Physiologia Plantarum 144: 289–301.

2012 Copyright © Physiologia Plantarum 2011, ISSN 0031-9317

Patterns of spatio-temporal distribution of winter chronic

photoinhibition in leaves of three evergreen Mediterranean
species with contrasting acclimation responses
Marı́a Carolina Silva-Cancino∗ , Raquel Esteban, Unai Artetxe and José Ignacio Garcı́a Plazaola

Department of Plant Biology and Ecology, University of Basque Country (UPV/EHU), Apdo. 644, E-48080 Bilbao, Spain

Correspondence High irradiance and relatively low temperature, which characterize

*Corresponding author, Mediterranean winters, cause chilling stress in plants. Downregulation of
e-mail: [email protected]
photosynthetic efficiency is a mechanism that allows plants to survive
Received 23 September 2011; these conditions. This study aims to address whether this process shows
revised 11 April 2011 a regular spatial pattern across leaf surface or not. Three species (Buxus
sempervirens, Cistus albidus and Arctostaphylos uva-ursi ) with contrasting
doi:10.1111/j.1399-3054.2011.01556.x responses to winter stress were studied. During 7 days, macro and micro
Fv/Fm spatial patterns were monitored by the use of chlorophyll fluorescence
imaging techniques. In the field, the strongest photoinhibition was found
in B. sempervirens, while there was almost no chronic photoinhibition in
C. albidus. In leaves of the first species, Fv/Fm decreased from base to tip while
in C. albidus it was uniform over the leaf lamina. An intermediate behavior is
shown by A. uva-ursi leaves. Spatial heterogeneity distribution of Fv/Fm was
found inside the leaves, resulting in greater Fv/Fm values in the inner layers
than in the outer ones. Neither xanthophyll-linked downregulation of Fv/Fm
nor protein remobilization were the reasons for such spatial patterns since
pigment composition and nitrogen content did not reveal tip-base differences.
During recovery from winter, photoinhibition changes occurred in Fv/Fm,
pigments and chloroplast ultrastructure. This work shows for the first time
that irrespective of physiological mechanisms responsible for development of
winter photoinhibition, there is an acclimation response with strong spatio-
temporal variability at leaf level in some species. This observation should be
taken into account when modeling or scaling up photosynthetic responses.

Introduction freezing stress by the ice formation within the tissues

(Guy 1990). Photochilling stress takes place mainly dur-
There are two main symptoms that indicate the affec-
ing bright and cold days (Wise and Naylor 1987), when
tions produced by low temperatures in plants: chilling
a large amount of light energy is trapped by chlorophylls
stress by the loss of metabolic activity (McKersie and
but cannot be safely dissipated through photosynthetic
Desker 1994) which under high light conditions occurs
processes. During these periods of stress and when-
as overexcitation of the photosynthetic apparatus (pho-
ever carbon assimilation is limited, there is a decrease
tochilling) (Garcı́a-Plazaola et al. 1999a, 1999b); and

Abbreviations – β-Car, β-carotene; A, antheraxanthin; Anx, anhydroeschscholtzxanthin; CCD, charge coupled device; Chl
a/b, chlorophyll a/b ratio; Fm, maximal fluorescence in dark-adapted samples; Fo, minimum fluorescence in dark-adapted
samples; Fv, variable fluorescence; Fv/Fm, maximal quantum yield of PSII; L, lutein; PAM, pulse amplitude-modulated;
PSII, photosystem II; ROS, reactive oxygen species; V, violaxanthin; VAZ, violaxanthin + antheraxanthin + zeaxanthin; Z,
zeaxanthin.

Physiol. Plant. 144, 2012 289

of photosynthetic efficiency (Huner et al. 1993), and the distribution of photosynthetic pigments or increased
this excess of energy can lead to the formation of red pigmentation (Hormaetxe et al. 2004) or they can
reactive oxygen species (ROS) in the photosynthetic be non-conspicuous and related to the spatial pattern
apparatus, such as singlet oxygen, superoxide radicals, of carbon gain (Nicotra et al. 2003). The techniques
hydroxyl radicals and hydrogen peroxide, that rapidly of the chlorophyll fluorescence imaging system enable
damage living tissues (Minkov et al. 1999). Photopro- the visualization of spatial and temporal variations in
tection mechanisms are induced to counteract these the photosynthetic capacity over the individual leaves in
harmful effects. They basically consist of the avoidance response to chilling (Gray et al. 2003) based on the natu-
of light capture, the downregulation of the efficiency of ral autofluorescence properties of chlorophyll (Schreiber
light energy conversion (or photoinhibition) and the acti- 1983). In this sense, the aim of this study is to char-
vation of antioxidant mechanisms. The first is achieved acterize the spatio-temporal patterns of recovery from
by a set of morphological changes, the second seems to winter photoinhibition in leaves of three coexisting ever-
be in connection with the operation of the xanthophylls green species with contrasting strategies to overcome
cycles and modifications in the reaction centers and the winter stress: Arctostaphylos uva-ursi Spreng, Buxus
third includes a set of enzymatic and non-enzymatic sempervirens and Cistus albidus. The first represents
reactions that directly detoxify ROS. a low-creeping woody evergreen shrub with a circum-
The term photoinhibition has been used to describe boreal distribution for which the study site represents its
either a conversion of photosystem II (PSII) to a photo- southern limit of distribution range; the second repre-
chemically inactive state or a degradation of PSII cores sents a highly stress-resistant Mediterranean species with
or both (Demmig-Adams and Adams 2006). Photoin- high contents of photoprotective compounds (Garcı́a-
hibition can be classified as chronic, which represents Plazaola et al. 2003) and the last one represents a
a sustained decrease of maximal photochemical effi- semi-deciduous species with photoprotective morpho-
ciency (Fv/Fm) without a complete overnight recovery logical characteristics such as leaf pubescence (Oliveria
that occurs during periods of prolonged stress, and as and Peñuelas 2000), but no induction of photoprotec-
a dynamic that corresponds to a fully reversible diurnal tive compounds (Garcı́a-Plazaola et al. 2003). To our
decline in Fv/Fm (Werner et al. 2002). The first is due knowledge, there is no information on how chilling
to a mix of photoprotection and photodamage (Müller acclimation under high light develops over the whole
et al. 2001) which corresponds to the maintenance of area of the leaf. Therefore, the use of chlorophyll fluores-
PSI II in primed state for thermal energy dissipation cence imaging techniques for patterning photoinhibition
(Adams et al. 2001). A common example of chronic at cell level and the whole leaf will help to resolve
downregulation of photosynthesis is overwintering ever- these gaps, in contrast, with standard fluorimeters that
green strategies, which maximizes the carbon gain or measure at a single small point of the leaf surface.
nutrient efficiency over a long term by using their leaves
for more than 1 year (Öquist and Huner 2003). This Materials and methods
strategy is energetically less costly, but there is a huge
Plant material, study sites and sampling design
risk of energy imbalance during stress periods since con-
tinued light absorption leads to an overexcitation of the The study site is a southern slope of Sierra de Cantabria
photosynthesis apparatus. located in Alava, Northern Spain (latitude 42◦ 36 , lon-
Adaptive mechanisms for winter acclimation have gitude 2◦ 28 and altitude 993 m above sea level).
been established in a wide range of ecosystems, among Vegetation is dominated by holm oak (Quercus ilex )
which Mediterranean ecosystems with two stress periods that coexists with other evergreen shrubs including the
(summer and winter) represent some of the most deeply three species in which this study is focused: A. uva-ursi,
studied (Larcher 2000). Indeed, these mechanisms play B. sempervirens and C. albidus. Samples were collected
a key role in plant fitness since they illustrate the fact immediately before (December 4, 2009), in the middle
that winter stress seems to be the main factor limiting the (January 25, 2010) and then immediately after (April
natural geographic distribution of many Mediterranean 7, 2010) winter season with mean temperatures dur-
plant species northwards and in altitude (Treitach et al. ing sampling days of 5.5, −2.3 and 2.6◦ C, respectively.
1997), and that winter photoinhibition may reflect Three different individuals of each species were used for
an adaptive photoprotection of the photosynthesis these studies.
apparatus (Demmig-Adams and Adams 2006). Small twigs from the outer part of the crown (sun-
Environmental factors that impose stress on plants exposed) were collected, placed in a loosely sealed
are likely to have spatially variable responses that can container with a wet paper towel and transported to the
either be conspicuous and associated with changes in laboratory where they were kept in a hydroponic solution

290 Physiol. Plant. 144, 2012

under controlled conditions: a photoperiod of 16/8 h high-sensitivity charge coupled device (CCD) camera
(day/night), temperature 24/20◦ C, respectively; 60% of equipped with an F4.5–10 mm, 1/1.6 that produced
the relative humidity and a photosynthetic photon flux images in a 12-bit scale. The opening interval of the
density 10 μmol m−2 s−1 at leaf level provided by white electronic shutter was limited to 20 μs. Images of Fo
fluorescent tubes. Previous experiments showed that and Fm were obtained using the CCD camera sensi-
these conditions were adequate to provide physiological tivity settings from 60 to 70%, which was adjusted for
recovery from winter photoinhibition (Hormaetxe et al. the acquisition of optimal signal based on the photo-
2004). The recovery process was followed at 1, 3 and synthetic state of the plant material. The CCD camera
7 days after field collection. This design allowed us to captures chlorophyll fluorescence images as a func-
evaluate the extent of the chronic photoinhibition on tion of time under different light sources and irradiance
the basis of the sustained decrease in maximal pho- (Nedbal et al. 2000). The other series of images were
tochemical efficiency (Fv/Fm) (Werner et al. 2002). All produced with an epifluorescence microscope from a
measurements were made on healthy and fully expanded small portion of three hand-cut sections across the leaf
leaves which were detached for measurements. corresponding to tip, middle and base of the lamina.
For biochemical analyses, tip and base leaf discs of Slides were prepared under low-intensity illumination,
B. sempervirens (ø = 3 mm) were collected from five placed over wet-white paper to avoid sample desicca-
different individual leaves at every evaluated recovery tion, and maintained in the dark until examination.
day and immediately frozen in liquid nitrogen and stored Measurements were made with a microscopy pulse
at −80◦ C. Finally, for cell structure analysis, leaf tissue amplitude-modulated (PAM) fluorimeter (WALZ, Effel-
samples from sun individuals of B. sempervirens with trich, Germany), coupled to an Axiostar plus microscope
recovery timing of 1, 3 and 15 days after field collection (Carl Zeiss, Göttingen, Germany) with WINCONTROL pro-
were harvested and immediately fixed. gram for image processing software used to control
the timing, settings and trigger signals for the saturat-
Chlorophyll fluorescence measurements ing pulse light resources and special detector-ocular
Initial (Fo) and maximal fluorescence (Fm) were mea- microscope. The epi-illuminator features contain a sin-
sured in dark-adapted leaves (30 min). The maximal gle blue MICRO-head Luxeon LED (Walz, Effeltrich,
photochemical efficiency of PSII was estimated by the Germany) that measures light source with a peak emis-
ratio Fv/Fm = (Fm − Fo)/Fm. This measurement can be sion at 470 nm, a short-pass filter, a diachronic beam
considered as inversely proportional to the degree of splitter and collimators optic, which provide pulse-
‘chronic photoinhibition’ (Werner et al. 2002), consid- modulated measuring light and actinic and saturation
ering 0.83 as the optimal value. The saturating pulse in pulse illumination integrated with a CCD Imag-K4 cam-
each system was required to reduce the plastoquinone era (1392 × 1040 pixel) (Walz, Effeltrich, Germany) and
pool and to obtain the Fm value that was measured a Zeiss fluor objective 20×/0.75. Fluorescence was
during the pulse. detected by the photomultiplier attached to the photo
Fluorescence measurements of leaves were made with port of the Axiostar plus microscope.
four different systems in order to obtain a complete image To process the captured images, false color images
of the extent of photoinhibition. In the first system, a field, of leaf chlorophyll fluorescence yield (Fv/Fm) were
upper and south-facing leaves were covered and kept established based on the assumption that pixel intensity
in dark for 30 min before using the measurements and values can be related to the physiological process (Aldea
a portable battery-powered fluorimeter FluorPen FP 100 et al. 2006). Data analysis was made integrating Fv/Fm
(PSI, Brno, Czech Republic) for chlorophyll fluorescence results over the surface of the whole leaf area and leaf
measurements in situ. In the second one, a laboratory, a area sliced with the intention of extracting the most
portable modulated fluorimeter OS 5-FL (Optisciences, relevant information: a circular area (6 mm diameter)
Tyngsboro, MA) was used. was drawn at the tip, central, lateral and base of
The third and fourth ones correspond to the two the Fv/Fm whole leaf picture and three transect lines
imaging systems measuring both adaxial and abax- were drawn through the Fv/Fm picture on the sliced
ial surfaces of the leaf and transversal sections across leaf image, establishing that the entire distance of the
it (three cuts). One series of chlorophyll fluorescence transect line corresponds to one ‘relative width’ measure.
images from the whole leaf was captured with FluorCam Photographs of the whole leaf and cuts were taken with
700MF chlorophyll fluorescence imaging system (PSI) a digital camera, Canon Power Shot SD1000 (Canon
as described in Nedbal et al. (2000) and the instrument Inc., Kawasaki, Japan), over the microscope.
was driven by FLUORCAM v. 3.5 software package for Standard and imaging systems used to obtain Fv/Fm
image processing. Fluorescence was detected by a values were strongly correlated (r2 = 0.9125; P < 0.001)

Physiol. Plant. 144, 2012 291

and the slope was close to 1. However, measuring at OsO4 ) and dehydrated with acetone. To enhance
a single and small point of the leaf surface (standard contrast, uranyl acetate (2%) was used. Later, leaf
fluorimeters) does not provide full information over the tissues were embedded in Durcupan ACM Fluka
heterogeneous situation of the whole leaf, which could resin (Sigma-Aldrich, St. Louis, MO, USA) Semi-thin
be the reason for the variation between fluorimeters. sections (3–5 μm) were sliced using Leica ULTRACUT
Additional analyses were made to B. sempervirens as UCT ultra-microtome (Leica Microsistems GmbH,
it points out the sharpest pattern in response to winter Wetzlar, Germany) stained with toluidene blue and
photoinhibition. viewed with a Zeiss Axioskop light microscope.
Pictures were captured with an Olympus OLY-220
Pigment analysis camera (Olympus Corporation, Tokyo, Japan). Ultra-thin
Photosynthetic pigments extracted from approximately sections (30–50 nm) were cut with Leica ULTRACUT
4 mg of leaf samples were homogenized in liquid N2 UCT ultra-microtome and observed with PHILIPS
with a Tissue-Tearor homogenizer (Model 395; Dremel, EM208S transmission electron microscope (Phillips,
Mexico D.F, Mexico) in pure acetone (100%) solution Eindhoven, The Netherlands) with a Morada digital
buffered with CaCO3 , centrifuged at 16000 g for 20 min, camera (Olympus Corporation, Tokyo, Japan). The
and syringe-filtered. Extracts were injected (15 ml) in a chloroplast ultrastructure was studied using the program
reverse-phase Waters (Milford, MA) HPLC system follow- called IMAGEJ (NIH, Bethesda, MD). Thirty cells were
ing the method of Garcı́a-Plazaola and Becerril (1999b) analyzed per recovery time.
with the modifications described in Garcı́a-Plazaola and
Becerril (2001). The 717 plus autosampler was equipped Statistics
with a thermostat which maintains temperature constant
Calculated P -values, coefficients and regression lines are
at 4◦ C avoiding pigment degradation or alteration. Pho-
indicated on the figures; every time it is significant at
tosynthetic pigments were measured with a PDA detector
α = 0.05. In order to test differences in pigments’ con-
(Waters model 996) in the range 250–700 nm. Peaks
tent upon the days of acclimation, one-way ANOVAs were
were detected and integrated at 445 nm for carotenoid
performed considering acclimation as a fixed factor. All
and chlorophyll content. Pigments were identified and
data was tested for normality and homogeneity of vari-
quantified by the method described by Garcı́a-Plazaola
ances and square root- or log-transformed, if necessary.
and Becerril (1999b). Retention times and conversion
Student–Newman–Keul tests were used to discriminate
factors for pigments were the same as described by
among different treatments after the significant F -test and
Garcı́a-Plazaola and Becerril (1999b, 2001).
Cochran’s test to evaluate the heterogeneity of variances.
In the case of chlorophyll a/b ratio (Chl a/b), in order
Organic nitrogen quantification
to standardize variances, two data were replaced by
The concentration of organic nitrogen was determined means of the group and 2 d.f. subtracted from Residual
by using the Micro-Kjeldahl method, in which all organic (Winer et al. 1991). The ultrastructural changes upon
nitrogen converts to ammonia (NH4 + ). Leaf discs of the days of acclimation were tested using one-way
B. sempervirens (ø = 3 mm) were dried for 5 days at ANOVAs, considering acclimation as a fixed factor. All
80◦ C. Approximately 8 mg of dry material with 5 ml data were tested for normality and homogeneity of vari-
of H2 SO4 (97%) were digested in Bloc-Digest (Selecta, ances, log-transformed, if necessary, and when failed to
Abrera, Barcelona, Spain) at 420◦ C for 1 h and 30 min. meet ANOVA assumptions, it was analyzed using nonpara-
Once temperature reached 250◦ C, K2 SO4 (2 g) and metric Kruskal–Wallis and Mann–Whitney tests. Fisher’s
CuSO4 ·5H2 O (0.5 g) were added as reaction catalyst. least significant difference tests were used to discrimi-
Later, once cooled, samples were filled up until 10 ml nate among different treatments. All statistical analyses
with Milli-Q water. The ammonia concentrations were were performed using SPSS 17.0 statistical package.
measured by color gradients with Nessler reactive over
12.5 μl of sample, 190 μl of water, 50 μl of NaH and
10 μl of Nessler reactive (Merck, Dammstadt, Germany)
at 410 nm using microplates (Hertog et al. 1998). The Results
patron solution used was NH4 Cl. Winter photoinhibition in Mediterranean
evergreens
Electron microscopy analyses
During the period of winter 2009–2010, temperatures
Leaf tissues of B. sempervirens were pre-fixed with in the study site showed five episodic cold waves with
glutaraldehyde (3%), fixed with osmium tetroxide (1% mean daily temperatures falling below −5◦ C. The field

292 Physiol. Plant. 144, 2012

 A

Fig. 1. Recovery from winter photoinhibition (measured as changes

in mean Fv/Fm) of studied species during 1(A), 3(B) and 7(C) days
after field sample collection. Measurements were done in dark-adapted
individuals in early winter (A), mid-winter (B) and late winter (C). Day 0
Fig. 2. False color images of chlorophyll fluorescence yield (Fv/Fm) of
corresponds to in situ measurements. These data were obtained from
the leaves of the three species evaluated after 1, 3 and 7 recovery days
standard fluorimeters (FluorPen FP 100 and OS 5-FL). Three replicates
regarding mid-winter samples. Numbers below the images represent
from different individuals of each species were used. Error bars represent
the corresponding mean of Fv/Fm of the whole leaf. The false color code
standard error of the mean. When not present, error bars are smaller
depicted at the right of figure ranges from 0.85 (red) to 0.50 (blue).
than symbol size. Only Buxus sempervirens was measured for late-winter
These data were obtained from images captured using PSI FluorCam
as Cistus albidus and Arctostaphylos uva-ursi did not show variation on
system. The experiment was repeated three times and the same results
their response to winter conditions.
were observed in all samples.

samples were collected before (the past autumn), dur- Qualitative analysis of Fig. 2 suggests spatial patterns
ing (mid-winter) and after (early spring) the period in of winter photoinhibition across the whole leaf. Two
which cold waves occurred. In situ Fv/Fm measure- main patterns were observed: B. sempervirens in which
ments revealed that A. uva-ursi and C. albidus leaves Fv/Fm values decreased from the base to the tip and the
were not affected by environmental conditions while other was represented by C. albidus in which no differ-
in B. sempervirens, Fv/Fm values decreased during the ences were observed. Arctostaphylos uva-ursi was inter-
course of the winter season (Fig. 1). mediate between both species. After 7 days of recovery,
Branches of these species were collected and trans- base to tip differences were attenuated in all species.
ferred to warm conditions to allow the recovery from Spatial patterns were quantified by comparing Fv/Fm
winter photoinhibition and two contrasting conditions across the two leaf axes (Fig. 3), confirming that in both
were observed. Thus, regardless of how advanced the B. sempervirens and A. uva-ursi photoinhibition fol-
winter was when the samples were collected; individuals lowed a longitudinal pattern (tip to base), with no lateral
of C. albidus reached a completely recovered status after differences across the leaf observed. After the recovery
one night beneath controlled conditions, as evidenced period, no spatial differences were observed and Fv/Fm
by high Fv/Fm values. However, B. sempervirens lasted was uniformly distributed across the leaf surface in A.
seven recovery days to reach this optimal status, and uva-ursi and C. albidus, while B. sempervirens showed
the A. uva-ursi response was intermediate between both a tendency to uniform Fv/Fm values and homogenous
species (Fig. 1). These results show that chronic pho- spatial pattern.
toinhibition caused by chilling stress under high light At tissue level, Fv/Fm false color images of leaf cross-
conditions is fully reversible at different time scales for section (Fig. 4) revealed a tendency for homogenous
the three studied species. Recovery process proceeded in images on day 7, implying similar photochemical effi-
two phases: fast and acute throughout the first night, and ciency in all photosynthetic layers. As described at
slow and sustained during the following recovery period. whole leaf level, noticeable changes were found during

Physiol. Plant. 144, 2012 293

 A B C

Fig. 3. Fv/Fm average measurements of selected area (tip, central, lateral and base) of the adaxial surface from the leaf lamina using the PSI FluorCam
system in the species studied during 1(A), 3(B) and 7(C) recovery days regarding mid-winter samples. Three replicates from different individuals of
each species were used. Error bars represent standard error of the mean.

Fig. 5. Light transmission micrograph images of the leaf cross-section

of the three fresh leaves from the species evaluated after 1, 3 and 7
Fig. 4. False color images of chlorophyll fluorescence (Fv/Fm) from the recovery days regarding mid-winter samples.
leaf cross-section images of the three species evaluated after 1, 3 and 7
recovery days regarding mid-winter samples. Numbers below the images
represent the corresponding mean Fv/Fm of the whole leaf section. The
contents in the adaxial face of B. sempervirens leaf
false color code depicted at the right of figure ranges from 0.85 (purple) during recovery. When fluorescence images were used
to 0.10 (red). Notice the difference of the false color code in this figure. to construct transects across the width leaf-cut (Fig. 6),
These data were obtained from images captured using Microscopy- it was evidenced that the inner layers of the leaf were
PAM. The experiment was repeated three times and the same results
less photoinhibited than outer layers as a tendency for
were observed in all samples.
the three species evaluated. The photoinhibition of the
abaxial side (spongy mesophyll) was higher than that
recovery in B. sempervirens cross-section images, minor found in the adaxial surface (palisade mesophyll). Fv/Fm
changes in A. uva-ursi and no differences in C. albidus. values decreased unexpectedly in C. albidus on day 7,
Light transmission micrograph images of the leaf cross- probably because of the effect of prolonged acclimation
section (Fig. 5) showed a decrease in red pigments at low light.

294 Physiol. Plant. 144, 2012

 A B C

D E F

G H I

Fig. 6. As can be seen here, Fv/Fm average measurement in a transect line (relative width) placed transversely on hand-cut sample. Adaxial mesophyll
layers correspond to 0–0.1 relative distance and abaxial mesophyll layers correspond to 0.9–1 relative distance. Inner layers correspond to 0.1–0.9
relative distance. Hand-cut slices were made in the tip, middle and base of the leaf during 1 (A,B,C), 3 (D, E, F) and 7 (G,H,I) recovery days regarding
mid-winter samples, to allow comparison among species and relative width was estimated considering the maximum leaf width as one. Three
replicates from different individuals of cross-section hand-cut leaves of each species and three transect lines drawn in each cross-section leaf image
were used for each species. Error bars represent standard error of the mean. When not present, error bars are smaller than symbol size.

Re-greening and recovery from winter A

photoinhibition in B. sempervirens
To gain light into the process of recovery from
winter photoinhibition we focused our attention on B.
sempervirens, the species on which this process was
more pronounced and conspicuous. B

Frequency distribution of Fv/Fm values in cross-

sections (Fig. 7) was markedly bimodal at photoinhibited
stage (Fig. 7A). It can be accounted by the distribution of
Fv/Fm between outer and inner mesophyll layers. After
C
the recovery time, the variation of the Fv/Fm distribution
across the leaf width decreased in the way that the ‘two
peaks’ of the bimodal distribution overlapped increas-
ingly, until they finally displayed a unimodal distribution,
which represented recovery Fv/Fm values (Fig. 7B, C).
During the process of recovery, pigment composi-
tion (Fig. 8) was studied in the tip and base of the
leaf, in order to explain the photoinhibitory distribu- Fig. 7. Distributions of frequencies of Fv/Fm (%) from leaf cross-section
tion patterns described previously in B. sempervirens images from Buxus sempervirens during 1(A), 3(B)and 7(C) recovery
leaves (lower Fv/Fm values at tip and higher Fv/Fm val- days regarding mid-winter samples. These data were obtained from
ues at base regions of the leaf). During recovery, total Microscopy-PAM. Three hand-cut sliced samples were made in the
middle of the leaf of each of the three replicates from different individuals
chlorophyll (chlorophyll a + b), Chl a/b and β-carotene
of each species.
on chlorophyll basis (β-Car/chl), did not change sig-
nificantly (Fig. 8A–C). But others such as lutein (L) and disappearance of the upper red pigments on mesophyll
anhydroeschscholtzxanthin (Anx) on chlorophyll basis, cells. In parallel, the VAZ (violaxanthin + antherax-
decreased from the initial values during the recov- anthin + zeaxanthin) pool decreased and its degree
ery process (Fig. 8D, E), which can also be observed of depoxidation (A + Z/VAZ) relaxed during recovery
in the light transmission micrographs images, by the (Fig. 8F, G, respectively). Despite the clear pattern of

Physiol. Plant. 144, 2012 295

Fig. 8. Total chlorophyll (Chl a + b), chlorophyll a/b ratio (Chl a/b), ratios of carotenoids to Chl a + b: β-carotene (β-Car), lutein (L),
anhydroeschscholtzxanthin (Anx), violaxanthin+ antheraxanthin + zeaxanthin (VAZ), the conversion state of the xanthophylls cycle [(A + Z)/(V + A + Z)]
and total chlorophyll (Chl a + b) present in the tip and the base of Buxus sempervirens leaves during 1, 3 and 7 recovery days regarding mid-winter
samples. Values mean ± standard deviations of five replicates. Different letters denote statistically significant differences at P < 0.05 among treatments
after Student–Newman–Keul test.

changes during winter de-acclimation, no significant Discussion

differences were observed between tip and base in any of
Spatial patterns of winter photoinhibition
the compounds analyzed (Fig. 8). The content of organic
nitrogen (no significant differences; data not shown) was When perennial species are exposed to prolonged
stable in different parts of the leaf. environmental stress, sustained photoprotection encom-
Chloroplast ultrastructure was studied throughout passed by thermal dissipation occurs implying no relax-
15 recovery days after field collection in order to ation of non-photochemical quenching upon darkening
allow a complete restoration of photosynthetic effi- of the leaves (Demmig-Adams and Adams 2006). Indeed,
ciency. Important variation in chloroplast structure was Mediterranean species are typically evergreen and are
observed (Fig. 9). The number and size of chloroplasts exposed to relatively low temperatures in winter. Conse-
per cell depicted significant decrease (Fig. 10A, B) after quently, this exposition under periodical chilling stress
the recovery process. Plastoglobuli were conspicuously in high light conditions activates photoprotective mech-
found in the micrograph images with a significant
anisms, among which thermal dissipation plays a key
decrease in their number during recovery; however,
role (Garcı́a-Plazaola et al. 2003).
the percentage of their area in chloroplast showed a sig-
In this study, the response to winter stress of three
nificant increase (Fig. 10C, D). The percentage of starch
species with different geographical and ecological distri-
granules area per chloroplast portrayed a significant
increase during the recovery process (Fig. 10E), although bution patterns was analyzed. These species also show
a tendency of increase in the number of starch gran- different responses to cope with low-temperature stress,
ules per chloroplast was established (data not shown). as found in the field study (Fig. 1). In accordance to other
Changes in thylakoid and stroma were also observed dur- studies, B. sempervirens with Mediterranean distribution
ing the recovery process (Fig. 9) with significant decrease and an altitudinal range in this area of 500–1800 m
of the area of free stroma (Fig. 10G) as there was a above the sea level, shows high concentration of
significant increase of the area occupied by thylakoids photoprotective compounds (such as the presence of red
(Fig. 10F) and a higher degree of organization (Fig. 9). carotenoids) and Fv/Fm sustained reduction (Hormaetxe

296 Physiol. Plant. 144, 2012

 reduce light absorbance. All these traits assure high
photochemical efficiency (Fig. 1) and low levels of pho-
toprotective compounds (Garcı́a-Plazaola et al. 2003)
throughout its relatively short leaf lifespan. Overall, these
results prove that Mediterranean evergreens have diverse
photoprotective strategies to cope with winter stress and
at the same time, extremely opposite strategies can be
found cohabiting and surviving the hostile climate.
Our results also show that photoinhibition was not
uniform across leaf lamina. In fact, two main and
opposite patterns of winter photoinhibition and recovery
were established (Fig. 2). These patterns are related
to the spatial distribution of Fv/Fm across the whole
lamina corresponding specifically to the contrasting
strategies to overcome winter stress by each coexisting
studied species. The first corresponds to B. sempervirens
leaves in which Fv/Fm decreases from base to tip
(Fig. 3) and progressively recovers when transferred
to warm conditions (Fig. 1). The mentioned pattern
performs as the conspicuous changes found in the
distribution of photosynthetic pigments or increased
red pigmentation (Hormaetxe et al. 2004). Curiously,
this represents the opposite pattern found in freezing
damage described in Arabidopsis thaliana by Ehlert
and Hincha (2008). However, this pattern is somehow
similar to the one found on senescing leaves (Wingler
et al. 2004), and may occur because of the inhibition
of assimilation transport to the tip of the leaf under
chilling temperatures (Sowinski et al. 1998). It may
be due to the existence of microenvironments at leaf
regions with colder or/and higher irradiation, principally
because of the position and orientation of the leaves
Fig. 9. Transmission electron micrograph images of chloroplast ultra- in situ. The second pattern is shown in C. albidus
structure of Buxus sempervirens sun-exposed individuals after 1, 3 and leaves in which Fv/Fm values were uniform across
15 recovery days after field collection. Note plastoglobuli (P), thylakoid the leaf area (Fig. 3) and no chronic photoinhibition
(T) and stroma (S).
was observed (Fig. 1) as expected from its functional
traits (semi-deciduous species, high trichome density
et al. 2004). In the case of A. uva-ursi, has a circum- and high leaf reflectance) that prevent excessive light
boreal and high-altitude distribution (altitudinal range energy from damaging the photosynthetic apparatus
500–2100 m) and different winter acclimation strategy (Oliveira and Peñuelas 2000). An intermediate behavior
depending on environmental conditions associated with between both photoinhibitory patterns is shown by
different altitudes. In fact, while likely all populations the leaves of A. uva-ursi (Fig. 3) probably because
experience suspension of photosynthetic activity during of its photosynthetic system intrinsically capable of
winter, only the high-altitude population responds with functioning at lower temperatures, as corresponds to its
a pronounced downsizing of PSII that is reversible in boreal and high-altitude distribution. The maintenance
spring (Zarter et al. 2006). We found that A. uva-ursi of high Fv/Fm ratio under high light and cold conditions
located in its warmer distribution limit showed lower reflects the capacity to recover after dark adaptation,
chronic photoinhibition compared with B. sempervirens suggesting a high energy-quenching capacity during
(Fig. 1). The third plant, C. albidus, is a semi-deciduous natural conditions as has been described in Antarctic
species that avoids stress by facultative defoliation, by plants (Bascuñán-Godoy et al. 2010). Interestingly, the
having two leaf populations, and by morphological three species reached a completely restored state after
characteristics such as high trichome density and high 7 days under warm conditions (Fig. 1). However, we
leaf reflectance (Oliveira and Peñuelas 2000) that cannot exclude the possibility that leaves are not only

Physiol. Plant. 144, 2012 297

 A

B C

D E

F G

Fig. 10. Area of chloroplast per cell (A), number of chloroplast per cell (B), number of plastoglobuli per cell (C), percentage of plastoglobuli area per
cell (D), percentage of starch granules area per cell (E), percentage of thylakoid area per cell (F) and percentage of stroma area per cell (G) present
in Buxus sempervirens sun-exposed individuals after 1, 3 and 15 recovery days after field collection. Values area means ± standard deviations of 30
cells. Different letters denote statistically significant differences at P < 0.05 among treatments after Student–Newman–Keul test and Mann–Whitney
tests when necessary.

recovering from winter stress, they are also acclimating photoinhibition resulted from a process of long-term
to deep shade as the recovery period is lengthy and acclimation and is not the result of a short and intense
occurs under low-light intensity (10 μmol m−2 s−1 ). treatment. The decrease in Fv/Fm observed at the end
A heterogeneous pattern of winter photoinhibition was of the recovery period in C. Albidus may indicate a
not only observed across the abaxial and adaxial leaf probable affection on leaves caused by the parallel
surfaces but also among different cell layers (Fig. 4–5). acclimation to shade conditions. It must be highlighted
In general, Fv/Fm values were higher in the upper than that the fluorescence signal detected by conventional
in the lower mesophyll (Fig. 6). This may be due to fluorimeters originates mostly from the first layers of cells
the protection provided by the outer cells, or in the while the microscope PAM analyses all layers, and this
case of B. sempervirens because of the presence of may explain the discrepancies between the fluorescence
red carotenoids in the upper mesophyll which may imaging systems studied.
act as passive light filters that protect the photosynthetic
apparatus (Hormaertxe et al. 2004). This pattern does not Photoinhibition and recovery from winter
photoinhibition: a case study in B. sempervirens
fit to that described in artificial light treatments in which
there is a photoinhibition gradient from the abaxial to In order to investigate further into the pronounced
adaxial side (Oguchi et al. 2011). This discrepancy may and conspicuous winter photoinhibition and recovery
arise from the fact that in nature leaves display variable patterns found in leaves of B. sempervirens, additional
positions with respect to light and that in this work research was conducted. After 7 days of recovery under

298 Physiol. Plant. 144, 2012

warm conditions, the spatial pattern of photoinhibition apparatus in a climate that experience relatively rapid
progressively disappeared as shown by visible pigment changes (Adams and Demmig-Adams 1994). Zeaxanthin
variation (Fig. 5). It occurred in parallel with a shift is involved in flexible and sustained thermal energy dissi-
from a bimodal frequency distribution of Fv/Fm (as a pation and is constantly associated with decreases in the
result of being able to resolve the two main types of maximal photochemical efficiency (Fv/Fm) (Demmig-
photosynthetic tissues in these leaves) (Hogewoning and Adams and Adams 2006). Regarding L, it may also
Harbinson 2007) to a unimodal frequency distribution be directly involved in heat dissipation, playing an
(Fig. 7). These results agree with Hogewoning and important photoprotective role reducing the risk of pho-
Harbinson (2007) who proposed that the frequency toinhibition during winter (Dall’Osto et al. 2006, Yan
distribution of maximal photochemical efficiency could et al. 2007). Consequently, its decrease during the recov-
be considered as a stress indicator due to its highly ery days studied means a lower photoprotective demand.
sensitive identifying photoinhibition recovery. Contrasting with photoprotective pigments, no changes
To delve deeper into the biochemical basis of the spa- were observed during the recovery process from winter
tial differences in the degree of photoinhibition, tip and photoinhibition in light-harvesting chlorophyll or β-Car
base of the leaves were compared. These results showed (Fig. 8B, C, respectively).
that xanthophylls, which mediated downregulation of In agreement with previous works (Stefanowska et al.
photosynthesis (Fig. 8) or protein remobilization, were 2002, Maslova et al. 2009, Muller et al. 2009), pro-
not directly implicated in patterning as we did not find nounced and reversible modifications in the ultrastruc-
significant differences in the pigment or nitrogen con- ture of leaf cells and chloroplasts occurred during winter
tents between tip and base regions of the leaf. The fact de-acclimation. Our results comprise evidence of this
that the parameters evaluated do not respond gradually postulated hypothesis since ultrastructural changes were
to the pattern does not mean that they do not play a found when winter-acclimated and recovered leaves
role during the photoinhibition process, which has been were compared (Fig. 9), suggesting that recovery was in
widely showed (Öquist and Huner 2003). part the consequence of structural reorganization. The
In some overwintering evergreens, the decrease of main difference observed during de-acclimation was an
Fv/Fm ratio has been related to strong accumulation of increase in the proportion and organization of thylakoids
red pigments on the sunlit surface of the overwintering that filled the free stroma space (Fig. 10F, G). Accord-
leaves (Nicotra et al. 2003, Hughes 2011). In fact, the ingly, Maslova et al. (2009) showed that the number
visible pigmentation in B. sempervirens leaves was due of grana decreased during winter for high light condi-
to the red carotenoids accumulated in cell layers just tions. Moreover, Bascuñán-Godoy et al. (2010) showed
below the epidermis (Fig. 5) as described by Hormaetxe that Andean ecotype of Colobanthus quitensis (Kunth)
et al. (2004). Moreover, Nicotra et al.’s (2003) data Bartl., which grows under cold temperatures and bright
indicated that differentiation in the degree of redness days, is able to decrease the probability of light capture
increased with exposure to direct light. developing smaller chloroplasts with a lower number of
Recovery from winter photoinhibition was character- stacked grana and stacked grana area per chloroplast.
ized by a conspicuous re-greening of B. sempervirens Furthermore, we expected a significant decrease of the
leaves, which occurred in parallel with attenuation of area of plastoglobuli per chloroplast, since higher size is
the pools of photoprotective pigments such as Anx, L interpreted as a stress acclimation or damage symptom
and VAZ (Fig. 8D–G, respectively). Anx corresponds (Bascuñán-Godoy et al. 2010) and these structures are
to the red carotenoids accumulated below the epider- the site of Anx accumulation (Hormaetxe, unpublished
mis already mentioned (Hormaetxe et al. 2004), so their data). However, our results contrast to the previous
decrease denotes re-greening and recovery from pho- assumption as there is an increase in the percentage of
toinhibition. Our results are congruent to Gray et al. the area of plastoglobuli in the chloroplast (Fig. 10D).
(2003) as they found epoxidation of Z in the xantho-
phyll cycle during the recovery process from winter
Concluding remarks
photoinhibition. Indeed, they also found two phases of
recovery: one fast and acute attributed to the relaxation For the first time we have shown that the process of win-
of non-photochemical quenching (Fig. 1) and the other ter photoinhibition is not uniform across leaf surfaces and
phase slow and sustained, related to the degradation depth profiles. Furthermore, we have shown the exis-
and re-synthesis of the D1 protein. The predawn conver- tence of contrasting spatial patterns in different species,
sion state of the xanthophylls cycle responded rapidly which reflect different photoprotective strategies in coex-
to day-to-day variation and such flexibility is important isting Mediterranean shrubs. Among the studied species,
in the regulation and protection of the photosynthetic B. sempervirens showed the most noticeable winter

Physiol. Plant. 144, 2012 299

photoinhibition and recovery. Important biochemical by in situ chlorophyll fluorescence and comparison with
and structural modifications seem to be the basis of drought stress. Env Exp Bot 60: 504–514
such responses, which are conspicuous in this species Dall‘Osto L, Cazzaniga S, North H, Marion-Poll A, Bassi R
because of the parallel accumulation of red carotenoids (2006) Lutein is needed for efficient chlorophyll triplet
in the outer cell layers. The spatio-temporal pattern quenching in the major LHCII antenna complex of
in the induction of winter photoinhibition described higher plants and effective photoprotection in vivo
here adds knowledge to previous studies which have under strong light. BMC Plant Biol 6: 32 doi:
found in-homogeneities of photosynthetic responses in 10.1186/1471-2229-6-32
relation to environmental stresses or physiological pro- Demmig-Adams B, Adams WW (2006) Photoprotection in
cesses such as leaf senescence (Wingler et al. 2004), an ecological context: the remarkable complexity of
thermal energy dissipation. New Phytol 172: 11–21
freezing stress (Nicotra et al. 2003, Ehlert and Hincha
Ehlert B, Hincha DK (2008) Chlorophyll fluorescence
2008, Gorsuch et al. 2010), heat stress (Petkova et al.
imaging accurately quantifies freezing damage and cold
2007), illumination responses (Lichtenthaler et al. 2007)
acclimation responses in Arabidopsis leaves. Plant
or pathogens (Christen et al. 2007). Taken together, all
Methods 4: 12 doi: 10.1186/1746-4811-4-12
these results show that the leaf cannot be treated as a
Garcı́a-Plazaola JI, Becerril JM (2001) Seasonal changes in
uniform photosynthetic system, and a deeper characteri-
photosynthetic pigments and antioxidants in beech
zation of photosynthetic responses is needed to perform (Fagus sylvatica) in a Mediterranean climate:
an accurate modeling of photosynthesis. implications for tree decline diagnosis. Aust J Plant
Physiol 28: 225–232
Acknowledgements – We would like to thank Beatriz Garcı́a-Plazaola JI, Artetxe U, Becerril JM (1999a) Diurnal
Fernández-Marin for her help during the sample collec- changes in antioxidant and carotenoid composition in
tion. Technical and human support provided by SGIker the Mediterranean schlerophyll tree Quercus ilex
(UPV/EHU, MICINN, GV/EJ, ESF) is gratefully acknowl- (L) during winter. Plant Sci 143: 125–133
edged. M. C. S. -C. received a fellowship from Fundación Garcı́a-Plazaola JI, Artetxe U, Duñabeitia MK, Becerril JM
Carolina and a master’s degree from UPV/EHU. This (1999b) Role of photoprotective systems of holm-oak
research was supported by research BFU 2007-62637 (Quercus ilex ) in the adaptation to winter conditions.
and BFU 2010-15021 from the Ministry of Education J Plant Physiol 155: 625–630
and Science of Spain and research project UPV/EHU-GV Garcı́a-Plazaola JI, Olano JM, Hernández A, Becerril JM
IT-299-07. We are very grateful to Jose Herazo for linguistic (2003) Photoprotection in evergreen Mediterranean
consultation of the manuscript. plants during sudden periods of intense cold weather.
Trees 17: 285–291
Gray GR, Hope BJ, Qin X, Taylor BG, Whitehead CL
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Edited by T. Vogelmann

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