biostimulants, as shown by Schiavon et al. (2010).
In the literature, the increase of phenolic compounds is often
reported in plants growing under N-deficient conditions (Fritz
et al., 2006; Giorgi et al., 2009). Fritz et al. (2006) showed
that the nitrate status of plants might induce a set of enzymes
functioning in the early steps of the phenylpropanoid meta-
bolic pathway. In our study, plants were not grown under N
limitation, but the nitrate concentration in plants after either
AP or BB application decreased (Fig. 1) as a result of the
enhanced N assimilation. Lower levels of nitrate would act as
a signal for the induction of PAL and phenolic accumulation.
Phenol compounds in AP and BB may have played an addi-
tional role in the induction of phenylpropanoid metabolism.
The hormone-like activity of several phenolic acids has been
previously reported (Pizzeghello et al., 2003), and the stimu-
lation of phenylpropanoid metabolism through auxin-mediate
signal transduction has been recently highlighted (Schiavon
et al., 2010). Phenol concentrations in the test products AP
and BB (Tab. 1) were not harmful for plants, otherwise toxicity
would have been noted, causing, for example, a retardation
in plant growth (Bergmark et al., 1992).
The improvement of nitrogen assimilation by AP and BB also
interacted with carbon metabolism (Tab. 2). The concentra-
tion of leaf sucrose may have diminished in plants treated
with the two products due to the higher consumption of glu-
cose in the respiratory process. As a result of a minimal
amount of glucose available to form sucrose, fructose accu-
mulated more in plants supplied with either AP or BB. Accord-
ing to Vaughan and Malcom (1985) as well as to Sidari and
Muscolo (2004), the greater concentration of phenols
recorded in plants after AP or BB treatment could be respon-
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J. Plant Nutr. Soil Sci. 2011, 000, 1–8
sible for a weak uncoupling of the oxidative phosphorylation,
which in turn would increase the metabolic processes requir-
ing glucose, such as glycolysis and citric acid cycle, and con-
sequently the production of proteins. The co-ordinated induc-
tion of N and C metabolic routes by biostimulants was previ-
ously proven by Schiavon et al. (2008) in a study where the
supply of low concentrations of an alfalfa protein hydrolyzate
to maize plants led to the increased activity and gene expres-
sion of enzymes involved in the tricarboxylic acid cycle and
nitrogen assimilation.
5 Conclusion
This work supports the potential use of recycled products
from agro-industry to improve plant productivity and the
synthesis of phenolic secondary compounds involved in sev-
eral physiological plant responses. The increase of phenolics
in plant tissues may enhance plant resistance to stress condi-
tions. Furthermore, it can provide a source of important anti-
oxidants for human health. Caffeic and gallic acids, for exam-
ple, inhibit carcinogenesis (Olthof et al., 2001; Raina et al.,
2008), and most analyzed phenolics seem to have antifungal
and antiviral properties (Haslam, 1989). Differences in effec-
tiveness between AP and BB may be related to the charac-
teristics of the starting matrixes as well as to the industrial
process used for their production. The cool-extraction meth-
od through which BB was obtained had the advantage of pre-
serving the organoleptic properties of the raw materials
(Machado, 2007), while the hydrolytic process employed for
AP production could cause chemical changes in thermolabile
bioactive compounds, including some phenolics (Buchanan,
2003).
Acknowledgments
This work was funded by ILSA S.p.A. We are grateful to Ms.
Marina Canapero and Prof. Hans-Werner Olfs for their pre-
cious help in editing the manuscript.
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