FT-Raman spectroscopy as a tool in evaluation the response of plants to drought stress

Abstract

The aim of study was to evaluate the usefulness of FT-Raman spectroscopy in assessing stress-induced metabolic changes in plants. 20-d-old optimally watered plants of soybean were exposed to drought. Metabolic changes in optimally watered and drought-stressed plants were monitored using FT-Raman spectroscopy. In parallel, analyses were carried out of fatty acid composition and pigment content using analytical methods. These compounds are associated with the response of plants to environmental stress. While fatty acid assays in study were inconclusive, the pigment content analysis gave promising results. FT-Raman experiment demonstrated a decrease in carotenoid content in leaf, as a result of drought, which was confirmed by spectrophotometric analysis. In addition to the analysis of aforementioned compounds, FT-Raman spectroscopy allowed the simultaneous observation of
a wider spectrum of compounds scattering the radiation in the leaves tested, and their subsequent comparative analysis. The impact of drought on metabolism of soybean was clearly visible on spectra and confirmed using cluster analysis. The technical problem of the influence of leaf water content on measurements, which appeared in studies, will be discussed. To conclude, FT-Raman spectroscopy may be a good complement to other non-invasive methods, e.g., fluorescent methods, in assessing the stress-induced damage of crops.

Full text

Open Chem., 2015; 13: 1091–1100
Research Article Open Access
Magdalena Rys*, Maciej Szaleniec, Andrzej Skoczowski, Iwona Stawoska, Anna Janeczko
FT-Raman spectroscopy as a tool in evaluation
the response of plants to drought stress
DOI: 10.1515/chem-2015-0121
received August 26, 2014; accepted June 1, 2015.
Abstract: The aim of study was to evaluate the usefulness
of FT-Raman spectroscopy in assessing stress-induced
metabolic changes in plants. 20-d-old optimally watered
plants of soybean were exposed to drought. Metabolic
changes in optimally watered and drought-stressed plants
were monitored using FT-Raman spectroscopy. In parallel,
analyses were carried out of fatty acid composition
and pigment content using analytical methods. These
compounds are associated with the response of plants to
environmental stress. While fatty acid assays in study were
inconclusive, the pigment content analysis gave promising
results. FT-Raman experiment demonstrated a decrease in
carotenoid content in leaf, as a result of drought, which
was conrmed by spectrophotometric analysis. In addition
to the analysis of aforementioned compounds, FT-Raman
spectroscopy allowed the simultaneous observation of
a wider spectrum of compounds scattering the radiation
in the leaves tested, and their subsequent comparative
analysis. The impact of drought on metabolism of soybean
was clearly visible on spectra and conrmed using
cluster analysis. The technical problem of the inuence
of leaf water content on measurements, which appeared
in studies, will be discussed. To conclude, FT-Raman
spectroscopy may be a good complement to other non-
invasive methods, e.g., uorescent methods, in assessing
the stress-induced damage of crops.
Keywords: Carotenoids, Drought stress, Fatty acids,
FT-Raman spectroscopy, Soybean
1 Introduction
Abiotic and biotic environmental stresses such as drought,
ooding, extreme temperatures or diseases, occurring
during the growing season, aect the metabolism of
plants, causing multidirectional biochemical changes in
cells [1]. The purpose of these alterations is the adaptation
of plants to the existing conditions and to counteract the
eects of stress. The mechanisms of plant responses to
various stress factors have been investigated for many
years as the occurrence of environmental stress is
a signicant problem in agriculture. Therefore, methods
are sought for rapid assessment of the post-stress damage
in individual plants and entire plantations. These
studies involved invasive but also non-invasive methods.
The major advantage of non-invasive techniques is their
non-destructive eect on plant tissue in comparison to
standard analytical methods. The most popular non-
invasive methods include infra-red analysis of metabolic
CO2 exchange [2], reectance techniques [3], and fast
kinetic chlorophyll a uorescence [4]. Isothermal
calorimetry is less known but is also applied [5,6]. These
non-invasive methods allow the assessment of the status
of individual leaves, whole plants or even entire crop [7],
providing information on broadly dened eciency
of photosynthesis (uorescence methods) or total
metabolic activity (calorimetry). Raman spectroscopy
with Fourier transformation (FT-Raman spectroscopy)
is also classied as non-invasive technique where
information on the chemical composition of a sample
can be obtained without any need to disrupt it [8].
If characteristic key bands of individual analyte
molecules are found in the spectrum, then FT-Raman
spectroscopy can be successfully applied to identify
various plant components [9,10]. The changes in
chemical composition of the plant can be observed on
the FT-Raman spectra in the intensities of visible bands
originating from various plant components. There is
*Corresponding author: Magdalena Rys: The Franciszek Górski
Institute of Plant Physiology, Polish Academy of Sciences,
Niezapominajek 21, PL-30-239 Krakow, Poland ,
E-mail: m.rys@ifr-pan.edu.pl
Maciej Szaleniec: Institute of Catalysis and Surface Chemistry,
Polish Academy of Sciences, Niezapominajek 8,
PL-30-239 Krakow, Poland
Andrzej Skoczowski, Iwona Stawoska: Institute of Biology,
Pedagogical University of Cracow, Podchorążych 2,
PL-31-054 Krakow, Poland
Anna Janeczko: The Franciszek Górski Institute of Plant Physiology,
Polish Academy of Sciences, Niezapominajek 21, PL-30-239 Krakow,
Poland
© 2015 Magdalena Rys et al., licensee De Gruyter Open.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.

1092 Magdalena Rys et al.
a simple correlation between the intensity of FT-Raman
signal and the concentration of the analyte in the
sample. The dierences in FT-Raman band on the spectra
indicate dierent chemical composition of the samples
investigated. Generally, FT-Raman spectroscopy allows
the study of analytes in situ in their natural environment.
Primary and secondary metabolites can be analysed,
such as mono- and oligosaccharides, fatty acids, amino
acids, proteins, alkaloids, avonoids, carotenoids,
terpenes and polyacetylenes. In the biological sciences,
FT-Raman spectroscopy has a wide range of applications,
for review see [11]. Until now, FT-Raman spectroscopy
has been used in plant stress physiology to study the
phenomenon of allelopathy [12,13], the eects of ozone,
light spectrum and pathogenesis on the metabolism of
plants [6].
The aim of this study was to evaluate the usefulness
of FT-Raman spectroscopy in assessing stress-induced
metabolic changes in plants. Soybean was selected as the
research object [Glycine max (L.) Merrill] (Legume), as it
is one of the most important crops in the world. Initially,
it was grown in China, then growth spread to other Asian
countries and currently it is cultivated worldwide because
of its usefulness in human and animal nourishment [14]
as well as industrial and medicinal applications [15].
Despite the relatively large geographical adaptability,
soybean plant is susceptible to cold stress but also, as the
whole family of leguminous plants, is sensitive to drought,
particularly during germination and early growth stages.
In the experiments conducted in this study, soybean was
exposed to water decit. As mentioned above – many
biochemical changes occur in plant cells, as a result of the
action of stress factors (including drought), in the content
and the chemical composition of the compounds such
as fatty acids or carotenoids that are detectable by FT-
Raman spectroscopy [16,17]. In parallel with the analysis
of the metabolic changes of plant tissue by FT-Raman
spectroscopy we have carried out biochemical analysis
of fatty acids and pigments (carotenoids) in the leaves
of plants watered optimally and drought-stressed plants
using standard analytical methods (gas-chromatography
and spectrophotometry).
2 Experimental procedure
2.1 Plant material and experimental design
The experiment was performed in pots (40 × 20 × 15 cm;
15 plants per pot). Seeds of Aldana and Augusta cultivars
were soaked 24 h in water on Petri dishes. Aer sowing
into the soil, seeds germinated in a growth chamber
(darkness, 25°C) for 5 days, next the pots were moved to the
greenhouse under natural light conditions (June; latitude:
50°03’ North, longitude: 19°55’ East) at a day/night
temperature, 24/20°C. On the 20th day of vegetation,
seedlings had a pair of the rst foliage leaves and
developed one compound leaf with three leaets. On the
20th day of growing, the plants were watered for the last
time, and then watering was ceased, which resulted in
the occurrence of drought symptoms during next 5 days
(gradual plant wilting was observed). At that time, only
compensating watering was applied in order to avoid
uneven water loss between pots.
The rst foliage leaves were collected from 20-d-old
watered plants and from 26-d-old drought-treated
plants to measure fatty acid composition and pigment
(carotenoids and chlorophylls) content. It was important
that plants were in comparable stage of development due
to the fact that the development during period of drought
was slowed down. FT-Raman spectroscopy analysis was
carried out before the drought stress (20-d-old-plants)
and aer drought stress (26-d-old-plants). The drought-
stressed plants were subsequently watered (rehydrated)
and FT-Raman spectroscopy was performed for the third
time.
2.2 FT-Raman spectroscopy measurements
FT-Raman measurements were performed on fresh
soybean leaves using a FT-Raman Spectrometer Nicolet
NXR 9650 equipped with a Nd:YAG laser, emitting at
1064 nm, and a germanium detector cooled with liquid
nitrogen. Spectrometer was provided with an xy stage,
a mirror objective and a prism slide for redirecting the
laser beam.
All spectra were collected in the range of
150–3700 cm-1, accumulated from 128 scans, measured
with the laser power of 0.6 W, with a spectral resolution
of 8 cm-1 using an unfocused laser beam of the diameter
of approx. 100 µm. FT-Raman spectra were registered by
the Omnic/Thermo Scientic soware. Ten spectra were
collected and averaged for each object. Despite the fact that
the spectra were recorded in a wide range of frequencies,
only the range of 720 to 1670 cm-1 was analyzed. Within this
region one can identify vibration modes characteristic of
these groups of compounds of particular interest (namely
fatty acids, carotenoids and chlorophylls).

FT-Raman spectroscopy as a tool in evaluation the response of plants to drought stress 1093
2.3 Chemometrics
Similarities between FT-Raman spectra were studied
using hierarchical cluster analysis in the Statistica
soware package 10.0 (StatSo Inc.). The spectra were
baseline corrected. The cluster analysis was performed
for the whole wavenumber range using Ward’s algorithm.
The spectral distances were calculated with the standard
algorithm.
2.4 Fatty acid analysis
Analysis of the fatty acid content was carried out according
to the procedure described by Janeczko et al. (2009)
[18]. Briey, samples (0.3 g FW) were homogenized in
chloroform/methanol mixture with addition of isopropanol.
The separation of lipids into three classes (monogalactosyl
diacylglycerols, digalactosyl diacylglycerols and
phospholipids) was conducted on a silica acid column
(Koch-Light Laboratories Ltd, England, type 5030h,
325 mesh activated for 18 hours at 110°C). Then the lipids
were subjected to transestrication with BCl3 catalysts in
methanol yielding fatty acid methyl esters (FAME). FAMEs
were spiked with methyl heptadecanoate as an internal
standard and extracted with n-hexane. The analysis of
FAME content was conducted with a GC (Hewlett Packard
5890, Series II), equipped with a FID detector using
a GS-Alumina capillary column (30 m length, 0.542 mm in
diameter) purchased from J&W Scientic. The quantitation
of the FAME content was carried out using external
standard calibrations corrected for internal standard. Each
analysis was conducted in triplicate.
2.5 Pigment content analysis
The content of photosynthetic pigments (carotenoids
and chlorophylls) in the leaves was determined
spectrophotometrically according to the modied
method of Lichtenthaler and Wellburn (1983) [19]. Aer
lyophilization, leaf samples were ground, 25 mg DW
was weighed and pigments were extracted with 1 cm3 of
acetone. The supernatant obtained aer centrifugation
was diluted and absorbance was measured at the
wavelengths: 470 nm (carotenoids), 645 nm (chlorophyll
b), 662 nm (chlorophyll a). Analyses were performed in
ve replicates (one replicate = lyophilized material from
one leaf).
3 Results
3.1 FT-Raman discrimination between
optimally watered, drought-stressed and
rehydrated soybean seedlings
Three characteristic bands of carotenoids are visible in
the FT-Raman spectra obtained for all studied objects
at the following wavelengths: 1005, 1157 and 1525 cm-1
(Fig. 1A and 1B). Carotenoids are well known from the
fact that they give a strong FT-Raman signal. They have
a characteristic long central chain in the structure with a
conjugated double bond system. The rst, most intense
C=C stretching vibration of β-carotene was observed at
1525 cm-1. The second, medium in intensity at 1157 cm-1
was attributed to C-C stretching vibration. The third, low
Figure 1: FT-Raman spectra of leaves of soybean (A) cv. Aldana and (B) cv. Augusta collected from optimally watered, drought-stressed and
rehydrated seedlings.

1094 Magdalena Rys et al.
intensity band at 1005 cm-1 reected CH3 groups attached
to the polyene chain coupled with C-C bonds. Some bands
of low intensity at 1604, 1328, 1287 and 744 cm-1 could
be assigned to chlorophyll. Bands, which appeared at
1454 and 1384 cm-1 could be associated with deformation
vibrations of CH, CH2 and CH3 groups and C-C stretching
vibrations of aliphatic carbohydrates, respectively.
Furthermore, the spectra showed bands associated
with saturated and unsaturated fatty acids at 1657 cm-1,
1295 cm-1 and 1630 cm-1, 1269 cm-1. There were also bands
visible on the spectra associated with disaccharides
at 1188 and 870 cm-1. Additionally, in the range of
1000−1140 cm-1 a dierence in the shape and intensity of
peaks was observed. They were identied as the symmetric
(C-O-C, 1122 cm-1 ) and asymmetric vibration (C-O-C,
1094 cm-1) modes characteristic for polysaccharides.
The observed changes are much more pronounced in cv.
Augusta comparing to cv. Aldana.
3.2 Cluster analysis
Cluster analysis was applied in order to nd the meaningful
and systematic dierences among the spectra of the leaves
of cv. Aldana and cv. Augusta, which were optimally
watered, drought-stressed and nally rehydrated (Fig. 2).
A distinct discrimination between these six objects
was achieved throughout the wavenumber range. The rst
group of objects investigated consisted of well watered
plants of cv. Aldana and cv. Augusta. The plants of these
two cultivars were very similar in terms of their chemical
Figure 2: Cluster analysis of the FT-Raman spectra of soybean
leaves (cv. Aldana and cv. Augusta) performed according to Ward’s
algorithm in the whole wavenumber range with standard algorithm
as data preprocessing.
Figure 3: Content of carotenoids (A) and chlorophylls (B and C)
[mg g D.W.-1] in the leaves of optimally watered and drought-stressed
soybean (cv. Aldana and cv. Augusta). Values marked with the same
letters (within columns) are not signicantly dierent according to
the Duncan’s multiple range test, P < 0.05.

FT-Raman spectroscopy as a tool in evaluation the response of plants to drought stress 1095
Table 1: The relative amounts (% of total) of fatty acids in monogalactosyl diacylglicerol, digalactosyl diacylglycerols and phosphiolipid
fractions isolated from leaves of soybean optimally watered and drought stressed. Values marked with the same letters (within columns) are
not signicantly dierent according to the multiple Duncan Test, P < 0.05.
Cultivar/treatment Monogalactosyl diacylglycerols Digalactosyl diacylglycerols Phospho-lipids
Palmitic acid (16:0)
ALDANA optimally
watered
4.0 a12.1 b24.3 ab
drought
stressed
3.4 a11.0 b25.8 a
AUGUSTA optimally
watered
4.4 a15.4 a22.3 b
drought
stressed
4.4 a10.6 b24.5 ab
Palmitoleic acid (16:1)
ALDANA optimally
watered
0.29 b0.31 b7.5 b
drought
stressed
0.27 b0.23 b6.8 b
AUGUSTA optimally
watered
0.77 a6.25 a11.2 a
drought
stressed
0.21 b0.25 b5.4 b
Stearic acid (18:0)
ALDANA optimally
watered
1.5 a2.2 a4.6 b
drought
stressed
1.1 a2.2 a4.8 ab
AUGUSTA optimally
watered
1.9 a2.5 a5.5 a
drought
stressed
2.0 a2.9 a4.6 b
Oleic acid (18:1)
ALDANA optimally
watered
1.0 a0.87 a4.4 a
drought
stressed
0.78 b0.61 b3.9 a
AUGUSTA optimally
watered
0.51 c0.67 b4.6 a
drought
stressed
0.83 b0.70 ab 3.1 b
Linoleic acid (18:2)
ALDANA optimally
watered
5.2 b4.1 a26.2 a
drought
stressed
5.7 a4.4 a20.3 b
AUGUSTA optimally
watered
3.1 c2.4 b18.6 b
drought
stressed
4.2 c3.7 a14.9 c
Linolenic acid (18:3)
ALDANA optimally
watered
87.9 a80.4 a32.9 b
drought
stressed
88.8 a81.6 a38.5 b
AUGUSTA optimally
watered
89.3 a72.8 b37.9 b
drought
stressed
88.3 a81.9 a47.5 a

1096 Magdalena Rys et al.
composition, particularly content and composition of
carotenoids, chlorophylls and fatty acids. The second
group comprised drought-stressed plants of cv. Augusta
and cv. Aldana. In this case, the variability between plants
was somewhat larger than in objects from the rst group.
The third group constituted plants of cv. Aldana and cv.
Augusta aer rehydration and the results in this group
were signicantly dierent compared to others.
The obtained dendrogram showed that dierent
water availability had an inuence on the plant metabolic
prole, especially for carotenoids, chlorophylls and fatty
acids compositions in the test soybean plants.
3.3 Content of carotenoids and chlorophylls
in the leaves of optimally watered and
drought-stressed soybean seedlings
The content of carotenoids and chlorophylls was similar
in the leaves of both studied cultivars growing under
optimal watering (Fig. 3A). Under the drought, carotenoid
content signicantly decreased in the leaves of cv.
Aldana (18%) and cv. Augusta (17%). Drought stress also
reduced the amount of chlorophyll in the leaves of both
varieties by 10−23%, even though the dierences were
statistically signicant only in the case of the cv. Augusta
(Fig. 3B, 3C).
3.4 Fatty acid composition in the leaves of
optimally watered and drought-stressed
soybean seedlings
Composition of fatty acids (FA) of phospholipids (PL) in
the soybean leaves, which constitutes main components of
cell membrane, diers from the composition of fatty acids
of fractions of monogalactosyl diacylglycerols (MGDG)
and digalactosyl diacylglycerols (DGDG), characteristic of
the membranes of chloroplasts (Table 1). In the FA pool
of the MGDG, DGDG fractions the largest percentage of
18:3 (linolenic acid) was found. However, in the case of
MGDG and DGDG, its contribution ranges from 72.8 to
89.3%, while in PL it averages at 40%. In addition, the PL
fraction had a relatively high percent (about 20%) of FA
18:2 (linoleic acid) and 16:0 (palmitic acid). The percent of
these FA in the fractions of MGDG and DGDG was ranging
from a few to a maximum of 15.4%. The MGDG and DGDG
fractions had the lowest (typically less than 1%) relative
amounts of 16:1 (palmitoleic acid) and 18:1 (oleic acid).
The percent of these acids in the case of phospholipids
averaged at the level of a few percent.
The soybean cv. Augusta and cv. Aldana varied in
fatty acid composition in individual groups of lipids. In cv.
Augusta in the MGDG fraction, there was higher percent of
16:1 than in cv. Aldana, while the percent of 18:1 and 18:2
was lower. In cv. Augusta in the DGDG fraction, there was
signicantly lower relative amount of 18:1, 18:2 i 18:3 when
compared to Aldana, while the relative amount of 16:0
Cultivar/treatment Monogalactosyl diacylglycerols Digalactosyl diacylglycerols Phospho-lipids
Ratio of unsaturated to saturated fatty acids (U/S)
ALDANA optimally
watered
17.4 a6.0 a2.5 a
drought
stressed
21.3 a6.6 a2.3 a
AUGUSTA optimally
watered
16.1 a4.6 b2.6 a
drought
stressed
14.8 a6.4 a2.4 a
18:3/18:2
ALDANA optimally
watered
16.8 bc 19.6 b1.3 c
drought
stressed
15.7 c18.4 b1.9 bc
AUGUSTA optimally
watered
29.3 a30.6 a2.0 b
drought
stressed
21.0 b22.4 b3.2 a
ContinuedTable 1: The relative amounts (% of total) of fatty acids in monogalactosyl diacylglicerol, digalactosyl diacylglycerols and phosphiolipid
fractions isolated from leaves of soybean optimally watered and drought stressed. Values marked with the same letters (within columns) are
not signicantly dierent according to the multiple Duncan Test, P < 0.05.

FT-Raman spectroscopy as a tool in evaluation the response of plants to drought stress 1097
was higher. Cv. Augusta had the higher relative amount of
16:1 and 18:0 compared to Aldana, whereas there was less
of the 18:2 acid in the pool.
Drought slightly modied the composition of FA
lipids in the leaves of cv. Aldana, causing amongst others
a decline of relative amount of 18:1 in MGDG and DGDG
fraction. A decrease of relative amount of 18:2 was recorded
in the PL fraction. An increase in the relative amount of
18:2 occurred in this cultivar in the MGDG fraction.
In the cv. Augusta, the drought triggered an increase in
the relative amount of 18:1 (MGDG), 18:2 and 18:3 (DGDG),
which was accompanied by a reduction of the percentage
of 16:0 and 16:1. In the PL fraction was observed the
largest increase in the proportion of unsaturated 18:3,
with declines in the percentage of almost all the other FA
in this fraction.
Trends in the percentage of FA (in the individual
fractions of lipids) that occurred as a result of drought
are synthetically expressed by the ratios: unsaturated
FA/saturated FA (U/S), and the ratio 18:3/18:2. Changes
resulting from the increase of FA unsaturation caused by
drought are statistically signicant in cv. Augusta, while
in cv. Aldana only a similar trend can be noticed.
4 Discussion
4.1 Evaluation of pigment content
The lower content of carotenoids and chlorophylls in
plants exposed to water decit is a typical phenomenon
[16,17]. However, there are exceptions from this rule
resulting from, e.g., interspecic or intercultivar
dierences as well as the intensity of the stress factor [20].
In general, it is thought that the decrease in the synthesis
and/or increased degradation of the pigments as a result
of stress is associated with a reduction in photosynthetic
eciency. In this study, increasing water decit during
ve days caused a decrease in hydration (wilting) of the
leaves of soybean and resulted in a signicant reduction
in the content of carotenoids and chlorophylls. The
content of pigments in the leaf samples collected before
and aer the drought was measured using one of the most
popular methods, i.e., the spectrophotometric method
described by Lichtenthaler and Wellburn [19], which
as all classical analytical method, is time-consuming,
laborious and requires the intake of reagents. Raman
spectroscopy with Fourier transformation (FT-Raman)
can serve as an alternative, non-invasive technique useful
for the characterization and identication of the pigment
content (especially carotenoids) in living tissue [21-25].
Our study has applied FT-Raman spectroscopy for the
rst time in the analysis of the eect of drought stress on
the chemical composition of plant tissue, including the
content of carotenoids and chlorophylls. The obtained
spectra showed that drought stress aected the chemical
composition of soybean leaves. Bands derived from
carotenoids (1005, 1157, 1525 cm-1 ) varied in height between
the rst two test objects, namely the leaves of optimally
watered plants and the leaves withered due to water decit
in soil. The observed changes are much more pronounced
in cv. Augusta, and are likely to be due to its greater
susceptibility to drought stress compared to cv. Aldana.
This method also allowed a quantitative estimation as
FT-Raman band intensity is directly correlated with the
content of a chemical compund in a sample [22]. However,
data on the content of carotenoids in the stressed
soybean, obtained spectrophotometrically and by
FT-Raman spectroscopy, were not entirely consistent. The
content of carotenoids measured spectrophotometrically
decreased in both cultivars as a result of drought stress.
However, data obtained using FT-Raman spectroscopy
demonstrated that the content of these compounds in cv.
Aldana increased during drought, while in cv. Augusta
it was lower. At the same time it should be noted that
the analysis of carotenoids by spectrophotometry was
performed on lyophilized material and the content of these
compounds was calculated based on the dry weight of the
tissue. It is a commonly used procedure because it avoids
the distortion of the amount of pigment that could occur
when estimating the quantity of carotenoids based on fresh
weight, susceptible to changes in plant tissue hydration.
Particularly in the case of experiments with drought, plant
leaves treated with this stress factor have a reduced water
content, which may appear to increase the concentration of
carotenoids and other pigments when fresh weight is used
in the calculations. Our results showed that the hydration
of fresh plant tissue, aecting surface structure of the leaf
and its optical properties, played similarly important role
in FT-Raman measurements. Inuence of morphological
properties of leaves on the intensity of the Raman signal
was also observed in Plantago media L. The thin leaves,
with a high water content, show a much weaker Raman
scattering intensity in comparison to the less hydrated
thick leaves (Skoczowski − unpublished data). The Raman
bands derived from water have low intensity, because
the low polarizability of water is reected in the low
intensity of scattered light. Nevertheless when comparing
various objects, uctuations in the amount of water in
the examined tissue seem to aect the sensitivity of the
method. Therefore, two approaches seems reasonable, i.e.,

1098 Magdalena Rys et al.
FT-Raman analysis on the lyophilized material or
rehydration of withered tissues before measurements.
In our experiment, to simplify the measuring procedure,
tissue was rehydrated by watering the plants. The results
of the FT-Raman measurements using leaves aer
rehydration showed that the quantity of carotenoids
signicantly decreased in both cultivars compared to
the content of these pigments in the leaves of the plants
watered optimally. Aer regaining the turgor by the
leaves, a decrease of the carotenoid content was observed
in the FT-Raman spectroscopy and correlated with
a decrease in the content of these compounds assayed by
spectrophotometric method. However, the quantitative
changes in the content of carotenoids in plants assayed
by spectrophotometry before and aer stress are low
(a few percent). The results of FT-Raman spectroscopy
indicated a two-, three-fold decrease in the amount of
carotenoids caused by stress. In our opinion, the results
of both analyses should not be directly compared in terms
of quantity, because the principle of the measurement
was dierent and the calibration was not carried out.
The amount of carotenoids in the spectrophotometric
method was determined based on the absorbance and
molar extinction coecient. For FT-Raman spectrometry,
carotenoid content was assessed on the basis of FT-
Raman intensity (FT-Raman scattering), which for these
compounds is distributed in three band frequencies (the
so-called carotenoid triplet). Furthermore, in addition to
the chemical diagnostics, absolute quantitative changes
of the selected metabolite are not necessarily needed to
describe the eects of stress on the plant. Signicantly
more important is the so-called trend of changes.
Variations in the pool of carotenoids caused by stress are
analyzed on the basis of the direction of change, i.e., a
decrease or increase in their content. Therefore, FT-Raman
spectroscopy is ideal for the assessment of changes in the
content of carotenoids in plant tissues caused by dierent
types of stress factors.
In addition to carotenoids, this study also analyzed
the content of chlorophyll using spectrophotometry
and comparative method of FT-Raman spectroscopy.
Spectrophotometric method allowed us to capture the
changes (a decrease in chlorophyll content) in both
cultivars studied. The optimally watered plants of cv.
Augusta and cv. Aldana had similar levels of chlorophyll.
Drought caused a decrease in the content of these pigments,
while this reduction was more prominent in the leaves
of more susceptible to drought (according to previous
studies) cv. Augusta when compared to cv. Aldana [27].
Unlike the carotenoid analysis, FT-Raman spectroscopy
did not provide unambiguous results in the analysis of
chlorophylls. Bands derived from chlorophylls (1604,
1328, 1287 and 744 cm-1) had very low intensity, because
chlorophyll in contrast to carotenoids is a compound
that demonstrates incomparably lower dispersion of the
radiation.
4.2 Fatty acid assay
Plant cell membranes are one of the main targets for
biotic and abiotic stresses. Fatty acids, as a structural
part of membranes, participate in maintaining membrane
integrity and regulating membrane uidity. The eect of
stress factors can modify the qualitative and quantitative
lipid composition of the cell (including the composition
of cell membranes). However, these changes very oen
depend on the genotype and on the type of stress.
Toumi et al. (2008) [27] studied grapevine under drought
stress, and observed changes in the composition of total
lipids towards the increase of the proportion of 18:3. The
increase in the percentage of 18:3 under the inuence
of drought was also reported in the study of Gigon et
al. (2004) [28] in certain lipid fractions (DGDG and
phosphatidylcholine fraction) in Arabidopsis thaliana L.
What is more, these authors found an overall decrease
in the amount of lipids reaching even 75% as a result
of drought. In turn, Zhong et al. (2011) [29] recorded a
decline in polyunsaturated fatty acids 18:2 and 18:3,
and an increase in saturated fatty acids 16:0 and 18:0 in
bermudagrass exposed to drought.
The FA changes in soybean have previously been
studied mainly in relation to the seeds. It was found that
the FA composition of the soybean seed is inuenced by
such factors as environmental conditions, fertilization and
genotype [30,32]. Moreover, the content of fatty acids in
the seeds involves mainly storage lipids (so-called neutral
lipids), while in leaves polar lipids are mainly found.
We have evaluated the eects of water stress on lipid
composition in the leaves of soybean. The results obtained
by gas chromatography indicated that the composition
of the FA polar lipids under drought in the leaves of cv.
Aldana did not change signicantly. There was an increase
in the percentage of 18:3 in cv. Augusta in digalactosyl
diacylglycerol and phospholipid fractions. This translated
into an increase of the U/S ratio. FA were also visible in the
FT-Raman spectra. The bands present were derived from
saturated (1657 and 1295 cm-1) and unsaturated fatty acids
(1630 and 1269 cm-1). The low intensity of the bands resulted
from the low content of the compounds analyzed in the
leaf tissue as well as high intensity of carotenoid bands,
which inuenced the visibility of the bands derived from

FT-Raman spectroscopy as a tool in evaluation the response of plants to drought stress 1099
the fatty acid spectrum. Since the bands derived from FA
in the spectra had low intensity, the U/S ratio could not be
determined with sucient accuracy. This parameter (U/S)
is sometimes calculated in order to determine the level of
plant stress [18,32]. Therefore, the results obtained in our
study showed, that the method of FT-Raman spectroscopy
was less suitable for the analysis of FA in leaves (due to
the small amounts of these compounds located mainly in
biological membranes), nevertheless it was also very well
suited for seeds high in fats [33,34]. Previous studies on
cotyledons of white mustard and rapeseed showed that
the lipid content determination by gas chromatography
was considerably less accurate as compared to an in situ
assay using FT-Raman spectroscopy [34]. The dierences
were observed in the spectra in the content of saturated
and unsaturated fatty acids caused by allelopathic
substances present in plant extracts, in which seedlings
were grown, while the chromatographic technique did not
demonstrate signicant statistical dierences [34].
4.3 General metabolic changes
For the purpose of this study, metabolites were
investigated, the quantitative changes of which were
expected in drought conditions. However, FT-Raman
spectroscopy allowed the simultaneous observation of a
wider spectrum of compounds scattering the radiation,
which were present in the objects tested, and a subsequent
comparative analysis between them. Cluster analysis
showed that the tested objects are grouped into three
clusters. The rst one was optimally watered plants. The
second constituted drought-stressed leaves, and third one
was composed of the leaves aer rehydration. The impact
of drought on metabolism was already visible in the
measurements performed on the withered leaves, but the
signicantly clearer eect of metabolic changes that have
occurred during the drought was demonstrated in plant
leaves exposed to drought aer rehydration. These results
constituted entirely separate branch of the dendrogram.
5 Conclusions
FT-Raman spectroscopy allows a detailed analysis of the
chemical composition of tissues and identies changes
in the qualitative content of various chemical compounds
scattering the radiation. Hence it seems to be a convenient
tool for monitoring the eect of environmental factors
on plants. The analysis can be preferably be carried out
based on the changes of carotenoids levels, even though
FT-Raman spectroscopy allows the simultaneous
observation of a wider spectrum of compounds scattering
the radiation present in the objects tested, and a subsequent
comparative analysis. This method can complement other
non-invasive methods, e.g., fast kinetic of chlorophyll a
uorescence, in assessing the stress-induced damage
of crops. However, in the measurements conducted
for the stress causing the loss of the leaf water content,
the problem of proper hydration of the tissue should be
bourne in mind and resolved by full rehydration of leaves
before analysis.
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