Physiological and Metabolomic Analysis of Punica Granatum (L.) Under Drought Stress
Physiological and Metabolomic Analysis of Punica Granatum (L.) Under Drought Stress
Physiological and Metabolomic Analysis of Punica Granatum (L.) Under Drought Stress
DOI 10.1007/s00425-015-2414-1
ORIGINAL ARTICLE
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were then irrigated at pot capacity. After 24 h, gas 15 min. To 0.5 ml aliquot of the supernatant, 0.5 ml of
exchange measurements, fluorescence parameters and leaf 10 mM potassium phosphate buffer (pH 7.0) and 0.75 ml
samples were collected again from control and stress-re- of 1 M KI were added, and the absorbance was measured at
covered plants (R). 390 nm. The relative absorbance (sample absorbance
minus the absorbance of the same supernatant aliquot
Chemicals without KI) was used to determine the H2O2 content
against a H2O2 standard curve. Data were expressed as
2-hexenal, 1-hexanol, pentane, proline, ninhydrin reagent lmol g-1 FW.
were purchased from Sigma-Aldrich (Milano, Italy).
Lipid peroxidation
Gas exchange and chlorophyll fluorescence
measurements Lipid peroxidation was measured in the term of malondi-
aldehyde (MDA) content (e = 155 mM-1 cm-1), a pro-
Steady-state net photosynthesis (A) and stomatal conduc- duct of lipid peroxidation, following the method of Heath
tance (gs), estimation of the quantum yield of photosynthetic and Packer (1968) with modifications. Briefly, leaf samples
non-cyclic electron transport (UPSII), and the photochemical (0.05 g) were homogenized in 1 ml of 0.1 % (w/v) tri-
quenching coefficient qP = (Fm0 - Fs)/(Fm0 - Fo0 ) were chloroacetic acid (TCA). The homogenate was centrifuged
determined in the laboratory under controlled conditions at 15,000g for 5 min. To the 0.5 ml aliquot of the super-
using a Li-6400 IRGA (LI-COR, Lincoln, NE, USA), by natant, 4 ml of 0.5 % (w/v) thiobarbituric acid (TBA) in
enclosing a portion of one leaf per plants in a 1 cm2 cuvette 20 % (w/v) TCA was added. The mixture was heated at
with a transparent Teflon window. A 300 lmol s-1 flow of 95 °C for 30 min and then quickly cooled in an ice bath.
non-contaminated air was provided to the leaves using a After centrifugation at 10,000g for 10 min, the absorbance
Teflon tube and mass flow controllers. The analyzed leaves of the supernatant was recorded at 532 nm. The absorbance
were exposed to a saturating photosynthetic photon flux at 600 and 440 nm of the same aliquot of leaf sample
density of 1000 lmol m-2 s-1 actinic white light, at a without TBA was subtracted to avoid overestimation of
temperature of 25 °C and with the relative humidity of the MDA. The MDA content was determined using a standard
air within the apparatus ranging between 45 and 55 %. In all curve and expressed as nmol MDA g-1 FW.
cases, only mature, fully expanded leaves were selected for
measurements from five different plants of pomegranate for VOCs analysis
each experimental condition. Two leaves per plant were
measured. VOCs analysis was done by Head Space Solid Phase Micro
Extraction sampling coupled with Gas Chromatography
Proline content analysis Mass Spectrometry (HS–SPME–GC–MS). For sample
preparation, 0.1 g aliquots of pomegranate leaf, finely
Extraction and determination of proline were performed ground with liquid nitrogen, were transferred to 2 ml screw
according to the method of Bates et al. (1973) with slight cap headspace vials and, for each sample, 0.5 ml of dis-
modifications. Briefly, leaf samples (20 mg) were extracted tilled water and approximately 0.15 g of NaCl were added.
with ethanol:water (70:30, v/v). Extracts were held for The volatile compound profile was obtained by SPME–
20 min a 95 °C, with 1 ml of and ninhydrin reagent: [1 % GC–MS technique. An Agilent 7820 GC-chromatograph
ninhydrin (w/v) in glacial acetic acid 60 % (v/v), ethanol equipped with a 5977A MSD with EI ionization operating
20 % (v/v)]. Proline content was measured with a spec- at 70 eV was used for analysis. A three-phase DVB/Car-
trophotometer (EASYSPEC SAFAS, UV-Vis spectropho- boxen/PDMS 75-lm SPME fiber (Supelco, Bellefonte, PA,
tometer) at 520 nm and calculated against a proline USA) was exposed in the head space of the vials at 60 °C
standard curve (5-2-1-0.5-0.2 mM of proline in 40:60 for 30 min for volatile compound sampling after a 5-min
ethanol:water, 40:60 v/v). Data were expressed as equilibration time. A Gerstel MPS2 XL autosampler
lmol g-1 fresh weight (FW). equipped with a magnetic transportation adapter and a
temperature-controlled agitator (250 rpm with on/cycles of
Hydrogen peroxide analysis 10 s) was used for ensuring consistent SPME extraction
conditions. This device ensured homogeneous sample
Endogenous H2O2 content was determined according to the mixing and favored the partitioning of VOCs into the head
method of Velikova et al. (2000), with slight modifications. space during SPME extraction. Chromatographic condi-
Briefly, leaves (0.25 g) were ground in 3 ml of 5 % TCA at tions were: column J&W Innovax (30 m, 0.25 mm, ID
4 °C. The homogenate was centrifuged at 12,000g for 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, (FTSW) in drought-stressed pomegranate pots reached, on
then 10 °C/min to 260 °C held for 5 min. Mass spectra average, 54 % (Fig. 1a, b). A significant decrease of CO2
were acquired within the 29–350 m/z interval with an assimilation to 0.02 lmol m-2s-1 was observed after
Agilent 5977 MSD spectrometer at a scan speed such as to 7 days of water deprivation, whereas gs dropped from
obtain three scans/s. The identification of volatile com- 0.296 measured in control plants to 0.029 mol m-2s-1,
pounds was done on the basis of both matching of the peak setting the end of the stress period (FTSW = 0). Interest-
spectra with library spectral database and matching of the ingly, after 24 h from irrigation after the end of drought
calculated Kovats retention indexes (KRI) with those experiment, both the values of net photosynthesis and
retrieved from literature. The data are expressed as percent stomatal conductance significantly increased, although
area of each compound over the sum of all the identified without reaching pre-stress values.
compounds.
Chlorophyll a fluorescence
Quantification of selected compounds
Three days after the beginning of drought stress, with
The compounds which resulted significantly different after FTSW close to 50 %, reductions of approximately 20 %
VOCs profile comparisons were quantitated after extraction were observed in the values of the effective quantum yield
with pentane according to Raffa and Smalley (1995) PSII (UPSII) (Fig. 1c) and the photochemical fluorescence
modified for a better quantitation of the compounds of quenching (qP) (Fig. 1d) in the drought-stressed pome-
interest and the use of GC–MS technique. The pentane granate plants compared to the control plants. At the end of
solution was supplemented with 5 methyl hexanol (at the drought stress experiment, when net photosynthesis
10 mg l-1) as internal standard (IS) instead of tridecane was below zero (Fig. 1a), the reductions were about 50 %
described in the original method. For extraction, 0.2 g for both UPSII and qP. Twenty-four hours after irrigation,
aliquots of the ground samples were soaked in 5 ml of IS UPSII, and qP increased significantly without reaching
pentane solution at room temperature for 24 h in 20 ml values similar to those of plants under treatment with
screw cap vials. The extracts were then filtered with irrigation (Fig. 1c, d).
0.45 lm PTFE syringe filters and injected in the GC–MS
system (1 ll in 1:10 split mode). Chromatographic condi- Proline content
tions were the same as for HS–SPME–GC–MS analysis.
Calibration lines, constructed with pure standards (hexanol, The proline content of drought-stressed pomegranate plants
2-hexenal, cis-3-hexenol, and hexenal) in the same ana- increased from 5.5 lmol g-1fresh weight (FW) in control
lytical conditions and in the range 2–50 mg l-1 allowed the leaves to 11.5 lmol g-1 FW, in drought-stressed leaves
calculation of the compound concentrations in the samples. and 11.01 lmol g-1 FW, 24 h after rehydration, respec-
tively (Fig. 2a).
Statistical analysis
Lipid peroxidation
Physiological data are mean ± SE of two leaves from each
of the five plants. Biochemical and metabolomics analysis The peroxyl radicals of polyunsaturated fatty acids
are means of ten leaves from five plants in each experi- (PUFAs) are usually formed in the membrane during lipid
mental condition. Means were analyzed using a one-way peroxidation and are subsequently decomposed, resulting
ANOVA with the Tukey post hoc test. The statistical in an instantaneous release of MDA and other volatile
analysis was performed by Statistica 10.0 (StatSoft soft- alkane and alkene compounds (Weber et al. 2004). Total
ware, Inc., USA). Statistical significance of the means MDA levels were measured in pomegranate leaves and
between control and treatments was evaluated at the 5 % found to be significantly increased of about threefold in
(P \ 0.05) probability level. 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).
Results
H2O2 content
Stomatal conductance and net photosynthesis
The H2O2 content of drought-stressed pomegranate plants
A significant decline in steady-state net photosynthesis increased from 10.7 lmol g-1 FW in control leaves to
(A) and stomatal conductance (gs) was observed after 25.9 lmol g-1 FW in drought-stressed leaves and
4 days, when the fraction of transpirable soil water 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 d Photochemical quenching of excitation energy (qP). Data points are
leaves. a Steady-state photosynthesis (measured in ambient air). means of two leaves from five plants for each experimental
b Stomatal conductance. c The effective quantum yield PSII (UPSII). condition ±SE
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Author contribution statement B. E. M., T. H., M. M., 04202006000200008
and Z. A. designed the research; B. E. M, S. C., and G. M. Loreto F, Pinelli P, Brancaleoni E, Ciccioli P (2004) 13C labelling
performed experiments; S. C., and G. E. analyzed data; B. reveals chloroplastic and extrachloroplastic pools of dimethylal-
E. M. wrote the manuscript. All authors contributed to lyl pyrophosphate and their contribution to isoprene formation.
Plant Physiol 135:1903–1907
editing and approving the final version of the manuscript.
Mano J, Tokushige K, Mizoguchi H, Fujii H, Khorobrykh S (2010)
Accumulation of lipid peroxide-derived, toxic a, b-unsaturated
Acknowledgments The authors kindly acknowledge Prof. Luca aldehydes (E)-2-pentenal, acrolein and (E)-2-hexenal in leaves
Calamai, UNIFI and CNR-ARCA Laboratory, for SPME analysis and under photoinhibitory illumination. Plant Biotech 27:193–197
Mrs Paola Bartolini for her skillful technical assistance in sample Matsui K (2006) Green leaf volatiles: hydroperoxide lyase pathway of
preparation and biochemical analysis. This work was supported by the oxylipin metabolism. Curr Opin Plant Biol 9:274–280
CNR-ANAS bilateral project MOXIVOL between National Research Medrano H, Escalona JM, Bota J, Gulias J, Flexas J (2002)
Council, Italy and Azerbaijan National Academy of Science, Azer- Regulation of photosynthesis of C3 plants in response to
baijan. The authors thank the anonymous reviewers for improving the progressive drought: stomatal conductance as a reference
manuscript with their comments. 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
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