1. ORIGINAL ARTICLE
Effects of soil water temperature on root hydraulic resistance of
six species of Iberian pines
P. ZUCCARINI1
, E. FARIERI1,2
, R. VA´ SQUEZ1,3
, B. GRAU1
, & R. SAVE´ MONSERRAT1
1
Environmental Horticulture Program – Ecophysiology Subprogram, IRTA – Institut de Recerca i Tecnologia
Agroalimentaries, Torre Marimon, C-59, Km 12.1, Caldes de Montbui, Barcelona 08140, Spain; 2
Department of
Agricultural and Food Science (DISPA), University of Catania, Via Valdisavoia 5, Catania 95023, Italy and 3
FAREM –
Universidad Autonoma de Nicaragua, Esteli, Las Segovias, Nicaragua
Abstract
The Iberian Peninsula hosts six native pine species, which are distributed according to an altitudinal gradient from coastal to
mountain areas, close to 1000 m a.s.l. Root hydraulic responses are the key factors of spatial segregation of trees in response
to environmental factors such as temperature and water availability, and they will be a determinant of future population and
species spatial dynamics in a changing climate scenario. Root hydraulic responses to soil water temperatures ranging from
308C to 08C were compared for young plants of these six aforementioned species. Hydraulic resistance (Rh) increased for all
species in response to temperature decrease. Mountain pines showed higher Rh values than coastal pines at all temperatures,
and showed a more prompt and marked hydraulic response when temperatures dropped down. Data point out that mountain
pines display a clear mechanism to avoid cold embolism and secondary water stress, while coastal species have a limited
responsiveness to temperature changes due to scarce hydraulic regulation. These differences in hydraulic behaviour support
the spatial segregation between mountain and coastal pines in the Iberian Peninsula, and will be one of the factors at the basis
of the future shifts of species and populations that will be associated to climate change.
Keywords: Climate change, drought stress, Iberian pines, root hydraulic resistance, soil temperature stress
Introduction
The genus Pinus contains 111 species (Richardson &
Rundel 1998), six of which have native populations
in the Iberian Peninsula: P. uncinata, P. sylvestris,
P. nigra, P. pinaster, P. pinea and P. halepensis. These
species show an important spatial segregation that
correlates with geomorphological and climatic
conditions (Alı´a et al. 1996; Prada et al. 1997;
Barbero et al. 1998): the first three (mountain pines)
grow in cold and mesic climates, in the highest parts
of the highest mountain ranges of the Iberian
Peninsula, while the other three (lowland pines)
grow in coastal drier Mediterranean environments.
A wide number of studies demonstrate that
Mediterranean woody species show different func-
tional strategies in response to abiotic stress factors
(see for example Villar-Salvador et al. 2004; Cuesta
et al. 2010; Ferna´ndez Calzado et al. 2012;
Kalajnxhiu et al. 2012). This response happens at
both inter- and intra-specific levels, and the different
ecophysiological behaviour correlates to different
tolerance, survival and growth capacity. These data
suggest that climatic and geomorphological con-
ditions determine plant species and populations
distributions through the differential plant ecophy-
siological responses to the environmental stresses.
Although a large number of studies have been
performed to identify functional differences among
populations within pine species (see for example Alı´a
et al. 1997; Lo´pez et al. 2007), there is a certain lack
of studies on comparative physiology of Iberian pines
species to support an ecophysiological theory of pine
distribution. A deeper knowledge of the ecophysio-
logical responses of the different species to environ-
mental constraints represents in fact a crucial tool for
medium- to long-term predictions of species and
q 2014 Societa` Botanica Italiana
Correspondence: R. Save´ Monserrat, Environmental Science and Global Change Area, Environmental Horticulture Program – Ecophysiology Subprogram,
IRTA – Institut de Recerca i Tecnologia Agroalimentaries, Torre Marimon, C-59, Km 12.1, Caldes de Montbui, Barcelona 08140, Spain. Tel: þ34 93 4674040
ext. 1326. Email: dimivilli@hotmail.com
Plant Biosystems, 2014
http://dx.doi.org/10.1080/11263504.2014.983206
Downloadedby[Plant&FoodResearch]at17:5616December2014

2. populations dynamics in a changing climate scenario
(Zavala 2004).
Several works show how spatial segregation of
Iberian pines, similarly to other genera of woody
plants, is correlated with summer drought and low
winter temperatures (e.g. Terradas & Save´ 1992;
Alı´a et al. 1996; Gil et al. 1996; Prada et al. 1997).
Climate change will promote important changes in
temperature and water availability, especially in the
Mediterranean region: this will also affect soil
temperature and associated aspects of plant metab-
olism such as water absorption (Sheffield & Wood
2008). In particular, it is predicted that winter frost
episodes will be less frequent and more limited in
duration, but still of comparable severity as now
(Giorgi & Lionello 2008).
Root hydraulic resistance (Rh) is an important
parameter describing plant behaviour towards water
and temperature conditions. High Rh values are
usually associated with drought tolerance (aniso-
hydric behaviour) and low Rh with drought avoidance
(isohydric behaviour) (Tardieu & Simonneau 1998),
but they are also associated, respectively, with better
and worse tolerance to frost-caused drought stress
(Tranquillini 1982), which mechanisms differ from
the one caused by high temperatures (Davis et al.
1999). The risk of winter stress cavitation depends in
fact on xylem water potential and on the diameter of
the bubbles that form in the xylem (Yang & Tyree
1992), which in turn depend on xylem conduit
diameter: bigger diameters mean higher susceptibility
to cold induced embolism and vice versa (Langan
et al. 1997). In this sense, a distinction can be made
between two strategies against cold stress. On one
side, we have trees that are more adapted to cold
climates (such as evergreens): they usually have
smaller conduit diameters, which give them better
resistance to cavitation even for very low values of
xylem water potential. The advantage is vegetative
growth throughout the whole year; the price to pay is
represented by the lower growth performances.
On the other side, we have trees that are less tolerant
(such as deciduous): their xylem conduit diameters
are bigger, providing them with better hydraulic
efficiency in the warm seasons, as big volumes of
transported water sustain higher gas exchange rates,
but when cold arrives, they undergo extensive
cavitation, lose their leaves and arrest the growth
season, restoring water transport when warm tem-
peratures return (Wang et al. 1992).
The carbon starvation hypothesis has been
invoked by some authors as an alternative and
complementary mechanism to explain tree growth
limitation and mortality in response to drought and
frost-caused drought stress (Ward et al. 2005;
McDowell et al. 2008; Adams et al. 2009), but this
explanation can be rejected in the case of trees of
small age and size. Plant–water relationships, with a
special care on the physiology of root systems, play
a central role in young plants’ response to winter
drought stress, because Rh regulation must be
considered as a mechanism to permit water absorp-
tion when this is available for the plant in order to
increase plant growth (Bazzaz & Morse 1991).
The aim of the present work was to compare the
hydraulic response of young plants of the six
aforementioned species to soil temperatures ranging
from 308C to 08C. The goal is to define a first
characterization of the hydraulic behaviour of the
different species of Iberian pines towards water and
soil temperature, in order to provide the ecophysio-
logical tools that are necessary to support a theory of
pine distribution and guide medium- to long-term
predictions of species and population dynamics in a
changing climate scenario.
Materials and methods
Pine plants were germinated on March 2012 in El
Serranillo (INIA, Alcala´ de Henares, Madrid, Spain)
and grown under greenhouse conditions in daily
irrigated 3.5-l pots filled with peat:sand 40%:60%,
at IRTA – Torre Marimon, Catalonia, Spain.
Temperature ranged between 168C and 288C; air
humidity ranged between 50% and 70%; maximum
midday PPFD (photosynthetic photon flux density)
ranged between 500 and 1200 mmol m22
s21
depending on cloud cover.
Cuticular transpiration rate (TRc) and specific leaf
weight (SLW) were measured in summer 2012 and
winter 2012–2013 as a preliminary test aimed at
monitoring the response of young pine needles to
aging and changes in temperature that are typical of
the Mediterranean seasonality. At both seasons, six
groups of 6–10 juvenile needles each were selected
for each species and suspended in water overnight.
After saturation, needles were weighted at 5-min
intervals during the first 50 min, 10-min intervals
during the next hour and every 15 min during the last
2.5 h. Partial cuticular transpiration (CT) was
calculated for each interval as fresh weight loss by
period of time on a dry weigh basis:
CT ¼
DFW
Dt £ DW
;
where DFW corresponds to the weight loss during
the measurement interval Dt.
TRc was calculated as the average of CT values
taken from the moment in which stomatal closure
occurred (i.e. when the CT vs. RWC slope suddenly
changed; RWC ¼ relative water content).
Area (A) of every group of needles was
determined with the software WinFolia2009 (Regent
2 P. Zuccarini et al.
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3. Instruments, Inc., Switzerland). Needles were oven-
dried at 608C until DW was stable, and SLW was
calculated as follows:
SLW ¼
DW
A
:
Five replicates were taken for each species. The
growth medium was removed, the roots carefully
washed and the plants were left in immersion in tap
water for approximately 24 h at 208C. The root
systems were then cut from the aerial part at 3 cm
above the point of emergence of the first lateral root.
The cut was performed under water to prevent air
from entering the stem.
Root Rh was measured on the entire intact root
system to provide better accuracy (see Mu et al.
2006). The measurements were taken following the
Ramos and Kaufmann (1979) method (see Save´
et al. 1995) at four different temperatures of water
(308C, 208C, 108C and 08C ^ 5%), which were
obtained with a thermostatic bath. Entire root
systems were immersed in water inside a Scholander
pressure chamber and the protruding stem was
connected to a flexible tube with a diameter between
2 and 5 mm (according to the individual stem
diameter). Air pressure at 0.5 MPa was applied, and
the volume of water flowing through the roots and
into the tube was measured during eight intervals
(2 min each). Rh was subsequently calculated with
the following formula:
Rh ¼
P £ L
J
;
where Rh is hydraulic resistance (MPa s cm22
), P is
the applied pressure (0.5 MPa), L is root length (cm)
and J is the flow of water (cm3
s21
).
Root length was measured with WinRhizo (Regent
Instruments, Inc., Quebec, Canada).
Rh was then corrected into Rhcorr, to take into
account the change of water viscosity at different
temperatures, according to the Sperry equation to
normalize conductivity at 208C:
K0
¼ Kð0:02698 £ T þ 0:456Þ21
;
where K0
is the corrected conductivity, T is the
temperature and K is the measured conductivity. K
was calculated from Rh as K ¼ (Rh £ L 21
)21
, since
Rh is normalized to root length but K is not,
therefore K0
¼ (Rhcorr £ L 21
)21
.
Statistical analyses were performed with GraphPad
Prism (GrapPad Software, Inc., La Jolla, CA, USA).
Two-way ANOVA followed by Bonferroni post-test
was performed on the Rh and Rhcorr ¼ funct (species
£ temperature) data; linear regression was performed
for each species for Rh versus temperature.
Results
For all species, TRc significantly decreased and SLW
significantly increased in the transition from summer
to winter (Figure 1). The highest decrease in TRc
was observed for P. pinaster and P. uncinata, and in
both cases it was associated with the highest increase
in SLW. A significant negative correlation was found
between DTRc and DSLW (data not shown).
Rh increased for all species in response to
temperature decrease, which explained the 19.1%
of the total variability (p , 0.001, two-way ANOVA)
(Figure 2).
Very significant differences in Rh were found
among all the species at all temperatures, the species
factor explaining the 60% of the total variability
(p , 0.001). In particular, it was possible to define a
clear segregation of mountain and coastal pines into
TRc
P.halepensis
P.pinaster
P.pinea
P.nigraP.sylvestris
P.uncinata
0.000
0.001
0.002
0.003
0.004
TRc[g*gDW–1
*min–1
]
SLW
P.halepensisP.pinaster
P.pinea
P.nigraP.sylvestrisP.uncinata
0.000
0.005
0.010
0.015
0.020
0.025
Summer 2012
Winter 2012–2013
SLW[g*cm–2]
Figure 1. TRc and SLW for each species, measured consecutively in summer 2012 and winter 2012–2013.
Effects of soil water temperature on pines 3
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4. two distinct groups, the former ones always showing
higher values of Rh. More specifically, the Rh
gradient among species followed the altitudinal
gradient at which the species are found in nature,
with the only exception represented by the inversion
between P. sylvestris (second highest habitat in nature
but highest Rh values) and P. uncinata (highest
habitat and second highest values of Rh).
The interaction factor (species £ temperature)
explained about the 10% of the total variability of
Rh ( p , 0.001), which indicates a different dynamic
response of Rh to temperature changes among
species. The average increase of Rh was signifi-
cantly more marked for mountain pines rather than
lowland ones (Figure 4), the former ones having
regression slopes from two to five times higher than
the latter ones, which responded very weakly to the
temperature decrease. Moreover, all mountain
pines showed an increased inflection point at
temperatures between 208C and 108C, while coastal
pines had a weak but constant increase of Rh for
lower temperatures (a slight inflection between
108C and 08C was detected for P. pinea and
P. pinaster).
When corrected for changes in water viscosity,
Rhcorr markedly dropped down for all species at all
temperatures (Figure 3), and no significant differ-
ences among temperatures were observed for any of
the investigated species. Significant differences were
still present, at each studied temperature, among the
three mountain pines and between each of them
and the three coastal pines. Coastal pines displayed
completely homogeneous values among them when
observed separately from the mesic ones.
Discussion
The significant negative correlation between DTRc
and DSLW points out the winter loss of TRc as a
direct consequence of leaf hardening due to aging
(Hadley & Smith 1994; Richardson et al. 2000); this
trend can be also partially explained by the TRc
decrease usually associated to lower temperatures
(Schreiber 2001).
P. uncinata (the most altitudinal species) showed
the highest average value of winter TRc, while
P. halepensis (the most coastal one) showed the lowest
average value. The remaining species, which in
nature have an intermediate altitudinal distribution
between the two extremes, showed intermediate and
comparable values among them. This significant
difference between P. halepensis and P. uncinata can be
explained as the consequence of the different
cuticular thickness as a species-specific adaptation
to more xeric and more mesic conditions, respect-
ively, aimed for P. halepensis to the reduction of
cuticular water losses. Indeed, TRc is negatively
correlated with leaf cuticle thickness (Hajibagheri
et al. 1983; Burghardt & Riederer 2003), being a
thicker leaf cuticle a typical xeromorphic feature
(Tipton & White 1995), usually associated with
plants adapted to hotter and drier environments.
Cuticle conductance in evergreens tends to increase
with altitude, both at intraspecific (DeLucia & Berlyn
1984) and interspecific levels (Anfodillo et al. 2002).
Winter temperatures can restrict the hydraulic
functions of trees (Sakai & Larcher 1987). In fact,
water uptake from soil water reservoirs is very limited
when upper soil layers are cool or frozen during
0
30 20 10 0
5.0×105
1.0×106
1.5×106
P.sylvestris
P.uncinata
P.nigra
P.pinaster
P.pinea
P.halepensis
A*
a**A
ab
A
bc A
c
A
c
A
c
B
a
AB
ab
AB
bc
A
c A
c
A
c
C
a
B
b
B
c
A
d A
d
A
d
D
a
C
b
C
c
A
d A
d
A
d
Temperature [°C]
Rh[MPa*s*cm–2
]
Figure 2. Measured values of root Rh for all species at all temperatures. *Values of the same species marked with the same letter are not
statistically different at P , 0.05, according to Bonferroni’s post-test. **Values of the same temperature marked with the same letter are not
statistically different at P , 0.05, according to Bonferroni’s post-test.
4 P. Zuccarini et al.
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5. winter months or early spring (Mellander et al.
2006).
The segregation between mountain and coastal
pines (higher and lower Rh values respectively)
indicates a better adaptability of mountain pines to
winter stress and high water availability than coastal
ones (Figure 2). Low temperatures of water and soil
can cause impairments of water status, leading to
cavitation and plant embolisms (Sperry & Sullivan
1992; Mayr et al. 2006). Actually both drought
and freezing can cause xylem cavitation, but the
mechanisms involved are totally different (Willson &
Jackson 2006). Drought stress induces cavitation
through a dramatic reduction of xylem water
potential, which makes air enter into the xylem
from adjacent air-filled and cavitated conducts
(Zimmermann 1983). In this sense, drought-
induced embolism is poorly or not correlated to
0102030
0
2.0×105
4.0×105
6.0×105
8.0×105
1.0×106
P.sylvestris
P.uncinata
P.nigra
P.pinaster
P.pinea
P.halepensis
Temperature [°C]
Rh[MPa*s*cm–2]
Correlation
parameters
P.sylvestris P.uncinata P.nigra P.pinaster P.pinea P.halepensis
Slope 15.1*103
10.3*103
7.7*103
3.9*103
3.0*103
3.0*103
r2
0.9631 0.9601 0.9968 0.9513 0.9560 0.9786
Figure 4. Rates of Rh increase as temperature drops down, for the different species. Linear regression data are presented in the table, showing
the slopes of Rh increase and r 2
values.
302010 0 302010 0 302010 0 302010 0 302010 0 302010 0
0
2.0×105
4.0×105
6.0×105
8.0×105
1.0×106
P.sylvestris
P.uncinata
P.nigra
P.pinaster
P.pinea
P.halepensis
A
a
A
a
A
a
A*
a**
B
a
B
a
A
a A
a
B
a
B
a
C
a
C
a
C
a D
a
D
a
D
a
C
a D
a
D
a
D
a
C
a D
a
D
a
D
a
Temperature [°C]
Rhcorr[MPa*s*cm–2
]
Figure 3. Values of measured root Rh, corrected according to the Sperry equation (Rhcorr), for all species at all temperatures. *Values of Rhcorr
for different species at the same temperature, when marked with the same letter, are not statistically different at P , 0.05, according to
Bonferroni’s post-test. **Values of Rhcorr for the same species at different temperatures, when marked with the same letter, are not
statistically different at P , 0.05, according to Bonferroni’s post-test.
Effects of soil water temperature on pines 5
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6. conduit diameter, while frost-caused embolism is
positively related to it (Maherali et al. 2004). In this
sense, wide-conduit species experience greater
hydraulic failure during frost periods, which is
confirmed by considerable evidence (see for example
Hacke & Sauter 1996; Langan et al. 1997; Peguero-
Pina et al. 2011).
For these reasons, trees living in dry environ-
ments tend to have higher specific hydraulic
conductivity (i.e. lower Rh) than montane trees,
primarily because of larger tracheid lumen diameters
(Maherali & De Lucia 2000), which ultimately
makes their hydraulic system more vulnerable to
winter stress.
The low temperature effect has been studied
here on the very short term, so that the observed
reduction in root hydraulic conductivity can be
mostly ascribed to the higher viscosity of water, as it
has been demonstrated that changes in water
viscosity between 258C and 58C can reduce root
conductivity up to 40% (Brodribb & Hill 2000). The
correction of Rh for changes in water viscosity at
different temperatures (Figure 3) allowed the direct
observation of the interspecific differences in the
hydraulic performances of the root apparatuses,
independently from the physical properties of water.
The only significance that was not lost after the
correction was the higher Rhcorr for mountain pines,
which provides clear support to the fact that tree
species adapted to colder climates have smaller
tracheid lumen diameters to prevent the risk of
winter stress embolism (Sperry et al. 2006).
The scarce adaptation of coastal species to low
temperatures is connected to a low hydraulic
regulation that causes a limited responsiveness to
temperature changes (Sakai & Larcher 1987). The
Rh slopes towards the 30–08C temperature gradient
are always higher for mountain pines (Figure 4),
which indicates a prompter hydraulic response to
temperature reduction. Besides, the three mountain
species show a different slope pattern which reflects
their different altitudinal areas, while the low values
of Rh responsiveness to water temperature for coastal
pines do not vary significantly among the three
species. This fact shows how mountain pines are able
to hydraulically respond to the onset of cold stress,
and their ability is higher for species that live at higher
altitudes. On the other side, all the studied coastal
pines are equally vulnerable to winter stress and to
the subsequent risk of embolism. Cold tolerance
widely varies among species and can determine the
distribution of perennial plants (Sakai & Larcher
1987; Cavender-Bares et al. 2005).
The ongoing process of climate change will
induce in the next future, a progressive mitigation of
the climate and a reduction of precipitations in the
Mediterranean regions. In this frame, it is predicted
that frost episodes at higher altitudes will be less
frequent but of the same severity (Giorgi & Lionello
2008). These factors will expose mountain pines,
naturally adapted to winter stress, to its combination
with drought stress, which can strongly augment the
vulnerability to cold induced embolism (Willson &
Jackson 2006). By making them more vulnerable to
cavitation, this will threaten their survival especially
in those zones of their areals that will be more
dramatically affected by the reduction of water
availability, producing important changes in the
spatial distribution of Iberian pines. For example,
P. sylvestris lives in Spain at the southern limit of
its natural range (Poyatos et al. 2007), in the “arid
altitudinal belts” of mountains of the Mediterranean
area (Breckle 2002; Peguero-Pina et al. 2011). This
makes P. sylvestris subject to the combination of cold
winter temperatures and a generally limited avail-
ability of water, and can explain its highest Rh values
at all temperatures, which indicates an extreme
adaptation against the risk of winter-/drought-caused
cavitation. It is estimated that any further increase in
aridity in the Mediterranean region could cause the
extinction of many populations of P. sylvestris along
the Iberian Peninsula (Poyatos et al. 2008). Further
research on the ecophysiological behaviour of Iberian
pines towards drought and winter stresses will be
necessary to develop a solid ecophysiological basis,
experimentally sustained, for explaining their distri-
bution pattern and make predictions for the future.
Conclusions
Data point out that mountain pines display a clear
mechanism to avoid cold-induced embolism and
secondary water stress. Coastal species, which are
not adapted to low temperatures, display a scarce
hydraulic regulation which causes a limited respon-
siveness to temperature changes.
This different hydraulic behaviour of mountain
and coastal pines supports the spatial segregation
that can be found in the Iberian Peninsula, and will
probably be one of the factors at the basis of the
future shifts of species and populations that will be
associated with climate change.
In particular, the lower precipitations, combined
with winter low temperatures, will make mountain
pines more vulnerable to cavitation than they are
nowadays and will cause important shifts in species
and population distributions in microthermal
environments.
Results clearly point out how the species
distribution is influenced by the typically Mediterra-
nean frost-drought double stress (Terradas & Save´
1992), and landscape composition will be the result
of a wide and complex interaction of environmental
factors (Bazzaz & Morse 1991). The physiological
6 P. Zuccarini et al.
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7. mechanisms involved in survival and growth, related
with this combined environmental stress, will be
articulated into frost adaptations at root level and
drought adaptations at canopy level (Mooney et al.
1991).
Funding
This research was partially funded by MINECO
Project [grant number AGL2011-24296] and Con-
solider-Montes Project [grant number CSD2008-
00040].
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