seasonal variations of lipophilic compounds in needles of two chemotypes of pinus pinaster ait
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ORIGINAL ARTICLE
Seasonal variations of lipophilic compounds in needles of twochemotypes of Pinus pinaster Ait.
Carlos Arrabal • Marıa Concepcion Garcıa-Vallejo •
Estrella Cadahia • Manuel Cortijo •
Brıgida Fernandez de Simon
Received: 14 March 2012 / Accepted: 27 July 2013 / Published online: 20 August 2013
� Springer-Verlag Wien 2013
Abstract Variation of monoterpene, sesquiterpene, neu-
tral diterpene, and fatty and resin acid composition was
determined in adult needles of two chemotypes of Pinus
pinaster Ait., sampled in two different seasons: summer
and winter. Throughout the year, the terpenic and fatty acid
composition of adult needles of P. pinaster showed a
seasonal variation, both at individual and at global level.
These seasonal variations in distribution pattern were not
produced in the same way in the needles of the two
chemotypes studied. Therefore, we consider that secondary
metabolism compounds do not present the same sensitivity
to environmental conditions, and genetically different trees
have different responses to these environmental conditions.
Keywords Terpenes � Resin acids � Fatty acids �Pinus pinaster � Seasonal variation
Introduction
In conifer species, different parts of the tree as the needles,
the cortex tissues, the wood, the seedlings or the seeds,
show different quantities of terpenes, resin acids and fatty
acids (Piovetti et al. 1980; Tobolski and Zinkel 1982;
Rowe 1989; Wolff et al. 1997; Sallas et al. 1999; Bre-
itmaier 2006). The species, the geographical origin and the
plant genotype can influence these relative quantities but
also other environmental factors, both biotic and abiotic.
Several abiotic factors, such as temperature, water and light
availability, have an influence on seasonal variation of
concentrations of compounds in different parts of the tree
(Merk et al. 1988; Kainulainen et al. 1992; Langenheim
1994; Johnson et al. 1997; Sallas et al. 1999). Nerg et al.
(1994) found seasonal changes in the concentrations of
monoterpene, resin acid and total phenolic compounds of
Scots pine (Pinus sylvestris) seedlings. Zavarin et al.
(1971) reported an increase of monoterpenoids in P. pon-
derosa needle oil in summer. Several studies of emissions
of monoterpenes from conifer species [P. pinea, Staudt
et al. (1997); Camellia japonica, Chamaecyparis obtusa, P.
koraiensis, Kim et al. (2005); P. densiflora, Lim et al.
(2008); P. taeda, P. virginiana, Geron and Arnts (2010)]
reported an increase of these compounds during spring and
summer, compared with autumn and winter.
Also resin acid concentrations increase in Scots pine
needles during the growing season (Buratti et al. 1990)
and changed considerably in the needles and cortex tis-
sues from P. sylvestris, P. nigra and P. strobus during
shoot growth and maturation (Tobolski and Zinkel 1982).
Moreover, Gref and Tenow (1987) observed that resin
acid concentrations are significantly higher in sun than in
shade needles of P. sylvestris and Gleizes et al. (1980)
found in maritime pine (P. pinaster) seedlings primary
leaves, that the biosynthesis of monoterpene hydrocar-
bons, mainly a- and b-pinene, is strongly activated by
light availability, whereas diterpene hydrocarbons (b-
caryophyllene and a-humulene) are easily synthesized
under light or darkness.
Therefore, when these compounds are used as genetic
markers, the above mentioned abiotic factors should be
considered, especially in the Mediterranean area, charac-
terized by a marked seasonality and long dry summers with
C. Arrabal (&) � M. Cortijo
ETSI Ingenieros de Montes Universidad Politecnica de Madrid,
Madrid, Spain
e-mail: carlos.arrabal@upm.es
M. C. Garcıa-Vallejo � E. Cadahia � B. Fernandez de Simon
Departamento de Industrias Forestales, INIA-CIFOR,
Apdo. 8111, 28080 Madrid, Spain
123
Plant Syst Evol (2014) 300:359–367
DOI 10.1007/s00606-013-0888-5
scarce precipitation coinciding with high solar irradiance
and temperatures.
As a part of a research project on genetic improvement
of Spanish P. pinaster, a survey of the neutral terpenes and
resin and fatty acids in needles of two chemotypes for this
species has been carried out to assess their potential use as
molecular markers (Arrabal et al. 2012). The aim of this
work is to study and compare the seasonal variation of
terpenes, fatty acids and resin acids contents in needles of
P. pinaster in field conditions, in Segovia (central zone of
Spain) over the course of a year.
Materials and methods
Plant material
All samples were collected in ortets and in ramets grafted in
a clonal bank located in Carbonero, Segovia province
(Central Spain). Grafting was made on rootstock of P.
pinaster of the same region, where all branches were
removed (graft 6 years old). Two-year-old needles were
collected in July 20th, (summer) and in February 5th,
(winter), in the same ortets and ramets of 54 trees each time.
The needles were frozen with liquid nitrogen at the moment
of sampling and kept at -70 �C until their analysis.
Extraction
The needles were cut into small pieces (2–4 mm). A known
weight (1.5–2 g) was extracted during 24 h, at 4 �C in the
dark, with 5 ml of diethyl ether/petroleum ether (1:1), to
which 200 g/ml internal standards (isobutylbenzene for
monoterpenes, heptadecane for sesquiterpenes and neutral
diterpenes, and heptadecanoic acid for fatty and resin
acids) had been added. The extract was then decanted and
the neutral terpenes were analyzed by GC in an aliquot of
this extract, without any further purification. The needle
pieces were washed in 2 ml of diethyl ether/petroleum
ether (1:1), the washing solution was added to the
remainder extract, and the solvent was removed from the
final solution, in a nitrogen stream. The dried extract was
redissolved with 1 ml of methanol and analyzed by GC,
after adding 100 ll of tetramethyl ammonium hydroxide,
as methylation agent (Song et al. 1993; Galletti et al. 1995;
Beverly et al. 1997).
GC/FID
The terpenic compounds and fatty acids were analyzed by
gas chromatography with flame-ionization detection (FID).
GC equipment: HP 5890 gas chromatograph. Column:
30 m 9 0.25 mm internal diameter, PTE-5 (0.25 lm film
thickness). Chromatographic conditions: Sample volume
0.5 ll, split 1:50, helium flow 0.5 ml min-1, oven tem-
perature: 60 �C (2 min), 4 �C min-1, 270 �C (10 min),
injector temperature 260 �C, FID detector temperature,
300 �C.
GC–MS equipment
HP 5890 gas chromatograph connected to a 5971A mass
detector. Column and chromatographic conditions were
similar to the ones used with GC/FID equipment.
The identity of the compounds was assessed by their
relative retention and EI mass spectra at 70 eV, comparing
them with those in the Wiley (2005) and Nist/Epa/Nih
(2005) spectral databases and in literature (Enzell and
Ryhage 1965; Enzell and Wahlberg 1969; Zinkel et al.
1971; Ekman 1979; Ramaswami et al. 1986; Adams 2007;
Lange and Weibmann 1987, 1989, 1991). Anticopalic,
imbricataloic and epiimbricataloic methyl esters were
identified by comparing their mass spectra with those of
authentic samples, provided by Dr. Duane F. Zinkel (Forest
Products Laboratory, Madison, USA).
Statistical analysis
Univariate analysis was carried out, using the BMDP-7D
(ANOVA) program (WJ Dixon, BMDP Statistical Soft-
ware, Software Release, 1990). Average and standard
deviation were calculated for each variable of the two
groups of samples, using a single variable model. The
Student Newman–Keuls Multiple Range Test was also
carried out to determine the significance levels of the dif-
ferences between means, at 95 % confidence level.
Canonical discriminant analysis was also carried out with
all components evaluated, using CANDISC.SAS procedure
(SAS Institute INC., SAS/STAT�, version 6, fourth edi-
tion, 1994).
Results and discussion
Global trends
The results obtained in relation to the total contents of each
type of lipophilic components, expressed as mg/g of fresh
needle, are showed in Table 1. Needles collected in winter
showed a decrease in the average overall contents, com-
pared to summer. All types of studied compounds, mono-
terpenes, sesquiterpenes, neutral diterpenes, fatty acids and
resin acids, and total organic decreased. To know if these
decreases were statistically significant, we applied a com-
parison mean test to each group of components in needles
of both chemotypes independently, and as a result,
360 C. Arrabal et al.
123
significant differences were found for all groups, except for
sesquiterpenes, diterpenes and total neutral terpenes in
needles of chemotype 2. If we compare the results obtained
in samples of two chemotypes, some differences in sea-
sonal variation can be seen. In needles from chemotype 1,
the differences in the neutral components were more sig-
nificant than in the acid components: monoterpenes
showed the highest average decrease (-53 %), followed by
fatty acids (-44 %) and resin acids (-37 %). On the
contrary, in needles of chemotype 2, the differences in the
acid compounds were more significant than in the neutral
compounds: fatty acids (-57 %) and resin acids (-50 %)
showed the highest average decreases, followed by mon-
oterpenes (-32 %).
The canonical multivariate analysis of data in Table 1
provides a significant grouping of samples of each
Table 1 Seasonal variation of neutral and acid global contents (mg/g fresh needle)
Chemotype 1 (0.67**) Chemotype 2 (0.92**)
Summer Winter C-P S/W Summer Winter C-P S/W
Monoterpene 3.44 ± 1.16 1.62 ± 0.88 0.85** -52.9 2.26 ± 0.85 1.53 ± 0.36 0.55* -32.3
Sesquiterpene 3.16 ± 0.89 2.30 ± 1.02 0.55** -27.2 3.15 ± 1.20 2.37 ± 0.86 0.40 -24.8
Diterpene 1.71 ± 0.60 1.20 ± 0.74 0.48* -29.8 9.23 ± 3.92 8.41 ± 3.49 0.12 -8.9
Neutral terpenes 8.30 ± 2.17 5.11 ± 1.99 0.80** -38.4 14.63 ± 5.55 12.30 ± 4.17 0.26 -15.9
Fatty acids 1.88 ± 1.14 1.04 ± 0.26 0.49* -44.7 1.26 ± 0.48 0.54 ± 0.16 0.79** -57.1
Resin acids 30.41 ± 17.01 18.98 ± 7.43 0.44* -37.6 11.58 ± 3.54 5.80 ± 1.54 0.71** -49.9
Acids 32.30 ± 17.58 20.02 ± 7.57 0.46* -38.0 12.84 ± 3.91 6.33 ± 1.66 0.74** -50.7
Total 40.60 ± 19.18 25.13 ± 9.23 0.52** -38.1 27.47 ± 5.16 18.64 ± 4.55 0.67** -32.1
Mean ± standard deviation; the data within bracket close to words chemotype 1 and chemotype 2 are the global correlation and significance
from canonical multivariate analysis
C correlation with total canonical structure, S/W percentage variation data between summer and winter
P = Significance level in comparison mean test; **0.01 C P; *0.05 C P C 0.01
Table 2 Seasonal variation of monoterpenes and other volatile compounds (%)
Chemotype 1 (0.81**) Chemotype 2 (0.89)
Summer Winter C–P S/W Summer Winter C–P S/W
a-Pinene 33.36 ± 5.28 44.02 ± 7.47 0.75** ?31.9 35.78 ± 4.83 44.04 ± 4.84 0.76** ?23.1
Camphene 0.27 ± 0.18 0.21 ± 0.04 0.06 -22.2 0.28 ± 0.06 0.45 ± 0.18 0.13 ?60.7
b-Pinene 41.95 ± 6.22 27.73 ± 9.10 -0.78** -33.9 39.13 ± 4.89 29.20 ± 6.57 -0.76** -25.4
Myrcene 12.34 ± 3.55 11.78 ± 3.03 -0.08 -4.5 14.56 ± 2.75 13.84 ± 3.29 -0.14 -4.9
a-Phellandrene 0.30 ± 0.11 0.18 ± 0.09 0.13 -40.0
d-3-Carene 5.04 ± 2.86 2.21 ± 3.04 0.33* -56.1 2.45 ± 1.78 4.98 ± 2.93 0.25 ?103.3
b-Phellandrene ? limonene 5.99 ± 2.70 4.27 ± 2.00 0.07 -28.7 4.99 ± 2.59 4.40 ± 2.84 -0.13 -11.8
(E)-b-ocimene 0.43 ± 0.19 1.19 ± 0.90 -0.33* ?176.7 0.82 ± 0.62 1.46 ± 0.78 0.41 ?78.0
Terpinolene 1.34 ± 1.58 1.61 ± 1.22 0.51** ?20.1 1.31 ± 0.78 1.54 ± 0.93 0.16 ?17.6
Linalool 0.40 ± 0.16 0.45 ± 0.24 -0.03 ?11.1 0.41 ± 0.26 0.80 ± 0.22 0.32 ?95.1
Cuminic acid 0.26 ± 0.12 0.23 ± 0.07 0.10 -11.5
Linalyl acetate ? geraniol 1.44 ± 1.33 2.93 ± 3.62 0.03 ?103.4 0.48 ± 0.10 1.10 ± 0.45 -0.27 ?129.2
Bornyl acetate 0.34 ± 0.22 0.14 ± 0.01 0.30 -58.8
n-Tridecane 0.70 ± 0.60 1.27 ± 1.36 -0.22 ?81.4
Geranyl acetate 0.38 ± 0.22 1.16 ± 0.77 0.48** ?205.3 0.50 ± 0.20 0.93 ± 0.47 0.38 ?86.0
Methyl-eugenol 0.38 ± 0.14 0.89 ± 0.57 0.62** ?134.2 0.37 ± 0.10 0.65 ± 0.35 0.20 ?75.7
Phenyl ethyl isovalerate 1.03 ± 0.66 3.39 ± 2.84 0.47** ?229.1 1.70 ± 2.09 2.57 ± 1.10 0.23 ?51.2
Mean ± standard deviation; the data within bracket close to words chemotype 1 and chemotype 2 are the global correlation and significance
from canonical multivariate analysis
C correlation with total canonical structure, S/W percentage variation data between summer and winter
P = Significance level in comparison mean test; **0.01 C P; *0.05 C P C 0.01
Seasonal variations of lipophilic compounds 361
123
chemotype, with regard to the collection season, being
monoterpenes the group of components with the highest
correlation (C = 0.85) with total canonical structure in
samples of chemotype 1, and fatty and resin acids
(C = 0.79 and 0.71) in samples of chemotype 2.
Speciated composition
In these same needles sampled in summer and winter, we
studied also the detailed composition of monoterpenes,
sesquiterpenes, neutral diterpenes, and fatty and resin
acids, to know if there were not only quantitative seasonal
variations but also qualitative variations. 147 different
compounds were detected, and 112 of them were identified
(Arrabal et al. 2012).
As it can be seen in Tables 2, 3, 4, 5 and 6, the per-
centages of the majority of detected compounds changed
during a year. In the statistical analysis, significant differ-
ences were found for many compounds, both main and
minor, in needles of chemotype 1. On the contrary in
needles of chemotype 2, only eighteen compounds pre-
sented significant differences (P \ 0.01) between summer
and winter needles. Among these eighteen compounds,
some of them (a- and b-pinene, caryophyllene, germacren
Table 3 Seasonal variation of sesquiterpenes (%)
Chemotype 1 (0.95**) Chemotype 2 (0.96**)
Summer Winter C-P S/W Summer Winter C-P S/W
a-Cubebene 1.01 ± 0.31 1.31 ± 0.35 0.39** ?29.7 0.48 ± 0.16 0.42 ± 0.13 -0.12 -12.5
a-Ylangene 0.41 ± 0.12 0.45 ± 0.14 0.28* ?9.7 0.37 ± 0.17 0.23 ± 0.07 -0.34 -37.8
a-Copaene 1.64 ± 0.38 1.87 ± 0.36 0.27 ?14.0 1.11 ± 0.33 1.09 ± 0.23 0.19 -1.8
b-Cubebene 0.61 ± 0.08 0.69 ± 0.17 0.21 ?13.1
Longifolene 0.69 ± 0.49 0.35 ± 0.38 0.01 -49.3
(E)-b-Caryophyllene 20.65 ± 6.57 24.16 ± 7.88 0.22 ?17.0 16.54 ± 2.40 19.00 ± 2.45 -0.47* ?14.9
b-Gurjunene 1.02 ± 0.28 0.86 ± 0.27 -0.28* -15.7 0.82 ± 0.20 0.33 ± 0.11 0.66** -59.8
a-Humulene 3.64 ± 0.94 4.21 ± 1.27 0.24 ?15.7 2.95 ± 0.32 3.30 ± 0.43 -0.44 ?11.9
c-Muurolene 0.43 ± 0.11 0.35 ± 0.11 0.34* -18.6 0.36 ± 0.18 0.40 ± 0.39 -0.12 ?11.1
a-Amorphene 4.86 ± 1.28 6.52 ± 1.89 0.47** ?34.2 2.98 ± 1.31 3.58 ± 1.39 -0.23 ?20.1
Germacrene D 37.16 ± 8.72 26.09 ± 9.59 -0.49** -29.8 45.85 ± 6.53 45.93 ± 6.90 -0.01 ?0.2
Sesquiterpen hydrocarbon (M 204) 2.17 ± 0.56 3.00 ± 0.77 0.52** ?38.2 1.39 ± 0.49 1.81 ± 0.82 -0.32 ?30.2
a-Muurolene 1.83 ± 0.43 2.19 ± 0.77 0.30* ?19.7 1.47 ± 0.31 1.92 ± 1.08 -0.28 ?30.6
Sesquiterpen hydrocarbon (M?204) 0.86 ± 0.21 1.11 ± 0.39 0.39** 21.7 0.67 ± 0.21 0.70 ± 0.29 0.07 ?4.5
c-Cadinene 6.14 ± 1.19 7.47 ± 1.90 0.40** ?29.1 3.92 ± 1.05 3.75 ± 1.22 -0.08 -4.3
d-Cadinene 7.02 ± 1.75 9.49 ± 2.64 0.49** ?35.2 4.90 ± 1.83 5.23 ± 2.16 -0.08 ?6.7
1,4-Cadinadiene 0.75 ± 0.20 0.80 ± 0.24 0.33* ?6.7 0.57 ± 0.15 0.38 ± 0.06 -0.04 -33.3
(F)-a-Bisabolene 0.92 ± 0.34 1.31 ± 0.55 0.32* ?42.4 0.83 ± 0.40 0.84 ± 0.33 -0.11 ?1.2
Germacrene D-4-ol 1.00 ± 0.63 0.86 ± 1.01 0.09 -14.0 0.94 ± 0.76 1.01 ± 0.97 0.28 ?7.4
Guaiol 1.08 ± 0.50 0.54 ± 0.35 -0.12 -50.0 0.77 ± 0.32 0.73 ± 0.20 -0.04 -5.2
T-Cadinol 0.62 ± 0.16 0.93 ± 0.22 0.51** ?50.0 0.59 ± 0.16 0.58 ± 0.14 0.31 -1.7
a-cadinol 1.26 ± 0.64 1.58 ± 0.55 0.16 ?25.4 1.19 ± 0.53 1.11 ± 0.31 0.07 -6.7
Sesquiterpenol 0.91 ± 0.58 0.28 ± 0.34 0.11 -69.2 2.05 ± 1.61 0.14 ± 0.04 0.27 -93.2
(E,E)-farnesol 1.11 ± 0.87 1.11 ± 0.81 -0.08 0.0 0.36 ± 0.06 0.11 ± 0.07 0.18 -69.4
Germacren D-4-ol acetate 5.08 ± 1.65 1.02 ± 0.61 0.86** -79.9
(Z,E)-Farnesol acetate 0.32 ± 0.14 0.22 ± 0.11 -0.32* -31.2 0.45 ± 0.11 0.41 ± 0.31 0.05 -8.9
(E,E)-Farnesol acetate 1.14 ± 0.78 1.56 ± 1.46 0.19 ?36.8 1.27 ± 0.62 0.70 ± 0.75 0.22 -44.9
(Z,E)-Farnesol propionate 1.39 ± 0.86 0.56 ± 0.37 -0.29* -59.7 5.73 ± 1.51 4.99 ± 1.96 0.22 -12.9
(E,E)-Farnesol propionate 0.74 ± 0.51 1.11 ± 0.95 0.31* ?50.0 0.95 ± 0.65 0.92 ± 0.65 0.02 -3.1
(E,E)-Farnesol isovaleranate 2.63 ± 1.08 0.59 ± 0.43 -0.70** -77.6 0.41 ± 0.17 1.19 ± 0.71 -0.63 ?190.2
Mean ± standard deviation; the data within bracket, close to words chemotype 1 and chemotype 2 are the global correlation and significance
from canonical multivariate analysis
C correlation with total canonical structure, S/W percentage variation data between summer and winter
P = Significance level in comparison mean test; **0.01 C P; *0.05 C P C 0.01
362 C. Arrabal et al.
123
D-4-ol acetate, 8(14),13(15) abietadiene, palmitic and
stearic acid, and an isomer of anticopalic acid) showed
percentages higher than 5 %.
Among all components studied, it is interesting to point
up that only a few of them undergo the same evolution in
the needles of both chemotypes. Thus, a-pinene (Table 2)
Table 4 Seasonal variation of neutral diterpenes (%)
Chemotype 1 (0.97**) Chemotype 2 (0.99**)
Summer Winter C-P S/W Summer Winter C-P S/W
8(17),12,14-Labdatriene 7.66 ± 3.52 5.51 ± 3.07 0.26 -28.1 0.98 ± 0.56 0.61 ± 0.26 0.41 -37.8
19-Nor-4,8,11,13-abietatetraene 1.59 ± 0.57 0.78 ± 0.44 0.51** -50.1 0.46 ± 0.13 0.30 ± 0.22 0.43 -34.8
7,13-Abietadiene 0.60 ± 0.14 0.62 ± 0.67 -0.41** ?3.3 4.68 ± 4.14 7.70 ± 3.55 -0.38 ?64.5
8(14),12-Abietadiene 1.23 ± 0.64 0.35 ± 0.32 0.26* -71.5 8.47 ± 5.02 12.04 ± 3.67 -0.39 ?42.1
Oxygenated diterpene (M?285) 0.36 ± 0.08 0.16 ± 0.14 0.80** -55.6
19-Nor-6,8,11,13-abietatetraene 1.74 ± 0.59 0.65 ± 0.29 0.63** -62.6 0.18 ± 0.08 0.19 ± 0.06 -0.07 ?5.6
8,11,13-Abietatriene 2.73 ± 1.00 1.44 ± 0.94 0.50** -47.2 3.05 ± 0.67 2.63 ± 0.90 0.27 -13.8
8,13-Abietadiene 7.00 ± 1.73 2.53 ± 2.57 0.72** -63.9 34.32 ± 8.62 36.53 ± 9.48 -0.13 ?6.4
Isoabienol 15.93 ± 12.52 22.45 ± 13.58 -0.23 ?40.9 1.66 ± 1.03 2.08 ± 1.29 -0.18 ?25.3
Anticopalol isomer 2.09 ± 1.31 2.02 ± 2.18 0.02 -3.3
Bienol 1.95 ± 0.92 1.76 ± 1.26 0.08 -9.7 0.41 ± 0.28 0.17 ± 0.05 0.07 -58.5
8(14),13(15)-Abietadiene 5.51 ± 1.85 7.91 ± 1.91 -0.56* ?43.6
8,15-Pimaradien-18-al 4.30 ± 1.15 2.38 ± 1.72 0.55** -44.7 0.41 ± 0.19 0.33 ± 0.17 0.24 -19.5
8(14),11,13(15)-Abietatriene 0.72 ± 0.17 0.29 ± 0.08 0.86** -59.7
Diterpene alcohol (M?288) 0.70 ± 0.33 0.52 ± 0.19 0.33 -25.7
Isopimaral 2.78 ± 1.04 0.79 ± 0.49 0.61** -71.6
Anticopalol 15.03 ± 5.32 10.61 ± 5.44 0.40 -29.4
Diterpene alcohol (M?288) 1.09 ± 0.37 5.18 ± 4.18 -0.76** ?375.2
Levopimaral 5.91 ± 2.99 4.62 ± 2.89 0.18 -21.8 1.40 ± 0.23 0.33 ± 0.30 0.64** -76.4
Pimarol 4.02 ± 2.06 3.47 ± 1.68 0.15 -13.7
Dehydroabietal 1.10 ± 0.75 1.23 ± 0.70 -0.21 ?11.8
Diterpene hydrocarbon (M?272) 0.25 ± 0.08 0.17 ± 0.09 0.15 -32.0
Oxygenated diterpene (M?302) 1.04 ± 0.46 0.46 ± 0.20 0.31* -55.8 0.60 ± 0.23 0.36 ± 0.38 0.36 -40.0
Abietal ? methyl
levopimaratea ? methyl
palustratea
15.63 ± 4.17 15.70 ± 3.95 -0.01 ?0.4 3.37 ± 0.98 1.67 ± 0.39 0.77** -50.4
Isopimarol 0.62 ± 0.35 2.42 ± 1.63 -0.57** ?290.3 1.08 ± 0.58 0.83 ± 0.52 0.23 -23.1
Oxygenated diterpene (M?302) 0.37 ± 0.14 0.57 ± 0.22 -0.24 ?54.0
Methyl dehydroabietate 9.66 ± 3.62 10.26 ± 6.05 -0.06 ?6.2 1.17 ± 1.36 3.18 ± 2.74 -0.44 -171.9
Neoabietal ? methyl
imbricataloatea3.47 ± 3.69 4.90 ± 4.07 -0.16 -41.2 0.42 ± 0.12 0.63 ± 0.24 -0.50* ?50.0
Oxygenated diterpene (M?302) 0.32 ± 0.13 0.34 ± 0.30 -0.11 ?6.2
Methyl abietate 8.43 ± 2.50 4.59 ± 2.31 0.47** -45.6 0.44 ± 0.40 1.18 ± 0.67 -0.63** ?168.2
Abietol 0.99 ± 1.54 4.03 ± 3.44 -0.66** ?307.0 3.15 ± 2.42 1.79 ± 1.22 0.35 -43.2
Oxygenated diterpene (M?302) 1.40 ± 0.62 0.13 ± 0.05 0.85** -90.7
Oxygenated diterpene (M?302) 1.37 ± 0.70 1.05 ± 0.79 0.25 -23.3 0.36 ± 0.15 0.25 ± 0.14 0.20 -30.6
Oxygenated diterpene (M?302) 2.80 ± 2.06 1.02 ± 0.83 0.33* -63.6 0.17 ± 0.09 0.35 ± 0.11 -0.14 ?105.9
Methyl neoabietate 5.42 ± 3.53 5.69 ± 2.89 -0.03 ?5.0 1.82 ± 0.98 0.16 ± 0.11 0.78** -91.2
Neoabietol 1.59 ± 1.86 1.98 ± 1.23 -0.62** ?24.5 0.65 ± 0.29 1.16 ± 0.36 -0.64** ?78.5
Mean ± standard deviation; the data within bracket, close to words chemotype 1 and chemotype 2 are the global correlation and significance
from canonical multivariate analysis
C correlation with total canonical structure, S/W percentage variation data between summer and winter
P = Significance level in comparison mean test; **0.01 C P; *0.05 C P C 0.01a Only detected in chemotype 1
Seasonal variations of lipophilic compounds 363
123
and caryophyllene (Table 3) increased considerably their
percentage, whereas b-pinene (Table 2) and stearic acid
(Table 5) decreased. A different evolution in each group of
samples was found for the rest of the components studied.
For example, in needles of chemotype 1 collected in win-
ter, a significant decrease was observed in the contents of
diterpene hydrocarbons and aldehydes, together with a
significant increase of alcohols (Table 4). However, in the
needles of chemotype 2, there is not a pattern of evolution
of compounds in connection to their chemical group.
The most distinctive fluctuations in terpene concentra-
tions are normally observed during needle development
(von Rudlolff 1975a, b; Hiltunen 1976; Schonwitz et al.
1990), and at the end of the first growing period, terpene
patterns similar to those of mature needles are reached.
Terpene composition is quite stable in mature needles, but
the absolute amounts of individual and total monoterpenes
fluctuate during seasons, and this has also been observed in
our study. As described by Schonwitz et al. (1990) for
monoterpenes, all total terpene levels increased in our
samples to the early summer and then drops towards
winter. With regard to acid components, data in literature
about a seasonal variation of fatty or resin acids in adult
needles of conifers were not found. However the data about
resin acids show that when light decreased and water
availability increased, also a resin acid content decrease
was found (Gref and Tenow 1987; Tobolski and Zinkel
1982; Buratti et al. 1990; Nerg et al. 1994). In our study,
the fatty and resin acid concentrations in needles have
shown a significant decrease in winter. During this season,
environmental conditions differ showing lower tempera-
tures and light availability but on the contrary, there is
higher water availability than during summertime.
The increase of lipophilic compounds in needles during
warm season might be due to the fact that terpenoids
increase the heat resistance of the photosynthetic process
by stabilizing the thylacoid membranes, and they also may
have a function in the drought resistance of plants in hot
and semiarid environments (Kylin et al. 2002). It might
also be a response to an oxidative stress due to the for-
mation of reactive oxygen species and photoinhibitory
damage especially under high solar radiation and temper-
atures occurring in summer. To neutralize these oxidative
species, plants increase antioxidant compounds, and this
may be the case for monoterpenes with likely antioxidant
properties (Llusia et al. 2006).
Table 5 Seasonal variation of fatty acids (%)
Chemotype 1 (0.92**) Chemotype 2 (0.91*)
Summer Winter C-P S/W Summer Winter C-P S/W
Decanoic C10:0 1.38 ± 0.96 1.47 ± 1.35 0.09 ?6.5 2.92 ± 0.97 3.32 ± 2.73 0.28 ?13.7
Lauric C12:0 2.81 ± 2.48 1.97 ± 1.22 0.23 -29.9 1.07 ± 0.75 1.98 ± 0.92 -0.70** ?85.0
Miristic C14:0 4.67 ± 3.81 4.10 ± 3.01 -0.05 -12.2 4.85 ± 0.90 4.00 ± 1.05 0.42 -17.5
Unidentified 2.76 ± 1.81 4.47 ± 1.74 -0.30 ?61.9
Pentadecanoic C15:0 1.58 ± 0.81 1.03 ± 0.86 -0.02 -34.8 3.55 ± 0.55 1.89 ± 1.03 0.73** -46.8
Palmitic C16:0 11.54 ± 7.43 10.88 ± 3.02 -0.03 -5.7 30.23 ± 5.71 20.67 ± 3.01 0.75** -31.6
Unidentified 1.77 ± 1.12 0.51 ± 0.52 -0.26 -71.2 4.04 ± 1.53 4.87 ± 0.98 -0.33 ?20.5
Linoleic C18:2 (9,12) 3.03 ± 2.56 7.36 ± 4.37 0.57** ?142.9
Octadecenoic C18:1 (10) 10.13 ± 4.50 20.34 ± 7.87 0.69** ?100.8
Oleic C18:1 (9) 13.26 ± 13.46 10.39 ± 10.42 -0.04 -21.6 10.98 ± 5.78 19.56 ± 8.43 -0.53* ?78.1
Stearic C18:0 23.73 ± 9.69 11.76 ± 4.65 -0.52** -50.4 22.83 ± 6.38 14.28 ± 5.80 0.60** -37.4
14-Hydroxy-10-octadecenoic 5.86 ± 3.13 6.28 ± 4.67 0.07 ?7.2
13-Hydroxy-9-octadecenoic 3.81 ± 2.26 3.77 ± 2.13 0.01 -1.0
Nonadecadienoic C19:2 (9,12) 0.16 ± 0.18 1.51 ± 0.88 -0.17 ?843.7
Nonadecenoic C19:1 (9) 0.96 ± 0.56 4.90 ± 2.32 0.89** ?410.4
Nonadecanoic C19:0 0.97 ± 0.75 1.82 ± 0.80 0.50** ?87.6 3.16 ± 1.07 3.65 ± 0.85 -0.58* ?15.5
Eicosanoic C20:0 4.81 ± 2.18 6.56 ± 5.25 0.28 ?36.4 5.49 ± 2.82 8.38 ± 4.35 -0.39 ?52.6
Eneicosanoic C21:0 5.74 ± 3.47 2.54 ± 2.29 -0.02 -55.7 2.12 ± 1.27 3.25 ± 1.89 -0.40 ?53.3
Behenic C22:0 8.19 ± 6.04 1.46 ± 1.97 -0.49** -82.2 3.35 ± 1.76 5.07 ± 4.36 -0.40 ?51.3
Lignoceric C24:0 5.01 ± 4.10 2.94 ± 0.90 -0.25 -41.3 4.88 ± 1.79 5.11 ± 1.92 -0.06 ?4.7
Mean ± standard deviation; the data within bracket, close to words chemotype 1 and chemotype 2 are the global correlation and significance
from canonical multivariate analysis
C correlation with total canonical structure, S/W percentage variation data between summer and winter
P = Significance level in comparison mean test; **0.01 C P; *0.05 C P C 0.01
364 C. Arrabal et al.
123
As it was outlined in the introduction, some authors
have described this different response of each compound to
the environmental conditions. Pimarane resin acids
gradually increased during the growing season while
abietane resin acids were maintained at the same level
(Buratti et al. 1990). In Nerg et al. (1994), some resin acid
Table 6 Seasonal variation of resin acids (%)
Chemotype 1 (0.94**) Chemotype 2 (0.89)
Summer Winter C-P S/W Summer Winter C-P S/W
Seco Ia 0.16 ± 0.17 0.31 ± 0.30 0.30* ?93.7
Seco IIb 0.21 ± 0.22 0.24 ± 0.61 0.01 ?14.3
Secodehydroabietic isomer 0.17 ± 0.17 0.35 ± 0.32 0.36** ?105.9
Anticopalic isomer 6.98 ± 3.06 8.17 ± 1.26 0.26 ?17.0
Eperuic 11.04 ± 2.44 11.64 ± 2.55 0.13 ?5.4
Pimaric 0.78 ± 0.64 0.98 ± 0.89 0.13 ?25.6 7.89 ± 5.13 4.98 ± 1.82 -0.37 -36.9
Anticopalic isomer 5.83 ± 2.60 8.91 ± 1.48 0.61** ?52.8
Sandaracopimaric 1.39 ± 0.19 1.48 ± 0.07 0.21 ?6.5 2.69 ± 0.88 3.42 ± 0.91 0.39 ?27.1
Anticopalic isomer 1.79 ± 0.89 2.95 ± 0.79 0.59* ?64.8
Isopimaric 0.35 ± 0.23 0.37 ± 0.18 0.03 ?5.7 0.85 ± 0.44 1.13 ± 0.32 0.35 ?32.9
Anticopalic 47.20 ± 6.37 47.01 ± 7.19 -0.01 -0.4
Levopimaric ? palustric 26.67 ± 8.25 35.80 ± 6.38 0.47** ?34.2
Dehydroabietic 6.02 ± 1.69 4.76 ± 1.29 -0.34* -20.9 2.95 ± 1.06 3.23 ± 1.75 0.10 ?9.5
Resin acid (M?316) 0.24 ± 0.15 0.28 ± 0.20 0.48** ?16.7
8,12-Abietadien-18-oic 0.17 ± 0.13 0.19 ± 0.24 0.17 ?11.8
Imbricataloic 11.71 ± 5.55 7.15 ± 6.06 -0.34* -38.9 0.87 ± 0.77 0.29 ± 0.21 -0.41 -66.7
Abietic 12.11 ± 2.93 12.76 ± 3.02 0.09 ?5.4 4.94 ± 2.92 4.68 ± 1.84 -0.06 -5.3
Resin acid (M?318) 0.39 ± 0.14 0.14 ± 0.10 -0.74** -64.1
Resin acid (M?314) 0.63 ± 0.31 0.43 ± 0.89 -0.18 -31.7
Epiimbricataloic 0.61 ± 0.47 0.87 ± 0.94 0.21 ?42.6
Neoabietic 22.99 ± 6.44 28.23 ± 6.26 0.35* ?22.8 4.07 ± 3.85 2.08 ± 0.60 -0.35 -48.9
Dihydroagatic 0.93 ± 0.49 0.64 ± 0.62 -0.30* -31.2 0.84 ± 0.37 0.55 ± 0.29 -0.27 -34.5
Pinifolic 0.42 ± 0.39 0.27 ± 0.25 -0.17 -35.7
Oxoresin acid (M? 330) 0.47 ± 0.27 0.37 ± 0.52 -0.03 -21.3
Hydroxyresin acid (M?334) 0.89 ± 0.59 0.50 ± 0.72 -0.39** -43.8
Oxohydroxydehydroabietic 0.77 ± 0.49 0.96 ± 0.89 0.22 ?24.7 0.34 ± 0.10 0.24 ± 0.06 -0.37 -29.4
Methoxyresin acid (M?346) 0.98 ± 0.95 1.67 ± 0.56 0.38** ?70.4
Oxohydroxydehydroabietic 3.32 ± 1.71 0.53 ± 0.52 -0.60** -84.0
Hydroxyabietic (M?332) 0.82 ± 0.67 0.29 ± 0.13 -0.36* -64.6
19-Nor-12-oxo-3,5,8-abietatrienoic 0.43 ± 0.34 0.17 ± 0.11 -0.22 -60.5
Hydroxyresin acid (M?330) 0.24 ± 0.23 0.22 ± 0.09 0.08 -8.3
Hydroxydehydroabietic 0.50 ± 0.42 0.11 ± 0.09 -0.37** -78.0
Dihydroxyresin acid (M?348) 0.40 ± 0.19 0.40 ± 0.42 0.06 0.0
Oxoresin acid (M?328) 1.09 ± 0.51 0.12 ± 0.09 -0.68** -89.0
15-hydroxydehydroabietic 2.46 ± 1.75 0.08 ± 0.06 -0.56** -96.7
Dihydroxyresin acid (M?348) 1.42 ± 0.87 0.05 ± 0.03 -0.61** -96.5
Dihydroxyresin acid (M?348) 1.39 ± 0.81 0.04 ± 0.05 -0.49** -97.1
Mean ± standard deviation; the data within bracket, close to words chemotype 1 and chemotype 2 are the global correlation and significance
from canonical multivariate analysis
C correlation with total canonical structure, S/W percentage variation data between summer and winter
P = Significance level in comparison mean test; **0.01 C P; *0.05 C P C 0.01a Seco I = 2a-[20(m-isopropylphenyl)ethyl]-1b.3a-dimethyl-cyclohexanecarboxylicb Seco II = 2b-[20(m-isopropylphenyl)ethyl]-1b.3a-dimethyl-cyclohexanecarboxylic
Seasonal variations of lipophilic compounds 365
123
concentrations (levopimaric and dehydroabietic) were at a
higher level in the autumn and some at a lower level
(palustric, abietic and neoabietic) than in previous spring.
Tobolski and Zinkel (1982) found that in pine needles the
concentrations of levopimaric/palustric, dehydroabietic and
neoabietic acids decreased while pinifolic acid concentra-
tion increased during the sampling period from June to
December. Gref and Tenow (1987) found only minor
seasonal changes in resin acid concentrations in needles
and cortex from pine grown in Sweden, but between sun
and shade needles some resin acids maintained the same
level while others showed significant decreases.
These observations suggest that secondary compounds
from genetically different trees have different response to
the environmental conditions.
Moreover, it is necessary to bear in mind that, except for
a- and b-pinene, 8,13-abietadiene (in needles of chemotype
1) and palmitic acid (in needles of chemotype 2), in general
the minor components showed the highest correlation with
the canonical structure of discrimination obtained in the
statistical analysis, and that significant differences in rela-
tion to sampling season were not found for many of the
main components. Therefore, from a chemosystematic
point of view, it is significant that the lipophilic patterns of
P. pinaster needles can have different sensitivity to envi-
ronmental factors, and the highest changes were showed by
the minor components.
Although the environmental factors can modify the
proportion of some compounds occurring in low amounts,
the chemotypes remain distinct.
Conclusions
The lipophilic composition (monoterpenes, sesquiterpenes,
neutral diterpenes, fatty acids and resin acids) of the adult
needle of P. pinaster undergoes a seasonal variation, both
at individual and at global levels. These seasonal variations
in distribution pattern of compounds were not produced in
the same way in the needles of the two chemotypes
detected. Therefore, among secondary metabolism com-
pounds there is a variation in the sensitivity to environ-
mental conditions, and the components from genetically
different trees have different response to these environ-
mental conditions.
To minimize associated errors, samples for genetic
studies should be taken from needles of equal age and at
the same moment of year.
Acknowledgments This work was financially supported by Project
SC97-118-C2-1 from MAPA (Ministry of Agriculture, Fisheries and
Food, Spain). We wish to thank Dr. Ricardo Alıa for his valuable
advice and Mrs. Rosa Calvo for her technical assistance in statistical
analysis.
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