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DOI 10.1007/s00425-015-2414-1

ORIGINAL ARTICLE

Physiological and metabolomic analysis of Punica granatum (L.)

under drought stress
Stefano Catola1,2,3 • Giovanni Marino3 • Giovanni Emiliani3 • Taravat Huseynova4 •

Mirza Musayev4 • Zeynal Akparov4 • Bianca Elena Maserti1

Received: 12 July 2015 / Accepted: 22 September 2015

Ó Springer-Verlag Berlin Heidelberg 2015

Abstract compounds extracted from the leaves of pomegranate

Main conclusion Punica granatum has a noticeable
adaptation to drought stress. The levels of the green leaf
volatile trans-2-hexenal increased in response to
drought stress suggesting a possible role of this com-
pound in drought stress response in pomegranate.
Punica granatum (L.) is a highly valued fruit crop for its
health-promoting effects and it is mainly cultivated in
semi-arid areas. Thus, understanding the response mecha-
nisms to drought stress is of great importance. In the pre-
sent research, a metabolomics analysis was performed to
evaluate the effects of drought stress on volatile organic
Electronic supplementary material The online version of this
article (doi:10.1007/s00425-015-2414-1) contains supplementary
material, which is available to authorized users.
& Bianca Elena Maserti
elena.maserti@ipsp.cnr.it
1
Dipartimento di Scienze Bio-Agroalimentari, Istituto per la
Protezione Sostenibile delle Piante, Area della Ricerca
Firenze, CNR-IPSP, Via Madonna del Piano 10, Florence,
Italy
2
Dipartimento di Scienze Agrarie, Alimentari e Agro-
Ambientali (DiSAAA-a), Università Degli Studi di Pisa, Via
del Borghetto 80, Pisa, Italy
3
Dipartimento di Scienze Bio-Agroalimentari, Istituto per la
Valorizzazione del Legno e delle Specie Arboree, Area della
Ricerca Firenze, CNR-IVALSA, Via Madonna del Piano 10,
Florence, Italy
4
Genetic Resources Institute, Azerbaijan National Academy of
Sciences (ANAS), Baku, Azerbaijan
plants grown under water shortage conditions. The time
course experiment (7 days of water deprivation and 24-h
recovery) consisted of three treatments (control, drought
stress, and rehydration of drought-stressed plants). Plant
weights were recorded and control plants were irrigated
daily at pot capacity to provide the lost water. Fraction of
transpirable soil water has been evaluated as indicator of
soil water availability in stressed plants. The levels of
proline, hydrogen peroxide and lipid peroxidation as well
as of the photosynthetic parameters such as photosynthesis
rate (A), stomatal conductance (gs), photosynthetic effi-
ciency of photosystem II, and photochemical quenching
were monitored after the imposition of drought stress and
recovery as markers of plant stress. Constitutive carbon
volatile components were analyzed in the leaf of control
and drought-stressed leaves using Head Space Solid Phase
Micro Extraction sampling coupled with Gas Chromatog-
raphy Mass Spectrometry. A total of 12 volatile com-
pounds were found in pomegranate leaf profiles, mainly
aldehydes, alcohols, and organic acids. Among them, the
trans-2-hexenal showed a significant increase in water-
stressed and recovered leaves respect to the well-watered
ones. These data evidence a possible role of the oxylipin
pathway in the response to water stress in pomegranate
plants.
Keywords Abiotic stress  Green leaf volatile  Plant
stress  Pomegranate  Trans-2-hexenal
Abbreviations
FTSW Fraction of transpirable soil water
GLV Green leaf volatile
MDA Malondialdehyde
VOC Volatile organic compounds

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Introduction
Pomegranate, a fruit native to the Caucasian area, has
gained widespread popularity as a functional food and
nutraceutical source. The health effects of the whole fruit,
as well as its juice and extract have been studied in relation
to a variety of chronic diseases. Promising results against
cardiovascular disease, diabetes, and prostate cancer have
been reported from human clinical trials (Johanningsmeier
and Harris 2011). Pomegranate plants are naturally grown
in arid and semi-arid areas. Thus, knowledge about their
response to drought stress is of importance from an eco-
nomical point of view, since drought stress may affect fruit
production and quality. Rodriguez et al. (2012) measured
the water relations in the leaves of pomegranate tree under
different irrigation conditions. Mellisho et al. (2012)
reported that pomegranate fruits subjected to drought stress
showed morphophysiological responses affecting the
quantity and quality of the final product. However, large
gaps remain in the understanding the response to drought
stress at molecular levels in pomegranate plants.
Abiotic stresses cause serious damage to plants; there-
fore, plants undergo a complex stress response through
signal transduction originating from environmental stimuli.
In wild or crop fields, water is often the most limiting
factor for plant growth. If plants do not receive adequate
rainfall or irrigation, the resulting drought stress can reduce
growth. Early responses to water stress occur at the leaf
level in response to stimuli generated in the leaf itself or
elsewhere in the plant. They have a negative influence on
carbon assimilation and growth. However, the integrated
response at the whole plant level, including carbon
assimilation and the allocation of photoassimilates to dif-
ferent plant parts and reproductive ability finally dictates
survival and persistence under environmental stress (Per-
eira and Chaves 1993). Thus, monitoring photosynthesis
parameters, such as gas exchange and chlorophyll fluo-
rescence is a widely used technique for tracking photo-
synthesis status and consequently the stress status in plants.
Plants produce a wide spectrum of volatile organic
compounds (VOCs) from above and below ground tissues.
The biosynthesis of most VOCs can be categorized into
three major pathways: terpenes, oxylipins (green leaf
volatiles), and shikimate and benzoic acid (Feussner and
Wasternack 2002; Dudareva et al. 2004). Most VOC are
released constitutively and the emissions can be observed
throughout the life cycle of the plant. However, environ-
mental stresses, mainly biotic, but also abiotic, such as
drought, heat, salinity, may induce de novo volatile syn-
thesis and emission including the production of specific
oxylipins, which have many different biological functions
(Blée 2002). Although the reduction of photosynthesis and
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the stomatal closure produced by drought and salt stress
was expected to negatively affect VOCs emission by
altering the carbon supply, it was reported that the induced
emission of VOC by salt and drought is made possible by
the induction of carbon sources alternative to photosyn-
thesis (Loreto et al. 2004).
The objective of this work is to shed new light on the
resistance mechanism to drought in the economic valuable
plant Punica granatum (L.). First, we measured the pat-
terns of the endogenous leaf volatile organic compounds
during drought stress by simultaneously measuring the net
photosynthesis, stomatal conductance, as well as stress
markers such as the quantum yield of photosynthetic non-
cyclic electron transport (UPSII), and the photochemical
quenching coefficient (qP) to determine the efficiency of
the photosynthetic apparatus. Additionally, the levels of
proline, hydrogen peroxide and lipid peroxidation levels
were assessed.
Materials and methods
Plant material
Ten two-year-old plants of Punica granatum L. were pur-
chased by a local nursery, transplanted in pots of 3L filled
with commercial soil (peat ? pumice, 1:1) and acclimated
in a growth chamber at control condition (T 25 °C; pho-
toperiod: light 16 h; dark 8 h. humidity 60–70 %) for
2 weeks. The plants were regularly watered to pot water
capacity before the beginning of the experiment. The day
preceding the initiation of the experiment, all the plants
were fully irrigated and allowed to drain the excess water
overnight. At Day 0, the pots were put into plastic bags
tightly closed at the basis of the plant (Suppl Figs. S1, S2,
S3) to avoid soil transpiration and weighted. Five plants
were water-stressed by withholding water. The pots were
daily weighted and each control plant was watered until
full pot capacity. Each day the position of the plants ran-
domized. Gas exchange and photosynthesis parameters
were measured daily to monitoring water stress. The water
stress was stopped when stomatal conductance in stressed
plants decreased nearly to 10 % of control plants values.
As indicator of soil water availability, the fraction of
transpirable soil water (FTSW; Sinclair and Ludlow 1986)
was calculated on each day n as:
FTSW ¼ ðPot weight at day n
 final pot weightÞ=ðInitial pot weight
 final pot weightÞ:
At the end of the stress period, leaves were harvested
from control and water-stressed plant and all the plants
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were then irrigated at pot capacity. After 24 h, gas
exchange measurements, fluorescence parameters and leaf
samples were collected again from control and stress-re-
covered plants (R).
Chemicals
2-hexenal, 1-hexanol, pentane, proline, ninhydrin reagent
were purchased from Sigma-Aldrich (Milano, Italy).
Gas exchange and chlorophyll fluorescence
measurements
Steady-state net photosynthesis (A) and stomatal conduc-
tance (gs), estimation of the quantum yield of photosynthetic
non-cyclic electron transport (UPSII), and the photochemical
quenching coefficient qP = (Fm0 - Fs)/(Fm0 - Fo0 ) were
determined in the laboratory under controlled conditions
using a Li-6400 IRGA (LI-COR, Lincoln, NE, USA), by
enclosing a portion of one leaf per plants in a 1 cm2 cuvette
with a transparent Teflon window. A 300 lmol s-1 flow of
non-contaminated air was provided to the leaves using a
Teflon tube and mass flow controllers. The analyzed leaves
were exposed to a saturating photosynthetic photon flux
density of 1000 lmol m-2 s-1 actinic white light, at a
temperature of 25 °C and with the relative humidity of the
air within the apparatus ranging between 45 and 55 %. In all
cases, only mature, fully expanded leaves were selected for
measurements from five different plants of pomegranate for
each experimental condition. Two leaves per plant were
measured.
Proline content analysis
Extraction and determination of proline were performed
according to the method of Bates et al. (1973) with slight
modifications. Briefly, leaf samples (20 mg) were extracted
with ethanol:water (70:30, v/v). Extracts were held for
20 min a 95 °C, with 1 ml of and ninhydrin reagent: [1 %
ninhydrin (w/v) in glacial acetic acid 60 % (v/v), ethanol
20 % (v/v)]. Proline content was measured with a spec-
trophotometer (EASYSPEC SAFAS, UV-Vis spectropho-
tometer) at 520 nm and calculated against a proline
standard curve (5-2-1-0.5-0.2 mM of proline in 40:60
ethanol:water, 40:60 v/v). Data were expressed as
lmol g-1 fresh weight (FW).
Hydrogen peroxide analysis
Endogenous H2O2 content was determined according to the
method of Velikova et al. (2000), with slight modifications.
Briefly, leaves (0.25 g) were ground in 3 ml of 5 % TCA at
4 °C. The homogenate was centrifuged at 12,000g for
15 min. To 0.5 ml aliquot of the supernatant, 0.5 ml of
10 mM potassium phosphate buffer (pH 7.0) and 0.75 ml
of 1 M KI were added, and the absorbance was measured at
390 nm. The relative absorbance (sample absorbance
minus the absorbance of the same supernatant aliquot
without KI) was used to determine the H2O2 content
against a H2O2 standard curve. Data were expressed as
lmol g-1 FW.
Lipid peroxidation
Lipid peroxidation was measured in the term of malondi-
aldehyde (MDA) content (e = 155 mM-1 cm-1), a pro-
duct of lipid peroxidation, following the method of Heath
and Packer (1968) with modifications. Briefly, leaf samples
(0.05 g) were homogenized in 1 ml of 0.1 % (w/v) tri-
chloroacetic acid (TCA). The homogenate was centrifuged
at 15,000g for 5 min. To the 0.5 ml aliquot of the super-
natant, 4 ml of 0.5 % (w/v) thiobarbituric acid (TBA) in
20 % (w/v) TCA was added. The mixture was heated at
95 °C for 30 min and then quickly cooled in an ice bath.
After centrifugation at 10,000g for 10 min, the absorbance
of the supernatant was recorded at 532 nm. The absorbance
at 600 and 440 nm of the same aliquot of leaf sample
without TBA was subtracted to avoid overestimation of
MDA. The MDA content was determined using a standard
curve and expressed as nmol MDA g-1 FW.
VOCs analysis
VOCs analysis was done by Head Space Solid Phase Micro
Extraction sampling coupled with Gas Chromatography
Mass Spectrometry (HS–SPME–GC–MS). For sample
preparation, 0.1 g aliquots of pomegranate leaf, finely
ground with liquid nitrogen, were transferred to 2 ml screw
cap headspace vials and, for each sample, 0.5 ml of dis-
tilled water and approximately 0.15 g of NaCl were added.
The volatile compound profile was obtained by SPME–
GC–MS technique. An Agilent 7820 GC-chromatograph
equipped with a 5977A MSD with EI ionization operating
at 70 eV was used for analysis. A three-phase DVB/Car-
boxen/PDMS 75-lm SPME fiber (Supelco, Bellefonte, PA,
USA) was exposed in the head space of the vials at 60 °C
for 30 min for volatile compound sampling after a 5-min
equilibration time. A Gerstel MPS2 XL autosampler
equipped with a magnetic transportation adapter and a
temperature-controlled agitator (250 rpm with on/cycles of
10 s) was used for ensuring consistent SPME extraction
conditions. This device ensured homogeneous sample
mixing and favored the partitioning of VOCs into the head
space during SPME extraction. Chromatographic condi-
tions were: column J&W Innovax (30 m, 0.25 mm, ID
0.5 lm DF); injection temperature 250 °C, splitless mode,
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oven programme 40° for 1 min then 5 °C/min to 200 °C,
then 10 °C/min to 260 °C held for 5 min. Mass spectra
were acquired within the 29–350 m/z interval with an
Agilent 5977 MSD spectrometer at a scan speed such as to
obtain three scans/s. The identification of volatile com-
pounds was done on the basis of both matching of the peak
spectra with library spectral database and matching of the
calculated Kovats retention indexes (KRI) with those
retrieved from literature. The data are expressed as percent
area of each compound over the sum of all the identified
compounds.
Quantification of selected compounds
The compounds which resulted significantly different after
VOCs profile comparisons were quantitated after extraction
with pentane according to Raffa and Smalley (1995)
modified for a better quantitation of the compounds of
interest and the use of GC–MS technique. The pentane
solution was supplemented with 5 methyl hexanol (at
10 mg l-1) as internal standard (IS) instead of tridecane
described in the original method. For extraction, 0.2 g
aliquots of the ground samples were soaked in 5 ml of IS
pentane solution at room temperature for 24 h in 20 ml
screw cap vials. The extracts were then filtered with
0.45 lm PTFE syringe filters and injected in the GC–MS
system (1 ll in 1:10 split mode). Chromatographic condi-
tions were the same as for HS–SPME–GC–MS analysis.
Calibration lines, constructed with pure standards (hexanol,
2-hexenal, cis-3-hexenol, and hexenal) in the same ana-
lytical conditions and in the range 2–50 mg l-1 allowed the
calculation of the compound concentrations in the samples.
Statistical analysis
Physiological data are mean ± SE of two leaves from each
of the five plants. Biochemical and metabolomics analysis
are means of ten leaves from five plants in each experi-
mental condition. Means were analyzed using a one-way
ANOVA with the Tukey post hoc test. The statistical
analysis was performed by Statistica 10.0 (StatSoft soft-
ware, Inc., USA). Statistical significance of the means
between control and treatments was evaluated at the 5 %
(P \ 0.05) probability level.
Results
Stomatal conductance and net photosynthesis
A significant decline in steady-state net photosynthesis
(A) and stomatal conductance (gs) was observed after
4 days, when the fraction of transpirable soil water
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(FTSW) in drought-stressed pomegranate pots reached, on
average, 54 % (Fig. 1a, b). A significant decrease of CO2
assimilation to 0.02 lmol m-2s-1 was observed after
7 days of water deprivation, whereas gs dropped from
0.296 measured in control plants to 0.029 mol m-2s-1,
setting the end of the stress period (FTSW = 0). Interest-
ingly, after 24 h from irrigation after the end of drought
experiment, both the values of net photosynthesis and
stomatal conductance significantly increased, although
without reaching pre-stress values.
Chlorophyll a fluorescence
Three days after the beginning of drought stress, with
FTSW close to 50 %, reductions of approximately 20 %
were observed in the values of the effective quantum yield
PSII (UPSII) (Fig. 1c) and the photochemical fluorescence
quenching (qP) (Fig. 1d) in the drought-stressed pome-
granate plants compared to the control plants. At the end of
the drought stress experiment, when net photosynthesis
was below zero (Fig. 1a), the reductions were about 50 %
for both UPSII and qP. Twenty-four hours after irrigation,
UPSII, and qP increased significantly without reaching
values similar to those of plants under treatment with
irrigation (Fig. 1c, d).
Proline content
The proline content of drought-stressed pomegranate plants
increased from 5.5 lmol g-1fresh weight (FW) in control
leaves to 11.5 lmol g-1 FW, in drought-stressed leaves
and 11.01 lmol g-1 FW, 24 h after rehydration, respec-
tively (Fig. 2a).
Lipid peroxidation
The peroxyl radicals of polyunsaturated fatty acids
(PUFAs) are usually formed in the membrane during lipid
peroxidation and are subsequently decomposed, resulting
in an instantaneous release of MDA and other volatile
alkane and alkene compounds (Weber et al. 2004). Total
MDA levels were measured in pomegranate leaves and
found to be significantly increased of about threefold in
drought-stressed plants, whereas MDA content dropped at
values similar to those measured in the control in the leaves
after 24 h rehydration (Fig. 2b).
H2O2 content
The H2O2 content of drought-stressed pomegranate plants
increased from 10.7 lmol g-1 FW in control leaves to
25.9 lmol g-1 FW in drought-stressed leaves and
12.6 lmol g-1 FW 24 h after rehydration (Fig. 2c).
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Fig. 1 Time courses of drought stress experiment in pomegranate
leaves. a Steady-state photosynthesis (measured in ambient air).
b Stomatal conductance. c The effective quantum yield PSII (UPSII).
Changes of leaf secondary volatile compounds
following drought stress and recovery
To investigate the effect of drought stress on secondary
metabolism, the levels of volatile organic compounds in
control, drought-stressed and 24 h-recovered plants were
analyzed using HS–SPME–GC–MS technique. In total, 12
metabolites were identified by matching their mass spectra
and retention time with known standards (Fig. 3 and Suppl.
Fig. S4).
Among them, two aldehydes, namely hexanal, trans-2-
hexenal and two alcohols, 1-hexanol and cis-3-hexenol
known to be potentially involved in abiotic and biotic stress
were quantified in control, drought-stressed and water-re-
covered pomegranate plants after pentane extraction and
GS-MS by matching its spectra area in each sample with
the areas of known concentrations of standards. Interest-
ingly, among the four compounds, only the trans-2-hexenal
levels showed significant accumulation after drought stress,
and dropped at values similar to that of control after 24 h of
irrigation (Fig. 4).
d Photochemical quenching of excitation energy (qP). Data points are
means of two leaves from five plants for each experimental
condition ±SE
Discussion
In the present study, we used an integrated physiological
and metabolomics approach to unravel the mechanisms
underlying mediated drought stress resistance in P.
granatum. The water deficit was gradually imposed until
the FTSW dropped to 0 to mimic what plants may expe-
rience in the field. In this way, the plant has time to adjust
its metabolism and better deploy its adaptive responses.
Therefore, this slowly developing drought stress may
increase the physiological relevance of the physiological
and metabolomic changes observed. The decrease of
stomatal conductance in drought-stressed pomegranate
leaves may be useful to diminish transpiration rate and
maintain turgescence, during drought stress. While the loss
of water vapor becomes slow due to the process of stomatal
closure, it also reduces the absorption of carbon dioxide
(CO2) and consequently, net photosynthesis (Chaves et al.
2011). In Minquartia guianensis seedlings subjected to
water deficiency, net photosynthesis and stomatal con-
ductance rates were reduced to about 50 % of those for
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Fig. 2 Levels of proline (a),

lipid peroxidation (b, measured
as malondialdehyde, MDA,
content) and H2O2 (c) in the
leaves of control (C), water-
stressed (WS) and 24 h after
irrigation (R) pomegranate
plants. Data points are means of
ten leaves from five plants for
each experimental
condition ±SE. Asterisk
significant differences
(P \ 0.05)

Fig. 3 Profile of the volatile

organic compound identified in
the leaves of control empty
square, water-stressed red
square and recovered blue
square pomegranate plants.
Data points are means of ten
leaves from five plants for each
experimental condition ±SE

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Fig. 4 Levels of trans-2-hexenal, hexanal, 1-hexanol and cis-3-
hexenol in the leaves of control empty square, water-stressed red
square and recovered blue square pomegranate plants. Data points are
means of ten leaves from five plants for each experimental
condition ±SE. Different letters significant differences (P \ 0.05)
control plants after drought imposition (Liberato et al.
2006). Medrano et al. (2002) suggested that stomata close
progressively as drought progresses and by parallel, net
photosynthesis decreases to keep the water balance and
minimize damage. Under these conditions that diminish
CO2 diffusion through the mesophyll, the photoinhibition,
a process that reduces the efficiency of UPSII and induces
photorespiration and H2O2 production might be occurred in
pomegranate leaves, in accordance with data reported by
Hossain et al. (2009). On the other hand, the photochemical
quenching of excitation energy (qP) is related to both the
proportion of electrons transported from PSII and stomatal
opening for absorption of CO2. The decline in values of
this variable may be indicative of an increase in quinone A
(QA) in the reduced state due to the increase in the pro-
portion of inactive PSII reaction centers (Genty et al.
1989). The fast recovery of the pomegranate photosyn-
thetic apparatus after irrigation suggests that other non-
assimilatory processes, such as Mehler reaction and/or
photorespiration may be enhanced to dissipate some of
excess excitation energy in the drought-stressed plants
(Chaves et al. 2011). However, whether and how the
Mehler reaction and/or photorespiration may operate in
pomegranate plant and contributes to avoid photoinhibitory
damage in drought-stressed plants remain to be
investigated.
Three compounds generally used as markers of stress,
such as proline, MDA, and H2O2 had interesting profiles
worth highlighting along with the drought stress experi-
ment in pomegranate. Proline has been widely recognized
as a drought-inducible proteinogenic amino acid with an
osmoprotective role, enabling the cell to retain more water.
In many plants species, an increase of proline content
during drought stress had been correlated with drought-
stress tolerance (Szabados and Savouré 2010). Besides its
role as an osmotic agent, proline has also been shown to
directly act as a ROS scavenger (Szabados and Savouré
2010) and as a regulator of the cellular redox status
(Sharma et al. 2011). In pomegranate plants under severe
drought stress, a significant proline accumulation was
observed in drought-stressed leaves, whereas no proline
accumulation was detected under well-watered, controlled
conditions implying that proline accumulation was specif-
ically induced by water withholding to minimize water
lost. Drought stress induced a noticeable accumulation of
H2O2 in pomegranate leaves, whereas the levels decreased
at control level after rehydration (Fig. 2). Plants are natural
producers of ROS, consequently superoxide and H2O2 are
synthesized at very high rates under normal conditions.
There is a frail balance between ROS production and
scavenging that defines the normal steady-state level of
intracellular ROS. Under drought stress, this balance suf-
fers an upward shift, ROS production being enhanced due
to stomatal closure and the concomitant limitation on CO2
fixation (Boo and Jung 1999). On the other hand, H2O2 can
act as signaling molecule promoting the accumulation of
several cellular protectants that may act directly or indi-
rectly in the regulation of the cellular redox status, and
consequently control the extent of the signal itself.
The accumulation of MDA may be indicative of
increased lipid peroxidation in drought-stressed pome-
granate leaves (Gunes et al. 2006). In plant cells, peroxi-
dation of free fatty acids can occur both in enzymatic and
non-enzymatic ways with the generation of a variety of
breakdown products such as aldehydes, alcohols, and their
esters. The process is considered as the main event
involved in oxidative damage to cell. However, it has been
suggested that reactive lipid species formed through lipid
peroxidation can benefit cells in a number of ways (Bhat-
tacharjee 2014).
The green leaf volatiles (GLV) are synthetized through
the hydroperoxide lyase pathway of oxylipin metabolism
and their synthesis is thought to be regulated at the step of
lipid hydrolysis, which provides free fatty acids to the
pathway (Matsui 2006). It is known that after wounding
GLVs are rapidly synthetized and emitted. The physiologi-
cal significance of the rapid formation of GLVs has been
mainly discussed in the context of defense against biotic
stresses, as insecticidal, fungicidal, and bactericidal activi-
ties have been reported for (Z)-3-hexenal and its related
aldehydes (Kishimoto et al. 2008). Also, increased emission
of GLVs is suggested to be important both for signaling
within and between plants and for allowing plants and other
organisms surrounding them to recognize or compete with
each other (Hammond et al. 2000; Niinemets et al. 2013).
On the other hand, Savchenko and Dehesh (2014) found that
in Arabidopsis drought stress induced production of 2-hex-
enal and 12-oxophytodienoic acid (OPDA), suppressing
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hexenol and maintaining jasmonic acid (JA) at the basal
levels. In pomegranate, although the profile of the volatile
carbon compounds in drought-stressed leaves showed no
qualitative changes with respect to the control ones, the
levels of endogenous trans-2-hexenal increased in drought-
stressed plants and decreased to control values after 24 h
from irrigation. This observation is in accordance with the
reported increased endogenous levels of (E)-2-pentenal and
(E)-2-hexenal in leaves of photo-sensitive and photo-tolerant
tobacco plants under photoinhibitory illumination found by
Mano et al. (2010). The authors suggested that the com-
pounds were involved in the photo-oxidative damage of leaf
cells, because they increased at early stages of the stress
treatment, and their increases were smaller in the photo-
tolerant lines. Also, Yamauchi et al. (2015) reported that
vaporized trans-2-hexenal on Arabidopsis plants rapidly
increased transiently the internal 2-hexenal concentration
and acted as signal chemical inducing the expression of
abiotic stress-related genes such as HSFA and DREB2. The
increased levels of endogenous trans-2-hexenal in drought-
stressed leaves of pomegranate plants may imply a role of
such compound as signaling compound in the response to
photo-oxidative damage produced by drought stress. How-
ever, the intriguingly question of what is the physiological
significance of 2-hexenal and in general of GLV in pome-
granate under drought stress should be dissected by further
investigations.
Author contribution statement B. E. M., T. H., M. M.,
and Z. A. designed the research; B. E. M, S. C., and G. M.
performed experiments; S. C., and G. E. analyzed data; B.
E. M. wrote the manuscript. All authors contributed to
editing and approving the final version of the manuscript.
Acknowledgments The authors kindly acknowledge Prof. Luca
Calamai, UNIFI and CNR-ARCA Laboratory, for SPME analysis and
Mrs Paola Bartolini for her skillful technical assistance in sample
preparation and biochemical analysis. This work was supported by the
CNR-ANAS bilateral project MOXIVOL between National Research
Council, Italy and Azerbaijan National Academy of Science, Azer-
baijan. The authors thank the anonymous reviewers for improving the
manuscript with their comments.
References
Bates LE, Waldren RP, Teare ID (1973) Rapid determination of free
proline for water stress studies. Plant Soil 39:205–207
Bhattacharjee S (2014) Membrane lipid peroxidation and its conflict
of interest: the two faces of oxidative stress. Curr Sci
107:1811–1823
Blée E (2002) Impact of phyto-oxylipins in plant defense. Trends
Plant Sci 7:315–321
Boo YC, Jung J (1999) Water deficit-induced oxidative stress and
antioxidative defenses in rice plants. J Plant Physiol
155:255–261
123
Planta
Chaves MM, Costa JM, Saibo NJM (2011) Recent advances in
photosynthesis under drought and salinity. In: Turkan I (ed)
Plant responses to drought and salinity stress: developments in a
post-genomic era. Adv Bot Res 57:49–104
Dudareva N, Pichersky E, Gershenzon J (2004) Biochemistry of plant
volatiles. Plant Physiol 135:1993–2011
Feussner I, Wasternack C (2002) The lipoxygenase pathway. Annu
Rev Plant Biol 53:275–297
Genty B, Briantais JM, Baker NR (1989) The relationship between the
quantum yield of photosynthetic electron transport and quenching
of chlorophyll fluorescence. Biochim Biophys Acta 990:87–92
Gunes A, Soylemezoglu G, Inal A et al (2006) Antioxidant and
stomatal responses of grapevine (Vitis vinifera L.) to boron
toxicity. Sci Hortic 110:279–284
Hammond G, Rangel S, Kubo I (2000) Volatile aldehydes are
promising broad-spectrum postharvest insecticides. J Agric Food
Chem 48:4410–4417
Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplast.
I. Kinetics and stoichiometry of fatty acid peroxidation. Arch
Biochem Biophys 125:189–198
Hossain Z, López-Climent MF, Arbona V, Pérez-Clemente RM,
Gómez-Cadenas A (2009) Modulation of the antioxidant system
in Citrus under waterlogging and subsequent drainage. J Plant
Physiol 166:1391–1404
Johanningsmeier SD, Harris GK (2011) Pomegranate as a functional food
and nutraceutical source. Annu Rev Food Sci Technol 2:181–201
Kishimoto K, Matsui K, Ozawa R, Takabayashi J (2008) Direct
fungicidal activities of C6-aldehydes are important constituents
for defense responses in Arabidopsis against Botrytis cinerea.
Phytochemistry 69:2127–2132
Liberato MAR, Gonçalves JFC, Chevreuil LR, da Rocha Nina A Jr,
Fernandes AV, Moreira dos Santos U, Jr (2006) Leaf water
potential, gas exchange and chlorophyll a fluorescence in
araraquara seedlings (Minquartia guianensis Aubl.) under water
stress and recovery. Braz J Plant Physiol. doi:10.1590/S1677-
04202006000200008
Loreto F, Pinelli P, Brancaleoni E, Ciccioli P (2004) 13C labelling
reveals chloroplastic and extrachloroplastic pools of dimethylal-
lyl pyrophosphate and their contribution to isoprene formation.
Plant Physiol 135:1903–1907
Mano J, Tokushige K, Mizoguchi H, Fujii H, Khorobrykh S (2010)
Accumulation of lipid peroxide-derived, toxic a, b-unsaturated
aldehydes (E)-2-pentenal, acrolein and (E)-2-hexenal in leaves
under photoinhibitory illumination. Plant Biotech 27:193–197
Matsui K (2006) Green leaf volatiles: hydroperoxide lyase pathway of
oxylipin metabolism. Curr Opin Plant Biol 9:274–280
Medrano H, Escalona JM, Bota J, Gulias J, Flexas J (2002)
Regulation of photosynthesis of C3 plants in response to
progressive drought: stomatal conductance as a reference
parameter. Ann Bot 89:895–905
Mellisho CD, Egea I, Galindo A, Rodriguez P, Rodriguez J, Conejero
W, Romojaro F, Torrecillas A (2012) Pomegranate (Punica
granatum L.) fruit response to different deficit irrigation
conditions. Agr Water Manag 114:30–36
Niinemets Ü, Kännaste A, Copolovici L (2013) Quantitative patterns
between plant volatile emissions induced by biotic stresses and
the degree of damage. Front Plant Sci 4:1–15
Pereira JS, Chaves MM (1993) Plant water deficits in Mediterranean
ecosystems. In: Smith JAC, Griffiths H (eds) Plant responses to
water deficits-from cell to community. Bios Scientific Publishers
Ltd, Oxford, pp 237–251
Raffa KF, Smalley EB (1995) Interaction of pre-attack and induced
monoterpene concentrations in host conifer defense against bark
beetle-fungal complexes. Oecologia 102:285–295
Rodriguez P, Mellisho CD, Conejero W, Cruz ZN, Ortuno MF,
Galindo A, Torrecillas A (2012) Plant water relations of leaves
Planta
of pomegranate trees under different irrigation conditions.
Environ Exp Bot 77:19–24
Savchenko T, Dehesh Z (2014) Drought stress modulates oxylipin
signature by eliciting 12-OPDA as a potent regulator of stomatal
aperture. Plant Signal Behav 9:e28304-1
Sharma S, Villamor JG, Verslues PE (2011) Essential role of tissue-
specific proline synthesis and catabolism in growth and redox
balance at low water potential. Plant Physiol 157:292–304
Sinclair TR, Ludlow MM (1986) Influence of soil water supply on the
plant water balance of four tropical grain legumes. Funct Plant
Biol 13:329–341
Szabados L, Savouré A (2010) Proline: a multifunctional amino acid.
Trends Plant Sci 15:89–97
Velikova V, Yordanov I, Edreva A (2000) Oxidative stress and some
antioxidant systems in acid rain-treated bean plants protective
role of exogenous polyamines. Plant Sci 15:59–66
Weber H, Chetelat A, Reymond P, Farmer EE (2004) Selective and
powerful stress gene expression in Arabidopsis in response to
malondialdehyde. Plant J 37:877–888
Yamauchi Y, Kunishima M, Mizutani M, Sugimoto Y (2015)
Reactive short-chain leaf volatiles act as powerful inducers of
abiotic stress-related gene expression. Sci Rep 5:8030. doi:10.
1038/srep08030
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