Photosynthetic responses of trees in high-elevation forests: comparing evergreen species along an elevation gradient in the Central Andes
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Photosynthetic responses of trees in high-elevation forests: comparing evergreen species along an elevation gradient in the Central Andes
Jose´ I. Garc´ıa-Plazaola 1
Duncan A. Christie 0 2
Rafael E. Coopman
Associate Editor: Astrid Volder
0 Laboratorio de Dendrocronolog ́ıa y Cambio Global, Instituto de Conservacio ́ n, Biodiversidad y Territorio, Facultad de Ciencias Forestales y Recursos Naturales, Universidad Austral de Chile , Casilla 567, Valdivia , Chile
1 Departamento de Biolog ́ıa Vegetal y Ecolog ́ıa, Universidad del Pa ́ıs Vasco UPV/EHU, Apdo. 644, E-48080 Bilbao , Spain
2 Center for Climate and Resilience Research , CR
Plant growth at extremely high elevations is constrained by high daily thermal amplitude, strong solar radiation and water scarcity. These conditions are particularly harsh in the tropics, where the highest elevation treelines occur. In this environment, the maintenance of a positive carbon balance involves protecting the photosynthetic apparatus and taking advantage of any climatically favourable periods. To characterize photoprotective mechanisms at such high elevations, and particularly to address the question of whether these mechanisms are the same as those previously described in woody plants along extratropical treelines, we have studied photosynthetic responses in Polylepis tarapacana Philippi in the central Andes (188S) along an elevational gradient from 4300 to 4900 m. For comparative purposes, this gradient has been complemented with a lower elevation site (3700 m) where another Polylepis species (P. rugulosa Bitter) occurs. During the daily cycle, two periods of photosynthetic activity were observed: one during the morning when, despite low temperatures, assimilation was high; and the second starting at noon when the stomata closed because of a rise in the vapour pressure deficit and thermal dissipation is prevalent over photosynthesis. From dawn to noon there was a decrease in the content of antenna pigments (chlorophyll b and neoxanthin), together with an increase in the content of xanthophyll cycle carotenoids. These results could be caused by a reduction in the antenna size along with an increase in photoprotection. Additionally, photoprotection was enhanced by a partial overnight retention of de-epoxized xanthophylls. The unique combination of all of these mechanisms made possible the efficient use of the favourable conditions during the morning while still providing enough protection for the rest of the day. This strategy differs completely from that of extratropical mountain trees, which uncouple light-harvesting and energy-use during long periods of unfavourable, winter conditions.
High mountain plants; light-harvesting; neoxanthin; photosynthesis; xanthophylls; zeaxanthin
One of the most challenging conditions that
highelevation plants experience is the combination of high
irradiance and low temperatures. Since enzymatic
reactions are sensitive to temperature, but light capture is
not, this stress combination generates a severe
imbalance between mechanisms’ high energy absorption
and low energy use through enzymatic carbon
assimilation (Ball et al. 1991). This imbalance causes an
over-excitation of the photosynthetic apparatus, which
triggers the formation of reactive oxygen molecules.
This reactive oxygen can provoke a large amount of
oxidative damage to proteins, lipids and nucleic acids,
leading to chronic photoinhibition (for a review, see Asada
2006). To counteract this effect, high mountain trees
and herbs have to exacerbate the expression of protective
(Streb et al. 1998; Streb and Cornic 2012)
thereby reaching a new equilibrium in the source/sink
balance, a concept referred to as ‘photostasis’ (O¨ quist and
Huner 2003). The photoprotective mechanisms that
high mountain plants display can be classified into five
major groups: (i) a decrease in light absorption by
increasing reflectance through morphological/structural
modifications such as trichomes or waxes
Griffiths 2006; Close et al. 2007)
, vertical positioning of
(Sanchez et al. 2014)
or through the accumulation
of anthocyanins in the upper cell layers which attenuate
light reaching mesophyll cells
(Williams et al. 2003)
(ii) energy-consuming sinks’ increase in metabolic
activity, such as CO2 assimilation, plastid terminal oxidase or
cyclic electron transport around photosystem I (PSI)
(Streb et al. 1998; Streb and Cornic 2012; Laureau
et al. 2013)
; (iii) thermal dissipation of the excess energy
absorbed by chlorophylls, a process modulated by the
activity of the xanthophyll cycle that is commonly measured
by the non-photochemical quenching (NPQ) of
chlorophyll a (Chl a) fluorescence
(for recent reviews, see
Demmig-Adams et al. 2012; Garc´ıa-Plazaola et al.
; (iv) quenching through the antioxidant
metabolism of reactive oxygen species generated by energy
imbalance (Wildi and Lu¨ tz 1996) and (v) repair of the
damage inflicted on photosystem II (PSII) reaction
(Streb et al. 1997)
. The operation of these
mechanisms has been characterized in high mountain plants in
temperate regions of Europe
(Ensminger et al. 2004;
Porcar-Castell et al. 2012)
, North America
(Zarter et al.
and Australia (Ball et al. 1991); in these cases, a
marked thermal seasonality defines a long winter and a
short growing period. In these environments, the forest
that extends up to the treeline is frequently made up
of evergreen trees (mostly conifers in the Northern
Hemisphere), which maintain light-harvesting antennae
in a state primed for energy dissipation during the
cold season. This mechanism is referred to as ‘sustained
energy dissipation’, due to its low rate of
disengagement when conditions return to their optimum state
Meanwhile, treelines in the high mountain areas of
North American and European temperate regions range
between 1000 and 3500 m above sea level (a.s.l.), while
trees in the tropical and subtropical mountains of South
America are able to develop at much higher elevations
(Ko¨ rner 2003)
, with Polylepis tarapacana Philippi forming
the highest treeline in the world at 5200 m a.s.l.
et al. 2009)
. At such high elevations, one of the most
challenging environmental factors that plants have to face is
the combination of large daily thermal amplitude with
strong irradiance. These conditions can easily be
summarized by the aphorism ‘summer every day and winter
(Lu¨ ttge 2008)
. In the case of the South
American Altiplano in the central Andes (158 – 248S),
which is a semi-arid plateau located at a mean elevation
of 4000 m a.s.l., the acclimation to thermal amplitude is
also superimposed to seasonality
(Garreaud et al. 2003)
In the Altiplano, in contrast to extratropical mountains,
the course of the seasons is mainly defined by periods
of water scarcity rather than by a very cold winter. In
such environments, plant species have to deal with
a harsh combination of unfavourable factors including
a long period of aridity, which leads to extremely low air
humidity, a high proportion of short-wave radiation, low
atmospheric pressure and year-round frosts
. Despite seasonal oscillations, the climate of the
Altiplano is predominantly cold and dry. It is
characterized by a dry season which corresponds to the coolest
period (winter) and a warmer and wetter season (summer)
when more than 80 % of the scarce total annual
(Garreaud et al. 2003)
. Compared with higher
latitudes, seasonal differences in total solar radiation are
small, just 1.7 times higher in the summer than in the
winter (Aceituno 1993). Under these unique
environmental conditions, only trees of the genus Polylepis are able to
(Gonza´ lez et al. 2002)
Species of Polylepis belong to the Rosaceae family,
and include 28 species of small- to medium-sized
evergreen trees. They grow at very high elevations in the
tropical and subtropical Andes of South America from
Venezuela to northern Argentina (88N – 328S)
and Schmidt-Lebuhn 2006)
. Among the Polylepis species,
P. tarapacana is a unique, long-lived tree that can reach
over 700 years in age
(Morales et al. 2012)
, and occurs
throughout the semi-arid ecosystem of the Altiplano in
the central Andes from 168 to 238S between 4000 and
5200 m a.s.l., forming the world’s highest elevation
forests (Braun 1997). Polylepis tarapacana has several
strategies to cope with such an unfavourable
combination of environmental factors. These strategies allow
this species to maintain positive assimilation rates (P) in
both the winter and the summer
(Garc´ıa-Nu´ n˜ ez et al.
: P. tarapacana also displays frost tolerance during
the cold period in addition to a super-cooling capacity
during the warm season
(Rada et al. 2001)
. Some of
the species’ other coping strategies include xerophytic
anatomical foliar traits
(Toivonen et al. 2014)
the elevation-dependent accumulation of ultraviolet
B-absorbing compounds and carotenoids
et al. 2007)
. However, the adaptations of P. tarapacana’s
photoprotective mechanisms (in particular the dynamics
of the xanthophyll cycle) have not yet been examined.
Given the unique environmental conditions where
P. tarapacana forests can occur, the main goal of this
study was to further examine how photostasis and
photosynthetic activity is modulated by the operation of
photoprotective mechanisms in the world’s highest forest.
To achieve this objective, we studied photosynthetic
responses in P. tarapacana along an elevation gradient
(from 4337 to 4905 m a.s.l.). The study was
complemented by a lower elevation Polylepis species growing at
3700 m a.s.l (P. rugulosa Bitter). We hypothesized that
the photosynthetic and photoprotective mechanisms of
Polylepis (in particular the operation of the xanthophyll
cycle) at high-elevation ranges differ from those
previously described for other temperate mountain systems
at lower elevations, 1500 m a.s.l.
(Zarter et al. 2006)
Study site and elevation gradient
This study was carried out in two high-elevation forest
sites in the Chilean Altiplano, one of P. rugulosa at
3777 m a.s.l. located at its lower elevation margin
(18824′S and 69830′W) and the other of P. tarapacana
comprising an elevation range from 4337 to 4905 m
a.s.l. (18856′S and 69800′W). To characterize the annual
cycle of monthly precipitation, and minimum, mean
and maximum temperatures for a long-term period
(1976 – 2007), we used data from two Chilean weather
service meteorological stations. These stations were
located at 3545 and 4270 m a.s.l. and 25 and 16 km
from the P. rugulosa and P. tarapacana sites, respectively
(Fig. 1). The total annual precipitation in each forest is
182 and 322 mm, respectively.
During the course of measurements (10 – 17 h period),
from 1 March through 20 March 2013, air temperature
(Ta, 8C) and relative humidity (RH) were evaluated along
the elevation gradient using dataloggers which recorded
these measurements every 15 min (U23-001, Onset, MA,
USA) in both the P. rugulosa (3777 m a.s.l.) and the
P. tarapacana sites (4337, 4624 and 4905 m a.s.l.). Air
vapour pressure deficit (VPD) was calculated, according to
: VPD ¼ Pv 2 ((RH/100) × Pv), where air
vapour pressure (Pv) is calculated as follows: Pv ¼ 0.611 ×
exp[17.27Ta/(Ta + 237.3)]. Daily environmental course
data from P. rugulosa forests were not included in the
study due to technical troubles with the RH recording
sensor. The diurnal course of photosynthetic photon flux
density (PPFD) was characterized at both sites with
microstation dataloggers recording every 10 s (H21-002
connected to two S-LIA-M003 PAR sensors, Onset, MA, USA).
Gas exchange, Chl a fluorescence and optical properties
Measurements were done in March 2013, which
corresponds to the beginning of the last third of the growing
season for both sites. Measurements of daily course of
gas exchange were performed at mid-elevation (4624 m
a.s.l.) in the P. tarapacana site, during 3 clear days.
North-facing and sun-exposed leaves from the upper
third of the canopy were measured in five different
trees per day. Current season fully expanded leaves were
clamped into the cuvette of an IRGA Li-6400XT with
an integrated fluorescence chamber head Li-6400-40
(Li-Cor, Inc., Lincoln, NE, USA) for simultaneous
measurements of gas exchange and Chl a fluorescence. The
environmental conditions in the leaf chamber were set to be
the same as the ambient conditions throughout the
course of the day, and the ambient CO2 concentration
(Ca) was set at 400 mmol mol – 1 air. Relative humidity
within the leaf chamber was equilibrated with the current
outside air RH every hour. The flow rate was adjusted to
300 mmol air min – 1 to ensure that CO2 differentials
between the reference and the sample IRGAs were
.5 mmol mol – 1 air. These deltas were achieved in all
cases at 400 mmol CO2 mol – 1 air. Daily course levels of
PPFD were reproduced in the chamber using the ‘track
ambient function’ of the Li-6400XT LED source with a
90 % red and 10 % blue light. From steady-state
measurements at an ambient CO2 concentration (Ca) of
400 mmol mol – 1 and at natural environmental
conditions, the net CO2 assimilation rate (AN), leaf conductance
to water (gl) and sub-stomatal CO2 concentration (Ci)
were recorded. When the leaf did not cover the entire
leaf cuvette surface (2 cm2), a digital photograph of the
leaf was taken immediately after its measurement
using an equal-sized foam gasket located in the same
measured area as previously marked; ImageJ software
(Wayne Rasband/NIH, Bethesda, MD, USA) was used to
estimate the actual leaf area. Gas exchange values
given by Li-6400XT were corrected using the ratio cuvette
area/actual leaf area as a correction factor.
Dark-adapted fluorescence signals were measured in
new, fully expanded leaves from the upper third of the
plant foliage. Maximal photochemical efficiency (Fv/Fm)
was measured in all sites before 9:00 am. The efficiency
was measured 30 min after the leaves adapted to the
dark. According to the terminology of
Van Kooten and
), minimal fluorescence (Fo) was determined
by applying a weak modulated light (0.4 mmol m22 s21)
and maximal fluorescence (Fm) was induced by a short
pulse (0.8 s) of saturating light ( 8000 mmol m22 s21).
The light energy partitioning at PSII was determined
after 5 min of actinic light to obtain fluorescence
parameters under steady-state photosynthesis. Saturating
pulses were applied after steady-state photosynthesis
was reached in order to determine Fm′ and Fs. Finally,
the actinic light was turned off and immediately a 2 s
far-red (FR) pulse was applied in order to obtain F′ .
Hence, the saturated PSII effective photochemical
quantum yield [FPSII], the yield of energy dissipation by
antenna down-regulation [F(NPQ)], where NPQ refers to
non-photochemical quenching, and the constitutive
nonphotochemical energy dissipation plus fluorescence of
PSII [F(NO)] were calculated according to the lake
model shown by
Kramer et al. (2004)
. Leaf absorptance
to the Li-6400 LED light was measured using a
spectroradiometer EPP2000-HR (StellarNet, Inc., Tampa, FL, USA).
Briefly, the method consists of measuring the incident
and transmitted radiation normal to the leaf surface
above and immediately below the lamina, respectively,
and the reflected radiation 1 cm above the leaf by placing
the sensor facing the leaf at a 458 angle from the leaf
surface. The results obtained with this technique were found
to be in excellent agreement with those taken with an
(Schultz 1996; Gago et al. 2013)
After taking the gas exchange measurements, leaves
were placed in a drying oven at 60 8C until they reached
a constant weight, which was taken by estimating the leaf
mass area (LMA) as dry weight/area. Leaf nitrogen (N)
content per dry mass (LNC, g g21 DM)] was determined
in the same tissue used for LMA according to the Kjeldahl
procedure (AOAC 1980).
Photosynthetic pigments were quantified in five plants
per elevation; where three leaf discs (3.8 mm diameter)
were collected from each plant. The same leaves used
for gas exchange were also used to measure
photosynthetic pigments. Samples were collected at predawn
and midday, immediately frozen in liquid nitrogen and
stored at 280 8C. Discs ( 30 mg fresh weight) were
pulverized in a cold mortar with liquid nitrogen. To avoid acid
traces, a spatula tip of CaCO3 was added before extraction
with 1 mL of 100 % high-performance liquid
chromatography (HPLC) grade acetone at 4 8C under a PPFD of
10 mmol m22 s21. Pigments were separated and
quantified by reverse-phase HPLC
(Garc´ıa-Plazaola and Becerril
, equipped with a quaternary pump with an
automatic degasification system and an automatic injector.
Signals from a diode matrix detector were integrated
and analysed with Agilent Chem Station B.04.01 software
(Agilent Technologies, Waldbronn, Germany). The
chromatography was performed in a reverse-phase
Spherisorb ODS-1 column (5 mm particle size; 4.6 × 250 mm,
Atlantil Hilit, Waters, Ireland) and a Nova-Pack C-18
guard column (4 mm; 3.9 × 20 mm) (Waters, Ireland).
The mobile phase was binary: solvent A, acetonitrile :
methanol : Tris buffer (0.1 M, pH 8.0) (84 : 2 : 14); solvent
B, methanol : ethyl acetate (68 : 32). Pigments were
eluted using a lineal gradient of 100 % A to 100 % B
within the first 12 min, followed by isocratic elution with
100 % B during the next 6 min. Absorbance was
monitored at 445 nm. Retention times and response factors
of Chl a, Chl b, neoxanthin (Neo), lutein (Lut), b-carotene,
violaxanthin (V), anteraxanthin (A) and zeaxanthin (Z)
were determined by injecting pure standards (DHI,
Hoershholm, Denmark). The epoxidation state was
determined as (V + A)/(V + A + Z ).
A one-way analysis of variance (ANOVA) was employed to
determine significant differences in environmental data
and descriptive foliar traits (LMA, Fv/Fm, absorptance,
reflectance and leaf nitrogen content) for P. tarapacana
along their elevation gradient. Gas exchange and a
daily course of environmental data at a mid-elevation
(4624 m a.s.l.) in the P. tarapacana site were compared
between the morning and the afternoon (10 – 13 vs.
14 – 17 h periods).
A two-way multivariate analysis of variance on all
xanthophylls and carotenes was used to evaluate
differences between elevation and daily changes in pigment
composition (predawn/noon). Next, we used a univariate
ANOVA (following the same pattern as previously
described, two-way with elevation and daily changes
as fixed effects). A least-square difference (LSD) test
(P , 0.05) was used to carry out post hoc analyses.
Normality and homogeneity of variance were evaluated
using the Shapiro – Wilk (P , 0.05) and Levene (P , 0.05)
tests, respectively. When appropriate, variables were
transformed to follow the former assumptions
. All the statistical analyses were performed
with Statistica V.7 Software (Statsoft, Tulsa, OK, USA).
The annual variations in the climatic parameters at two
nearby meteorological stations (located at 3545 and
4270 m a.s.l.) for which a long data record is available
are shown in Fig. 1. Annual changes in rainfall and
temperatures correspond to the typical high-elevation
subtropical climate, characterized by small thermal
seasonality; precipitation concentrated in the summer
months and night frosts occurring year-round (Fig. 1).
When comparing both stations, higher elevation in this
particular tropical climate corresponds with higher
precipitation, along with much lower mean
temperatures. At higher elevations, thermal amplitude also
increases, reaching more than 30 8C in the winter at the
upper station. Meteorological data collected during the
gas-exchange measurement period along the elevation
gradient (Fig. 2) were consistent with these long-term
measurements. Thus, the mean air temperature was
14 % lower at the higher elevation than the average
of the lower elevations (P ¼ 0.067). In contrast with
temperature, the mean VPD decreased by 19 % at
the higher elevation (P , 0.001). Thermal amplitude
during the measurement period was remarkably 64 %
higher in the P. tarapacana site than in the P. rugulosa
site (P , 0.001). In the P. tarapacana site, the
temperature oscillated during the study period between 18 and
9 8C. Conversely to temperature, the amplitude of VPD
oscillations was 52 % higher in the lower P. tarapacana
forest than the average of the higher elevations (P ¼ 0.024).
To characterize how P. tarapacana leaves counteract
environmental stresses associated with elevation, several
descriptive foliar traits were measured in this study
(LMA, Fv/Fm, absorptance, reflectance and leaf N content)
along the elevation gradient (Table 1). However, all of
them were remarkably constant in P. tarapacana along
the elevation gradient, and no elevation-dependent
trend was observed in any of the foliar traits studied
(lower P ¼ 0.189). Although Fv/Fm did show statistical
differences (P ¼ 0.005), the biological meaning of this
variation is negligible. Irrespective of the elevation,
P. tarapacana leaves were thick, highly absorptive and
Fv/Fm was slightly lower than 0.8.
To gain information about the photosynthetic
performance of P. tarapacana, daily cycles of gas exchange
were studied at the mid-elevation (4624 m a.s.l.) of the
P. tarapacana site (Fig. 3). Two periods of activity were
clearly separated (10 – 13 vs. 14 – 17 h periods): morning
when low VPD favoured a 99 and 83 % higher stomatal
opening and carbon assimilation, respectively (Fig. 3A,
higher P , 0.001) and afternoon, when a 24 % higher
VPD (Fig. 3D; P , 0.001) caused stomatal closure
that led to a drop in AN. This trend was confirmed by
the light energy partitioning at PSII (Fig. 3D), which
showed a progressive decrease in photochemistry
throughout the course of the day, reaching a 19 %
lower FPSII in the afternoon (FPSII; P , 0.001), and
a parallel enhancement of 10 % in the dissipative
non-photochemical heat emission (FNPQ; P , 0.001).
Electron transport rates (ETRs) calculated from these
data, from irradiance (Fig. 3B) and from measured leaf
absorptance (Table 1), were as high as 150 mmol
electrons m22 s21 during the highest irradiance period.
According to ETR/AN stoichiometry, ETR values are high
enough to supply NADPH+ and ATP for the observed rate
(Flexas et al. 2012)
To further understand the biochemical mechanisms
responsible for the adaptation of the photosynthetic
apparatus in P. tarapacana to the prevailing conditions at
such high elevations, daily changes in pigment
composition were analysed along the elevation gradient. In
P. tarapacana, no elevation-dependent trend was
observed in relation to the total chlorophyll content or the
Chl a/b ratio (Fig. 4; lower P ¼ 0.131), although the
lower elevation species P. rugulosa reached a 34 % higher
total chlorophyll content than that of P. tarapaca.
However, a tendency of the net synthesis of Chl took
place during the course of the first half of the day in
P. rugulosa (24 % enhancement; P ¼ 0.122), while a
significant reverse trend was observed in P. tarapacana
(16 % decrease; P ¼ 0.022). As is shown by the relative
changes in Chl a/b, these daily changes in the Chl content
appear to be due to de novo synthesis of both Chl a and
Chl b in P. rugulosa (Chl a/b did not change; P ¼ 0.713),
which occurred throughout the morning hours. On
the other hand, the relative changes in Chl a/b in
P. tarapacana seemed to be due to the degradation of
Chl b (Chl a/b increased in 9 %; P ¼ 0.001). When
carotenoid composition was analysed in P. tarapacana, a different
response was observed in carotenes and xanthophylls
(Fig. 5). Thus, the b-carotene content did not change in
response to elevation (P ¼ 0.233). In contrast, the
xanthophyll composition differed between elevations; for
example, the highest elevation site showed the lowest
Neo and highest Lut contents (higher P ¼ 0.025). The
Lut content was 14 % higher in P. tarapacana than in
P. rugulosa, whereas P. tarapaca contained a 15 % smaller
VAZ pool. When the carotenoid composition was
compared between predawn and noon, no changes were
observed in P. rugulosa (lower P ¼ 0.282), while substantial
variations were observed in P. tarapacana. Basically, a
16 % decrease in Neo and a 32 % enhancement in VAZ
occurred (higher P ¼ 0.002). These patterns were more
marked in the sites at higher elevations. Apart from
the higher synthesis of VAZ pigments in the two
more elevated sites (4624 and 4905 m a.s.l.; higher
P , 0.001), a concomitant 37 % higher de-epoxidation
index [(A + Z)/(V + A + Z )] was observed at noon (Fig. 6;
P , 0.001). Interestingly, this was accompanied by a
significant overnight retention of de-epoxidized xanthophylls
(P ¼ 0.010), evidenced by the 52 % higher predawn values
of (A + Z)/(V + A + Z) in these two more elevated sites.
Since P. tarapacana is an evergreen tree, its leaves have
to be able to not only tolerate freezing temperatures
and dry conditions throughout the year, but also must
conclude the annual cycle with a positive carbon balance.
In addition, during the relatively short growing season
(November – April)
(Sol´ız et al. 2009)
, photosynthesis is
constrained by the high midday and afternoon VPDs in
addition to the low night temperatures. All of these
environmental limitations imply that P. tarapacana’s leaves
must take advantage of this short gap of semi-favourable
conditions in order to maximize carbon gain. For example,
during the growing season, photosynthesis is able to
proceed at temperatures close to zero (Fig. 3B) due to,
among other factors, the accumulation of compatible
solutes (proline and sugars) which increases its
supercooling capacity, preventing the freezing of leaves
et al. 2001)
. High resistance to low-temperature
photoinhibition has been described in other high-elevation plants
(Germino and Smith 2000)
, and probably relies on a large
(Laureau et al. 2013)
, but also on
the kinetic properties of the Calvin Cycle enzymes
and Cornic 2012)
. Therefore, despite the occurrence of
extremely low night temperatures, in P. tarapacana the
potential for carbon assimilation is maintained high,
reaching maximum rates close to 15 mmol m22 s21
(Fig. 3A), a value which is in the upper-part of the range
of photosynthetic capacity in high mountain trees and
fits better with the high values reported for alpine herbs
(Ko¨ rner 2003)
or cushion plants like the sympatric
(Kleier and Rundel 2009)
. This agrees
with other authors
(Hoch and Ko¨ rner 2005; Young and
Leo´ n 2007)
who found no evidence of carbon gain
limitation along the highest treeline of P. tarapacana. Lower
diurnal mean values of CO2 assimilation for the same
species were found in the Sajama volcano (Garc´ıa-Nu´ n˜ ez
et al. 2004; Azo´ car et al. 2007). This site is located 90 km
north of the P. tarapacana site, and has the opposite
exposition; its slope faces south which makes it a shadier
and colder environment. The geographical location and
topographical differences between these two sites were
found to be consistent with the 39 and 40 % lowest
maximum PPFD and air temperature reported in both of the
previous studies and this study. Further argumentation
regarding differences in gas-exchange values is restricted
by the lack of methodological information in
Garc´ıaNu´ n˜ ez et al. (2004) and Azo´ car et al. (2007).
Another remarkable foliar property observed in this
study was the null phenotypic plasticity of P. tarapacana
leaves in response to elevation, evidenced by the lack
of response when several foliar attributes (LMA, foliar N,
Fv/Fm, PPFD reflectance) were compared along the
elevation gradient (Table 1). This contrasts with the general
pattern described in other high mountain species in which the
same parameters (LMA, leaf N, reflectance) responded
differently (positively or negatively) to the elevation gradient
(Meinzer et al. 1985; Filella and Pe n˜uelas 1999; Taguchi and
. The low responsiveness of P. tarapacana’s
foliar traits to elevation gradients has been previously noted
(Gonza´ lez et al. 2002), although its biochemical
composition has shown to be more responsive to these gradients
(Gonza´ lez et al. 2007)
. The importance of genetically
determined traits in the acclimation capacity of the genus
Polylepis has recently been addressed by
Toivonen et al. (2014)
in a common garden experiment. This study showed that,
in this genus, some important leaf functional traits have
been strongly selected during evolution, restricting their
Throughout the day, when high VPD forces stomatal
closure in P. tarapacana, high solar radiation increases
the energy excess and photochemistry is not enough to
channel all of the reducing power; the re-establishment
of photostasis requires the activation of alternative
mechanisms able to reduce the effective absorptive
cross-section of the antennae. These mechanisms are
basically two: an up-regulation of NPQ and a reduction
of antenna size. To evaluate the relative contribution of
both mechanisms, daily changes in pigment composition
were studied along the elevation gradient.
In agreement with the foliar traits shown in Table 1,
the chlorophyll content was constant in P. tarapacana,
regardless of elevation (Fig. 4A). The absence of
elevation-related changes in P. tarapacana’s chlorophyll
content was previously reported by Gonza´ lez et al.
(2007) who interpreted it as an adaptive mechanism
that might allow this species to maintain its
photosynthetic capacity along an elevation gradient. However, in
the present study, the comparison between predawn
and noon values, together with the more accurate
analysis of photosynthetic pigments by HPLC, has allowed
us to move a few steps forward in this interpretation.
First, considering only the daily variations, a marked
decrease in Chl b and Neo took place at the two higher
elevations (Figs 4B and 5A). The same pattern of midday
decreases in Neo has been reported in other treeline
species, such as Pinus canariensis
(Tausz et al. 2001)
Both pigments are mostly bound to the PSI and PSII
antenna proteins. Specifically, most of the Neo pool is
bound in the N1 site of the lhcb1-3 proteins
2008; Morosinotto and Bassi 2012)
, which constitute the
outer trimeric light harvesting complexes of photosystem
II (LHCII). Therefore, Neo molecules play multiple roles at
structural, light-harvesting and photoprotective levels
(Dall’Osto et al. 2007)
. Thus, the decrease in Neo can be
plausibly explained as a result of a down-regulation of
antenna size during the first part of the day. This is
consistent with the fact that most of the flexibility of the
light-harvesting apparatus relies on the synthesis and
degradation of trimeric LHCII, while the stoichiometry
of minor LHCII antenna and of LHCI to their respective
reaction centres is maintained stable (Ballottari et al.
2007). In agreement with this hypothesis, no daily
changes in Neo were observed in the lower elevation
species P. rugulosa.
Concomitant with the decrease in the effective
antenna cross section, an elevation-dependent de novo
synthesis of the VAZ pool occurred, reaching 40 % at the site
with the highest elevation (Fig. 5D). Increases throughout
the course of the day in the content of VAZ pigments have
been described in other high mountain plants, such as
(Streb et al. 1997)
. Since there is no
evidence of the additional formation of new antenna
proteins or the replacement of other xanthophyll (Lut) by
newly synthesized VAZ pigments, it is likely that most of
these new VAZ molecules remained unbound in the
thylakoid. The existence of such a pool of free xanthophylls and
its antioxidant effects have been recently demonstrated
(Dall’Osto et al. 2010; Havaux et al. 2007)
and most likely
contribute to reinforcing the antioxidant defences of
P. tarapacana in the upper limit of its distribution.
Interestingly, changes in the VAZ pool were not matched by
lutein, which is also involved in photoprotection (Dall’Osto
et al. 2006), suggesting that, under these extreme
conditions, the biosynthetic b-pathway that leads to the
formation of VAZ pigments prevails (Beisel et al. 2010). As
occurred with Neo, no daily changes in VAZ pigments or
Lut occurred in P. rugulosa, reinforcing the
photoprotective interpretation of these changes in P. tarapacana.
Nevertheless, despite all of these mechanisms, the
decrease in b-carotene, independent of elevation but
consistent throughout the day, may denote that these
mechanisms are not enough to prevent some damage
to the PSI and PSII reaction centres, as this carotenoid
is basically bound to both reaction centres (Croce and
van Amerongen 2011).
As another signal of stress, midday de-epoxidation of
the VAZ pool, expressed as the (A + Z )/(V + A + Z ) ratio,
was also elevation dependent (Fig. 6), with the lowest
value found in P. rugulosa. The parallel increase in energy
allocated to thermal dissipation (Fig. 3C) supports the
idea that the xanthophyll cycle’s activity is involved in
the regulation of NPQ. However, more importantly, in
the most elevated sites, 30–40 % of the VAZ components
were retained overnight in the de-epoxidated state
(A and Z ). Overnight retention of the complete VAZ pool
in the de-epoxidated form is a widely described
phenomenon in temperate woody plants exposed to cold winters
(Demmig-Adams et al. 2012) and is related to the
maintenance of a photoprotective, dissipative state which
does not require light for its activation. This mechanism
is then able to cope with the excess of energy from the
earliest hours of light, when temperatures are close to
the minimum value. In the case of P. tarapacana and
P. rugulosa, as has been observed in other Andean plants
(Bascu n˜a´ n-Godoy et al. 2010), this retention is much
lower than in the previously mentioned temperate
evergreens, in which more than 80 % of the VAZ pool
remained de-epoxidated overnight in the winter months
(Demmig-Adams et al. 2012). Considering P. tarapacana’s
high rates of photosynthesis throughout the morning, it
is unlikely that this retention plays the same dissipative
role that has been described in temperate evergreens
The only option for successful survival in such a harsh
environment for P. tarapacana trees is to take advantage of
every favourable window for carbon gain. In this sense,
contrasting with other high-elevation plants, which
show a more conservative strategy such as Lobelia
rhynchopetalum (Fetene et al. 1997), the photosynthetic
apparatus is remarkably well adapted to cope with low
temperatures, and the maximum rates of carbon
assimilation are remarkably high. The limiting factor is then
imposed by the high VPD that occurs from noon onwards.
Consequently, throughout the morning, photoprotection
relies on high photochemical activity, while the activation
of photoprotective mechanisms occurs in the afternoon,
along with stomatal closure, and the decrease of carbon
assimilation. During this period, changes in antenna
pigments such as Neo and Chl b suggest that photostasis
could be achieved by a process of antenna size
readjustment, which is complemented by de novo synthesis of a
pool of the xanthophyll cycle pigments. Some of these
xanthophylls remain overnight, but their involvement in
a state of sustained dissipation as occurs in temperate
evergreens is unlikely considering the high rates of
assimilation during the early morning. All of these
mechanisms act in coordination to reduce photodamage
and to allow the maintenance of a positive carbon
balance in P. tarapacana at the world’s highest treeline.
This strategy contrasts with that of temperate treelines,
dominated by conifers, which are characterized by the
activation of a process of sustained energy dissipation
during the cold season
Sources of Funding
This research was carried out with the aid of grants
from the Chilean Research Council (FONDECYT 1120965
and FONDAP 15110009) awarded to D.A.C., and BFU
2010-15021 from the Spanish Ministry of Economy
and Competitiveness (MINECO) and the European
Regional Development Fund ERDF(FEDER) and the Basque
Government (UPV/EHU-GV IT-299-07) awarded to J.I.G.-P.
Contributions by the Authors
J.I.G.-P. was involved in research design and manuscript
preparation. R.R. carried out data processing and field
measurements. D.A.C. collected samples and
participated in manuscript preparation. R.E.C. was involved in
research design and sample collection, and participated
in manuscript preparation.
Conflict of Interest Statement
The authors thank Leo´ n A. Bravo for photosynthetic
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