Seasonal variations of lipophilic compounds in needles of two chemotypes of Pinus pinaster Ait

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: [email protected]

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




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 (%)



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





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 (%)



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




P = Significance level in comparison mean test; **0.01 C P; *0.05 C P C 0.01a Only detected in chemotype 1


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*)


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






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)


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




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


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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Seasonal variations of lipophilic compounds in needles of two chemotypes of Pinus pinaster Ait