Nutritional traits of bean (Phaseolus vulgaris) seeds from plants chronically exposed to ozone pollution

Article abstract of DOI:10.1021/jf802819m

The effect of chronic exposure to ozone pollution on nutritional traits of bean (Phaseolus vulgaris L. cv. Borlotto Nano Lingua di Fuoco) seeds from plants grown in filtered and nonfiltered open-top chambers (OTCs) has been investigated. Results showed th

Full text of DOI:10.1021/jf802819m

J. Agric. Food Chem. 2009, 57, 201–208 201
Nutritional Traits of Bean (Phaseolus vulgaris) Seeds
from Plants Chronically Exposed to Ozone Pollution
,
†,‡
§
#
MARCELLO IRITI,*
ANTIMO DI MARO, SILVANA BERNASCONI,
⊥
|
‡
∇
NEDDA BURLINI, PAOLO SIMONETTI, VALENTINA PICCHI, CINZIA PANIGADA,
O
§
,†,‡
GIACOMO GEROSA, AUGUSTO PARENTE, AND FRANCO FAORO*
Istituto di Patologia Vegetale, Universit a` di Milano, via Celoria 2, 20133 Milano, Italy; Dipartimento
di Scienze delle Vita, II Universit a` di Napoli, via Vivaldi 43, 81100 Caserta, Italy; Dipartimento di
Chimica Organica ed Industriale, Universit a` di Milano, via Venezian 21, 20133 Milano, Italy;
Dipartimento di Scienze Biomolecolari e Biotecnologie, Universit a` di Milano, via Celoria 26, 20133
Milano, Italy; DiSTAM, Sezione Nutrizione, Universit a` di Milano, via Celoria 2, 20133 Milano, Italy;
CNR, Istituto di Virologia Vegetale, Dipartimento Agro-alimentare, via Celoria 2,
2
0133 Milano, Italy; DISAT, Laboratorio di Telerilevamento e Dinamiche Ambientali, Universit a` di
Milano-Bicocca, Piazza della Scienza 1, 20126 Milano, Italy; and Dipartimento di Matematica e
Fisica, Universit a` Cattolica del Sacro Cuore, via dei Musei 41, 20125 Brescia, Italy
The effect of chronic exposure to ozone pollution on nutritional traits of bean (Phaseolus vulgaris L.
cv. Borlotto Nano Lingua di Fuoco) seeds from plants grown in filtered and nonfiltered open-top
chambers (OTCs) has been investigated. Results showed that, among seed macronutrients, ozone
significantly raised total lipids, crude proteins, and dietary fiber and slightly decreased total free amino
acid content, although with a significant reduction of asparagine, lysine, valine, methionine, and glycine,
compensated by a conspicuous augmentation of ornithine and tryptophan. Phytosterol analysis showed
a marked increase of ꢀ-sitosterol, stigmasterol, and campesterol in seeds collected from nonfiltered
OTCs. With regard to secondary metabolites, ozone exposure induced a slight increase of total
polyphenol content, although causing a significant reduction of some flavonols (aglycone kaempferol
and its 3-glucoside derivative) and hydroxycinnamates (caffeic, p-coumaric, and sinapic acids). Total
anthocyanins decreased significantly, too. Nevertheless, ozone-exposed seeds showed higher
antioxidant activity, with higher Trolox equivalent antioxidant capacity (TEAC) values than those
measured in seeds collected from filtered air.
KEYWORDS: Bean seeds; ozone pollution; free amino acids; sterols; polyphenols; antioxidant activity
INTRODUCTION
carbons (1). Therefore, ozone is produced on bright sunny days
over areas with intense primary pollution, due mainly to vehicle
exhausts, fossil fuel burning, and industrial processes, in the
so-called photochemical cycle (2). Furthermore, meteorological
conditions can move the pollutant, or its precursors, from these
areas toward less polluted ones, such as rural zones, with
detrimental effects on natural and cultivated plant species (2).
Because of its strong oxidizing potential (+2.07 eV), ozone is
a powerful oxidizing agent capable of reacting with virtually
any biomacromolecule, namely, lipids, proteins, nucleic acids,
and carbohydrates, and producing reactive oxygen species (ROS)
(3). This, in turn, causes an array of biological effects at
molecular, biochemical, and physiological levels, the last
affecting biomass formation and crop yield (4). Ozone can also
activate the plant immune system (5), and it influences the
biosynthesis of plant secondary metabolites by changing either
the transcription or the activity of key enzymes of secondary
metabolic pathways (6). These compounds, to a large extent
deriving from three biosynthetic routes, the phenylpropanoid,
isoprenoid, and alkaloid pathways, include a plethora of
3
Tropospheric ozone (O ), a constituent of photochemical
smog, is a secondary pollutant produced through sunlight-
catalyzed reactions involving primary pollutants, such as
nitrogen oxides, sulfur oxides, carbon monoxide, and hydro-
*
Author to whom correspondence should be addressed (telephone
+
390250316786; fax +390250316781; e-mail marcello.iriti@unimi.it
or franco.faoro@unimi.it).
†
Istituto di Patologia Vegetale, Universit a` di Milano.
CNR, Istituto di Virologia Vegetale.
Dipartimento di Scienze delle Vita, II Universit a` di Napoli.
‡
§
#
Dipartimento di Chimica Organica ed Industriale, Universit a` di
Milano.
⊥
Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita`
di Milano.
|
DiSTAM, Sezione Nutrizione, Universit a` di Milano.
DISAT, Laboratorio di Telerilevamento e Dinamiche Ambientali,
∇
Universit a` di Milano-Bicocca.
O
Dipartimento di Matematica e Fisica, Universit a` Cattolica del Sacro
Cuore.
1
0.1021/jf802819m CCC: $40.75  2009 American Chemical Society
Published on Web 12/04/2008

202 J. Agric. Food Chem., Vol. 57, No. 1, 2009
Iriti et al.
Figure 1. Phytosterol and phenylpropanoid secondary metabolites analyzed in bean seeds chronically exposed to atmospheric ozone pollution. Among
phenylpropanoids, both phenolic acid (hydroxycinnamates and hydroxybenzoates) and flavonoid (isoflavonoids, flavonols, and anthocyanins) contents
were determined.
protectant molecules able to improve the plant’s tolerance to
environmental pollutants, as well as the plant’s resistance to
pathogens and phytophagy. Phytochemicals arising from these
pathways include not only compounds with broad-spectrum
antibiotic activity but also powerful antioxidants able to
efficiently scavenge ozone-induced ROS (7-9). As the precur-
sors of secondary metabolic routes are products of the primary
metabolism, a severe or long-lasting stress factor, such as ozone
exposure, may induce an excessive shift between primary and
secondary metabolism and, consequently, a diversion of essential
available resources from growth to stress tolerance could occur.
Plants are exposed to either acute and chronic ozone doses
according to the gas concentration and exposure time. An acute
exposure consists of relatively high ozone concentration (>80
of both the weight of pods per plant and seeds per pod (11). In
this work, we investigated the quality traits of bean seeds
harvested from plants grown in filtered and nonfiltered OTCs
in order to assess the impact of chronic ozone exposure on their
nutritional value. For this purpose, we evaluated the difference
in the content of macronutrients (carbohydrates, proteins, and
lipids), dietary fiber, free amino acid, phytosterols and phenyl-
propanoids (hydroxycinnamates, hydroxybenzoates, collectively
named simple phenols or phenolic acids and flavonoids) (Figure
1), as well as their antioxidant activity. So far, with the exception
of a few published works on wheat and potatoes (12-14), scarce
attention has been paid to possible modification of plant
foodstuff quality induced by one of the most widespread
pollutants, the background concentration of which has doubled
since preindustrial times and has continued to rise over the past
three decades with a 0.5-2% yearly rate at the mid-latitudes
of the northern hemisphere (15). Moreover, the above-mentioned
works mostly deal with starch and crude protein content,
whereas this is the first study reporting a detailed analysis on a
3
ppb, or 80 nL/L, or 160 µg/m ) for a few consecutive hours,
whereas a chronic exposure involves a relatively low gas
3
concentration (<40 ppb, or 40 nL/L, or 80 µg/m ) for the entire
life of a plant, with intermittent episodes of high concentration,
either periodically or accidentally (10). Symptoms of injury
appear typically in the leaves, and pathophysiological conditions
related to the ozone-induced damages include the impairment
of stomatal function, the decrease of photosynthetic activity,
and the induction of senescence, resulting in dysfunction of
transpiration and water use efficiency, reduction of dry matter
production, detrimental effects on flowering and pollen tube
extension, and yield losses (reviewed in ref 4).
set of quality parameters influenced by O
species.
3
pollution in a crop
MATERIALS AND METHODS
Experimental Facilities, Plant Material, Ozone Exposure, and
Seed Harvest. Open-top chamber (OTC) facilities were localized within
the Regional Forest Nursery of Curno (Bergamo), on the northern edge
of the Po Valley. In this region, ozone levels are among the highest in
Europe, and the critical levels for crops are frequently exceeded (11).
Seeds of common bean (P. Vulgaris L. cv. Borlotto Nano Lingua di
Fuoco) were sown in July 2006 in two filtered OTCs (OTCs-F) and in
Previously, we carried out experiments in open-top chamber
(OTC) facilities to assess the crop yield parameters of bean
(Phaseolus Vulgaris) plants, growing in charcoal-filtered OTCs
(OTCs-F) and in nonfiltered OTCs (OTCs-NF), meanwhile
two nonfiltered OTCs (OTCs-NF). O
3
concentrations in the chambers
3
assessing the real O uptake by leaves (11). Those results
were continuously monitored by an ozone automatic analyzer model
showed a significant reduction of crop yield in OTCs-NF, mainly
due to the detrimental effect of ozone on photosynthetic activity.
Particularly, plants grown in OTCs-F showed a higher number
of pods per plant and of seeds per pod as well as an increment
1
1
108 RS (Dasibi Italia, Milano, Italy). During the growing season (July
1-October 5), the cumulative exposures of plants over an O
3
concentration of 40 ppb (AOT40, accumulated exposure over a
threshold of 40 ppb) were 535 ppb h in OTCs-F and 4675 ppb h in

Nutritional Traits of Beans Exposed to Ozone
J. Agric. Food Chem., Vol. 57, No. 1, 2009 203
OTCs-NF. Similarly, ozone stomatal fluxes referred to the projected
leaf area (AFST0, a measurement of the amount of ozone effectively
and amyloglucosidase (pH 4.5, 60 °C, 30 min) to remove proteins and
starch. Total dietary fiber was precipitated with ethanol and, after
washing and drying, the residue was weighed. Results were corrected
for protein and ash contents.
-
2
absorbed by plants) were 9055 mmol of O
mmol of O m
3
3
m
in OTC-F and 20548
in OTC-NF (for details, see ref 11). After harvesting,
in October, part of the seeds were frozen in liquid N , powdered in a
-
2
2
Phenylpropanoid Determination. For anthocyanin extraction, bean
seeds (5 g) were previously dehulled as described in reff 21. After
separation from cotyledons, hulls were homogenized with 100 mL of
methanol and maintained overnight at 4 °C. Samples were then
centrifuged and pellets extracted again with 50 mL of methanol, for
2 h. After desiccation in a rotary evaporator at 40 °C, residues were
dissolved again in 10 mL of methanol/0.3% perchloric acid (23:73),
and the final extracts were stored in foil-wrapped glass vials at -20
°C. HPLC analysis of anthocyanins was performed as previously
described (17).
precooled mortar, and stored at -80 °C until analysis. Another part
was left to dry and stored in desiccators at room temperature.
Starch Determination. The total starch content was determined
according to the American Association of Cereal Chemists (AACC)
(
1983) Official Standard Method 76-13, using the Total Starch Assay
Kit by Megazyme International, Ireland Ltd. (Bray Business Park,
Wicklow, Ireland).
Crude Protein Content. Crude protein content was measured by
nitrogen determination, using the Kjeldahl procedure (16), by a Mineral
Six digestor and an Autodisteam semiautomatic steam-distilling unit
Flavonoids and phenolic acids were extracted from 5 g of whole
bean seeds, as described in ref 22. HPLC analysis was performed with
a Shimadzu instrument, constituted by an LC-10 AD vp pump, an FCV-
(
International PBI, Milan, Italy). Crude protein content was then
calculated as % N × 6.25. All chemicals and solvents for the Kjeldahl
method were provided by Carlo Erba Reagents (Milan, Italy).
Free Amino Acid Analysis. Free amino acids were analyzed with
a Biochrom 20 amino acid analyzer, as previously reported (17, 18).
All chemicals and solvents for the amino acid analysis were obtained
from Biochrom.
1
0 AL vp mixer, an SIL-10 AD vp auto sampler, and an SPD-10 A vp
UV detector set up on a wavelength of 320 nm. A Nova-Pak C18
column (300 mm × 3,9 mm, particle size ) 4 µm) at room temperature
was used. The mobile phase was constituted by (A) a solution of water/
acetic acid (98:2, v/v) and (B) a solution of 0.5% of acetic acid in
water/acetonitrile (1:1, v/v), maintained at a flow rate of 1 mL/min.
Class VP 3,4 software was employed for chromatographic analysis.
Total Lipid Content and Phytosterol Analysis. Seeds powdered
in liquid N
Stone Ridge, NY). The dried powders (∼2.5 g each) were extracted
by the Soxhlet apparatus for 4 h with CHCl , and the obtained solutions
2
were lyophilized by an FTS-System Flex-Dry instrument
(
Spectrophotometric detection of total polyphenols and total antho-
cyanins was performed according to the Folin-Ciocalteu assay on
whole seeds, as previously described (14), and results were expressed
as gallic acid (GAE) and malvidin (ME) equivalents, respectively.
Folin-Ciocalteu reagents were obtained from Sigma-Aldrich (St. Louis,
3
were dried in an evaporator to obtain the crude extracts.
Acid Hydrolysis and Saponification for Free Phytosterol Deter-
mination. Seeds powdered in liquid N (2 g) were transferred into a
2
flask with 25 mL of ethanol (25 mL) and dried under vacuum.
Cholesterol was added to each sample (1 g), as internal standard (2
mL of a solution 1:1 w/v in ethanol), together with 2 mL of ethanol
and 10 mL of 6 N HCl. The suspension was stirred at 80 °C for 60
min. After cooling, 10 mL of ethanol was added, and the mixture was
stirred for a further 5 min. Finally, 50 mL of hexane/diethyl ether (1:1
v/v) was added and, after 10 min of stirring, 35 mL of the organic
layer was removed and evaporated under vacuum by a B u¨ chi Rotavapor
R-114 (temperature not greater than 40 °C). The residue was treated
with ethanolic pyrogallol (16 mL of a solution 3% w/v) and KOH (8
mL of a solution 4.3% w/v). After 15 min at 80 °C, the solution was
cooled, and 24 mL of water and 40 mL of cyclohexane were added
therein. The mixture was centrifuged at 6500 rpm for 5 min at 4 °C.
Part of the organic layer (30 mL) was removed, and the solvent was
evaporated in a vacuum by a B u¨ chi Rotavapor R-114 (temperature not
greater than 40 °C). Dry samples were stored at -20 °C (19).
MO).
ABTS•+ Radical Cation Scavenging Activity. The 2,2′-azinobis(3-
•+
ethylbenzothiazoline-6-sulfonic acid (ABTS ) radical cation decolori-
zation test assesses the ability of hydrogen- or electron-donating
•+
antioxidants to scavenge this radical (23). The ABTS radical cation
was produced by reacting 7 mM ABTS with 2.45 mM potassium
persulfate (final concentration) and maintaining the mixture in the dark
•+
at room temperature for 12-16 h before use. The ABTS solution
was diluted with ethanol to an absorbance of 0.70 ( 0.02 at 734 nm
and equilibrated at 30 °C.
A weighed amount (ca. 200 mg) of dried seed sample was added to
5
mL of methanol/acetone/water (40:40:20, v/v/v) containing 0.1%
acetic acid, and the mixture was stirred at room temperature for 12 h
and centrifuged at 2000g for 10 min. Dilutions (10 µL) of sample
surnatant, or ethanol (negative control), or standard solution of the
synthetic antioxidant 6-hydroxy-2,5,7,8-tetramethychroman-2-carboxy-
lic acid (Trolox, positive control) were mixed for 30 s with 1 mL of
Gas Chromatographic Analysis of Phytosterols. Dry samples
obtained after acid hydrolysis and saponification were analyzed by GC
and GC-MS to determine the content of sterols and their structure.
Standards of the principal phytosterols (campesterol, stigmasterol, and
•+
diluted ABTS solution, and the absorbance at 734 nm was taken at
30 °C 1 and 10 min after the initial mixing. The percentage of inhibition
was calculated and reported as Trolox activity (Trolox equivalent
antioxidant capacity, TEAC) at time points of the reaction.
Presentation of Results. Extraction of metabolites, spectrophoto-
metric, HPLC, GC, GS-MS, and amino acid analyses were repeated at
least three times for each replicate; mean and standard deviation (SD)
of values are reported, and the evaluation of the statistical significance
of results was based on Student’s t test for paired data analysis, with
a probability value of <0.05 (p < 0.05) considered to be statistically
significant.
ꢀ
-sitosterol) and cholesterol were transformed into the corresponding
trimethylsilyl ethers, according to the following general procedure: 1
mg of a single sterol and 0.5 mL of a mixture of bis(trimethylsilyl)
trifluoroacetamide and dry pyridine (1:1 v/v) were left at room
temperature for 1 h. The extracts were treated and analyzed with the
same procedure previously described for the standards. Trimethylsilyl
ethers were directly analyzed by GC with a Dani 86.10 instrument,
equipped with a fused silica capillary column WCOT-CP-Sil-5 CB
(
(
Chrompack, 25 m × 0.32 mm i.d., film ) 0.11 µm), carrier helium
3
0.45 kg/cm ), injector, 300 °C, detector, 320 °C, oven, 180 °C (4 min)
RESULTS
and then raised to 300 at 5 °C/min. The sterol structures were confirmed
by injection of the same samples and of the standards in a Dani 3800
gas chromatograph, equipped with the same capillary column and
connected to a VG 7070 EQ mass spectrometer. Retention times of
sterols were 18.97 min for cholesterol, 19.11 min for campesterol, 20.20
min for stigmasterol, and 21.03 min for ꢀ-sitosterol (19).
Moisture and Total Dietary Fiber. Moisture was evaluated after a
further drying of seeds for 24 h at 80 °C. Total dietary fiber was
measured according to the Prosky method (20). Samples were previ-
ously delipidized with chloroform/methanol (3:1, v/v); afterward, they
were gelatinized with a heat-stable R-amylase (pH 6.0, 100 °C, 30 min)
and then sequentially digested with protease (pH 7.5, 60 °C, 30 min)
Macronutrient Composition and Dietary Fiber. On the
whole, the amount of macronutrients was higher in beans
exposed to ozone pollution (Table 1). In particular, total lipid
content rose by 28.5%, crude protein content by 7.88%, and
dietary fiber by 14.54%. In contrast, starch content and moisture
did not change significantly.
Free Amino Acid Profile. Total free amino acid content
decreased (-5%) from 490 ( 9.83 to 465 ( 25.44 mg/100 g
in seeds collected from filtered and nonfiltered facilities,
respectively (Table 2). However, ozone exposure only partially

204 J. Agric. Food Chem., Vol. 57, No. 1, 2009
Iriti et al.
Table 1. Macronutrient Composition, Dietary Fiber, and Moisture of Seed
Extracts from Beans Grown in Charcoal-Filtered (F) and Nonfiltered (NF)
Open-Top Chambers
particularly the plant secondary metabolites occurring in foods
and the food chain and responsible for the health benefits arising
from a high consumption of fruits and vegetables. In fact, these
bioactive phytochemicals are compounds involved in plant
resistance/tolerance against biotic/abiotic stresses (4, 24).
a
F (g/100 g)
NF (g/100 g)
% change
starch
35.69 ( 0.93
15.98 ( 0.75
1.96 ( 0.24
27.5 ( 0.4
35.99 ( 1.31
17.24 ( 0.30
2.52 ( 0.14
31.50 ( 0.8
8.64 ( 0.08
+0.84
+7.88
+28.57
+14.54
-0.23
Legumes are an important component of the Mediterranean
diet, and beans, in particular, are good sources of macronutrients
(mainly proteins and carbohydrates). Furthermore, beyond their
basic nutritional value, they provide health benefits by virtue
of their minor components. Our data on macronutrients showed
that starch was not influenced by ozone pollution, unlike crude
proteins and total lipids, the concentrations of which increased
significantly, as already reported by other authors (25). The
functional significance of these changes may be due to the
necessity of the plant, to improve the fitness of the next
generation, to ensure higher contents of proteins and lipids,
besides synthesizing defense metabolites. Nevertheless, in a
study on seed nutritional quality of 34 bean accessions with
different ozone sensitivity no correlation was found between
ozone sensitivity and protein, lipid, and carbohydrate contents
of the accession (26).
crude proteins
total lipids
dietary fiber
moisture
8.66 ( 0.04
a
Data (mean ( SD) derive from three separate seed extracts. Results are
expressed as grams of constituent per 100 g of seeds.
modified the whole free amino acid profile of the samples, with
significant decreases of the following five amino acids: aspar-
agine (-13.5%), lysine (-10%), valine (-6%), methionine
(
-7%), and glycine (-22%). Contrary to the general trend, the
concentrations of two amino acids significantly rose as a
consequence of the ozone exposure, with increases of 55.5%
in ornithine and 33.5% in tryptophan (Table 2).
Phytosterol Content. All three main phytosterols markedly
increased in seeds from plants grown in OTCs-NF compared
to those from OTCs-F (Table 3). In particular, ꢀ-sitosterol, the
most abundant, rose 76.83%, stigmasterol, 65.69%, and campes-
terol, 52.17%.
Although ozone exposure did not markedly modify the whole
amino acid profile of bean seeds, we previously reported a deep
change of free amino acid composition, in grapevine treated
with benzothiadiazole, an inducer of systemic acquired resistance
(SAR), and in transgenic Arabidopsis transformed with a Myb
transcription factor. In both cases, the variation of free amino
acid content was related to an enhanced resistance to pathogens
and tolerance to abiotic stresses, including ozone (17, 18). Here
we showed significant decreases of asparagine, lysine, valine,
methionine, and glycine concentrations after ozone exposure.
In leguminous plants, asparagine is the major assimilation
product of nitrogen fixation in nodules and an important
compound for the transport and storage of nitrogen resources,
because of its high nitrogen to carbon ratio (27). Consequently,
the reduction of asparagine in ozone-exposed seeds may be due
to an impairment of nitrogen uptake from soil or its fixation in
radical nodules, besides transport and storage in plant. The
essential amino acid lysine belongs to the aspartate family of
amino acids, arising from aspartate after seven consecutive
enzymatic reactions started by dihydrodipicolinate synthase
Phenylpropanoids. Ozone induced a slight increase (+3.18%)
of the total polyphenols in whole seeds, their contents being
3
450 ( 21.1 and 3563 ( 33.4 µg/g in samples from OTCs-F
and OTC-NF, respectively. However, a pronounced decrease
-43.15%) of total anthocyanins, from 95 ( 4.1 µg/g in OTCs-F
(
to 54 ( 7.3 µg/g in OTCs-NF, was observed. Among the
different anthocyanins, detected by HPLC only in the seed coat
fraction, the concentrations of the 3-glucoside derivatives of
delphinidin, cyanidin, and peonidin were significantly lower in
seeds grown in polluted environment (OTCs-NF), whereas
ozone exposure significantly raised the content of petunidin-3-
glucoside and pelargonidin-3-glucoside (Figure 2A; Table 4).
With regard to the polyphenolic compounds, no significant
difference was reported in the isoflavonoid (daidzein, genistein,
and coumestrol) content, whereas an increase of both flavonol
aglycone kaempferol and its 3-glucoside derivative concentra-
tions was significantly detected in samples collected from
OTCs-F (Figure 2B; Table 4). Finally, among hydroxycin-
namates, ozone exposure significantly lowered the content of
caffeic, p-coumaric, and sinapic acids, in whole seeds from
OTCs-NF, whereas the levels of ferulic and syringic acids, the
latter a hydroxybenzoic acid, did not change (Figure 2C; Table
(
DHDPS) (Figure 4) (28). Therefore, the low content of lysine
following ozone exposure may be due to a down-regulation of
the key enzyme DHDPS or an up-regulation of enzymes
involved in lysine catabolism and degradation, such as lysine
2
-oxoglutarate reductase and saccharopine dehydrogenase, at
4
).
the level of mRNA transcription, protein translation, or enzy-
matic activity (29). In this view, the decrease of the sulfur-
containing essential amino acid methionine, arising from the
aspartate family as well (Figure 4), could be also explained
Antioxidant Activity. Extracts from ozone-exposed seeds
showed higher TEAC values than those measured in seeds
collected from filtered chambers, at both time points considered
(
(
Figure 3). After 1 min of reaction, TEAC values were 0.0323
0.0014 and 0.0439 ( 0.0030 mmol equiv of Trolox/g in seeds
(
30). However, methionine decrement may be the consequence
of direct ozone oxidation of methionine to methionine sulfoxide
31). The branched essential amino acid valine is the precursor
from OTCs-F and OTCs-NF, respectively, with the higher
TEAC in the latter indicative of a stronger antioxidant activity
(
of an array of plant defense secondary metabolites named
phytoanticipins, including glucosinolates, isothiocyanates, and
cyanogenic glucosides, mainly involved in defense against
pathogens and phytophagy (32), whereas glycine is an osmolyte
precursor of betains, other osmolytes protecting plant against
water stress caused by drought and salinity (33). Hence, the
low levels of both these free amino acids may represent an
unfavorable trait for the ozone-exposed beans.
(
Figure 3). When extracts had completed the reaction, after 10
min, TEAC values rose in both samples, reaching 0.0518 (
0
.0028 and 0.0693 ( 0.0028 mmol equiv of Trolox/g in seeds
from OTCs-F and OTCs-NF, respectively (Figure 3).
DISCUSSION
The main aim of this work was to evaluate the effect of
exposure of bean plants to atmospheric ozone pollution in terms
of quality and nutritional traits of seeds. In general, the exposure
to a stress can affect the nutritional properties of plant foodstuffs,
Ornithine and tryptophan were the only amino acids of which
contents improved because of ozone exposure. Ornithine is a
nonprotein amino acid precursor of polyamines, polycationic

Nutritional Traits of Beans Exposed to Ozone
J. Agric. Food Chem., Vol. 57, No. 1, 2009 205
a
Table 2. Free Amino Acid Profile of Seed Extracts from Beans Grown in Charcoal-Filtered (F) and Nonfiltered (NF) Open-Top Chambers
amino acid
F (mg/100 g)
NF (mg/100 g)
P
functions
Glu (glutamic acid)
Gln (glutamine)
Asp (aspartic acid)
Asn (asparagine)
21.10 ( 0.60
0.93 ( 0.10
11.15 ( 0.26
119.48 ( 1.62
21.54 ( 0.32
0.96 ( 0.07
11.35 ( 0.13
103.15 ( 1.80
NS
NS
NS
**
nitrogen assimilation, stress induced
Ser (serine)
Ethan (ethanolamine)
0.97 ( 0.01
0.51 ( 0.04
1.06 ( 0.10
0.53 ( 0.03
NS
NS
phospholipid precursors, stress induced
Arg (arginine)
Orn (ornithine)
17.74 ( 0.19
0.18 ( 0.02
18.13 ( 1.30
0.28 ( 0.05
NS
**
arginine metabolism, polyamine precursors, stress induced
aspartate metabolism
Lys (lysine)
Thr (threonin)
0.60 ( 0.02
1.93 ( 0.03
0.54 ( 0.01
1.87 ( 0.07
**
NS
Ile (isoleucine)
Leu (leucine)
Val (valine)
1.73 ( 0.05
2.02 ( 0.06
12.05 ( 0.36
1.54 ( 0.16
1.90 ( 0.14
11.35 ( 0.35
NS
NS
*
branched amino acids, secondary metabolite precursors
Tyr (tyrosine)
Phe (phenylalanine)
Trp (tryptophan)
1.17 ( 0.04
2.36 ( 0.05
6.25 ( 0.16
1.13 ( 0.12
2.16 ( 0.22
8.34 ( 0.23
NS
NS
**
aromatic amino acids, secondary metabolite precursors
Cys (cysteine)
Met (methionine)
0.26 ( 0.02
15.02 ( 0.40
0.28 ( 0.03
13.98 ( 0.59
NS
*
sulfured amino acids
Sarc (sarcosine)
Gly (glycine)
Ala (alanine)
113.66 ( 3.26
2.63 ( 0.13
9.26 ( 0.18
0.65 ( 0.04
4.79 ( 0.50
136.39 ( 3.63
0.28 ( 0.02
0.34 ( 0.04
110.43 ( 9.12
2.05 ( 0.16
8.82 ( 0.38
0.60 ( 0.07
4.19 ( 0.33
131.75 ( 9.35
0.29 ( 0.01
0.37 ( 0.03
NS
**
osmolytes, osmoprotectants
NS
NS
NS
NS
NS
NS
ꢀ
-Ala (ꢀ-alanine)
Pro (proline)
Hypro (hydroxyproline)
AABA (R-aminobutyric acid)
GABA (γ-aminobutyric acid)
His (histidine)
2.43 ( 0.10
5.39 ( 0.15
6.46 ( 0.29
2.56 ( 0.37
5.30 ( 0.27
7.26 ( 0.78
NS
NS
NS
metal chelator
AAAA (R-aminoadipoic acid)
urea
a
Data (mean ( SD) derive from three separate seed extracts. Results are expressed as milligrams of amino acid per 100 g of seeds [*, P e 0.05; **, P e 0.01; NS,
not significant (Student’s t test)].
Table 3. Phytosterol Content in Seed Extracts from Beans Grown in
a
Charcoal-Filtered (F) and Nonfiltered (NF) Open-Top Chambers
more, it has been suggested that free tryptophan in legume seeds
can be easily absorbable at the stomach level and more apt to
cross the blood-brain barrier than protein tryptophan, thus
influencing the synthesis of serotonin (39).
phytosterol
F (mg/100 g)
NF (mg/100 g)
% increment
ꢀ
-sitosterol
stigmasterol
campesterol
cholesterol
27.2 ( 2.8
17.2 ( 1.0
2.3 ( 0.2
ND
48.1 ( 3.5
28.5 ( 1.7
3.5 ( 0.4
ND
76.83
65.69
52.17
Our results on phytosterols (plant sterols) showed that ozone
pollution greatly improved ꢀ-sitosterol, stigmasterol, and campes-
terol, the main plant sterols present in seed tissues (40). Sterols
determine the functional and structural properties of cell
membrane, such as fluidity and permeability, by modulating the
physical state of the lipid bilayer (reviewed in ref 41). At the
cellular level, the membrane represents the first structure directly
exposed to ozone injury, its polyunsaturated fatty acids being
the primary target. Ozone-induced lipid peroxidation, known
as the Criegee ozonation pathway, involves the ozonolysis of
a
Data (mean ( SD) derive from three separate seed extracts. Results are
expressed as milligrams of phytosterol per 100 g of seeds. ND, not detected.
nitrogenous compounds of low molecular weight ubiquitous in
all living organisms. In plants, both free and conjugated
polyamines improve ozone tolerance either by inhibiting the
biosynthesis of ethylene, the phytohormone partly responsible
for ozone damages, or by direct ROS scavenging, respectively
alkenes of polyunsaturated chains, that is, the electrophilic O
3
addition across the carbon-carbon double bonds, impairing
membrane structure and function (4). Thus, it seems to be
conceivable that the enhancement of the phytosterol pools, in
ozone-exposed plants, could assume a functional role to
counteract cell membrane damage. Certainly, from a nutritional
point of view, this alteration may be of interest, because of the
well-known cholesterol-lowering properties of plant sterols (42).
About phenylpropanoid content, ozone exposure reduced the
concentration of caffeic, p-coumaric, and sinapic acids, the three
main hydroxycinnamates in bean seeds (43), as well as the
content of both flavonol aglycone kaempferol and its 3-gluco-
(
34, 35). Additionally, apoplastic free polyamines can form
conjugates with hydroxycinnamates and phenolic acid deriva-
tives, effective in ROS detoxification (36). In some model plants,
tobacco and Arabidopsis, ozone-induced oxidative stress stimu-
lates both prechorismate and anthranilate biosynthetic pathways
(
9), raising the level of tryptophan, but not of phenylalanine
and tyrosine, the other aromatic amino acids arising from
chorismate via the branch point of prephenate/arogenate (37).
Tryptophan, besides being the precursor of the plant growth
hormone auxin, is the precursor of melatonin, a powerful
antioxidant significantly present in seed tissues (38). Further-

206 J. Agric. Food Chem., Vol. 57, No. 1, 2009
Iriti et al.
Figure 3. Antioxidant power of seed extracts from beans grown in
charcoal-filtered (F) and nonfiltered (NF) open-top chambers. Trolox
•+
equivalent antioxidant capacity (TEAC) is based on the ABTS [2,2′-
azinobis(3-ethylbenzothiazoline-6-sulfonic acid] radical cation decolorization
assay at specific time points. A higher TEAC is indicative of a stronger
antioxidant activity.
Figure 4. Aspartate pathway: lysine, threonine, and methionine belong
to the aspartate family of amino acids via the common intermediate
aspartate semialdehyde. Dihydrodipicolinate synthase (DHDPS) is the key
enzyme of the branch leading to lysine. See the text for more details.
recently reported in P. Vulgaris, was the most abundant
anthocyanin we found, instead of delphinidin-3-glucoside, as
indicated by previous studies (45). Flavonoid content was
reduced, too, after ozone exposure, despite the observed
increment of total polyphenols. This apparent discrepancy could
be due to the spectrophotometric detection of total polyphenols
that include proanthocyanidins (condensed tannins), polyphe-
nolic compounds arising from the polymerization of catechin
units. Interestingly, in a recent paper on the influence of
preharvest ozone exposure on the quality of strawberries, no
difference was reported in total anthocyanins and phenolic
compounds (46). The increase of total polyphenols could be
somehow in agreement with the higher content of the total
dietary fiber found in beans grown in an ozone-polluted
environment, which could be partly due to a higher content of
insoluble lignin, a phenol-derived polymer itself.
Finally, with regard to the antioxidant power, the above-
reported changes in phenylpropanoid composition do not
contribute to an explanation of the higher TEAC of ozone-
exposed seeds that can be possibly attributed to other com-
pounds, such as tocopherols or other lipid-soluble metabolites,
abundant in seed tissues and elicited by ozone (47).
In conclusion, although we did not take into account the
effects of ozone on some unfavorable traits of bean seeds,
including antinutritional factors (phytates, lectins, R-amylase and
Figure 2. Anthocyanin (A), flavonoid (B), and hydroxycinnamate (C)
contents in seed extracts from beans grown in charcoal-filtered (F) and
nonfiltered (NF) open-top chambers (OTCs). Data (mean ( SD) are
expressed as micrograms per gram of seeds; coumestrol values in B are
0
.012 µg (F) and 0.011 µg (NF) [/, P e 0.05; //, P e 0.01; NS, not
significant (Student’s t test)].
Table 4. Percentage Variation of Phenylpropanoidic Compounds in Seed
Extracts from Beans Grown in Nonfiltered Open-Top Chambers (OTCs) in
Comparison with Seeds Grown in Filtered OTCs
phenylpropanoid
% variation
phenylpropanoid
kaempferol
kaempferol-3-glucoside
caffeic acid
p-coumaric acid
ferulic acid
sinapic acid
% variation
delphinidin-3-glucoside
cyanidin-3-glucoside
peonidin-3-glucoside
petunidin-3-glucoside
pelargonidin-3-glucoside
daidzein
-68.41
-37.57
-61.23
+98.36
+343.37
-28.51
-0.32
-19.15
-13.12
-32.90
-17.26
-0.02
-23.54
-6.12
genistein
coumestrol
syringic acid
-8.33
side derivative, abundant in different Italian ecotypes (44). Total
anthocyanins were also reduced, although two of them, petuni-
din-3-glucoside and pelargonidin-3-glucoside, increased in
ozone-exposed seeds. Actually, peonidin-3-glucoside, only

Nutritional Traits of Beans Exposed to Ozone
J. Agric. Food Chem., Vol. 57, No. 1, 2009 207
protease inhibitors, tannins), flatulence promoters (the oligosac-
charides raffinose, stachyose, and verbascose), and structural
modifications detrimental for cooking (seed coat hardness) (48),
it seems that their nutritional value is not particularly affected,
if not possibly ameliorated, by the pollutant. In fact, some
important traits, such as dietary fiber, phytosterol, and polyphe-
nol contents appear to be enhanced, and the overall antioxidant
power is higher. The levels of two important free amino acids,
ornithine and tryptophan, are also higher, as is the total protein
content. However, with regard to the latter, it must be take into
account that seed storage proteins (globulins in legumes),
enzyme and protease inhibitors, and pathogenesis-related pro-
teins synthesized in response to abiotic and environmental
stresses can be potentially allergenic (49), thus exacerbating an
immunoglobulin (Ig) E-mediated hypersensitive response in
atopic patients. A set of experiments are now ongoing to
evaluate this important aspect.
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,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid); AOAC,
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