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Diversity of winter photoinhibitory responses: A case study in co-occurring lichens, mosses,
herbs and woody plants from subalpine environments.
Corresponding author: Míguez Fátima (fatimamiguezcano@gmail.com)
Department of Plant Biology and Ecology. University of Basque country (UPV/EHU) Apdo 644.
48080 Bilbao, Spain
Fernández-Marín Beatriz (beatriz.fernandezm@ehu.eus)
Institute of Botany and Alpine Space Innsbruck. University of Innsbruck. Sternwartestrasse 15. 6020
Innsbruck, Austria
Becerril José Maria (josemaria.becerril@ehu.eus)
Department of Plant Biology and Ecology. University of Basque country (UPV/EHU) Apdo 644.
48080 Bilbao, Spain
García-Plazaola José Ignacio (joseignacio.garcia@ehu.eus)
Department of Plant Biology and Ecology. University of Basque country (UPV/EHU) Apdo 644.
48080 Bilbao, Spain
This is the pre-peer reviewed version of the following article: Míguez, F. , Fernández‐Marín,
B. , Becerril, J. and García‐Plazaola, J. (2017), Diversity of winter photoinhibitory
responses: a case study in co‐occurring lichens, mosses, herbs and woody plants from
subalpine environments. Physiol Plantarum, 160: 282-296. doi:10.1111/ppl.12551, which
has been published in final form at https://doi.org/10.1111/ppl.12551. This article may be
used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use
of Self-Archived Versions.
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ABSTRACT (<250 words)
Evergreens living in high mountainous areas have to cope with hard conditions in winter as there is a
combination of high light and low temperatures. Under this situation, there is an imbalance between
light collection and energy use. As a response evergreens activate a photoprotective process which
consists on the down-regulation of photosynthetic efficiency, referred to as winter photoinhibition
(WPI). WPI has been classified in dynamic and chronic depending on the rapid (<14 h) or slower (>14
h) recovery kinetics. With the aim of characterize whether WPI is a general trait in evergreen high
elevation photosynthetic organisms and its dependence to the deepoxidation state of xanthophyll
cycle, WPI was analyzed in the field in 50 species including woody species, herbs, lichens and
mosses. Recovery kinetics were studied in detail in one model species from each group. Results show
that high levels of WPI are much more frequent (but not exclusive) among woody plants than in any
other group. Changes in AZ/VAZ were not related to the activation/deactivation of WPI in the field
and do not follow changes in photochemical efficiency during recovery treatments. Thylakoid proteins
seasonal changes differ among different functional groups. The obtained results highlight the diversity
of physiological solutions to winter stress.
ABBREVIATIONS
ΔpH: Transthylakoid proton gradient; A: Antheraxanthin; AZ/VAZ: De-epoxidation degree of
xanthophylls cycle pigments (antheraxanthin+zeaxanthin)/(violaxanthin+antheraxanthin+zeaxanthin);
Chl: Chlorophyll; F
m
: Maximum chlorophyll fluorescence; F
0
: Minimum chlorophyll fluorescence;
F
v
/F
m
: Maximum quantum yield of PSII; L: Lutein; Lhc: Light harvesting complex of photosystem;
NPQ: Non-photochemical quenching of chlorophyll fluorescence;
1
O
2
: Singlet oxygen; PS:
Photosystem; ROS: Reactive oxygen species; V: violaxanthin; V-cycle: Xanthophyll cycle; WPI
all
:
Total winter photoinhibition; WPI
>12h
: Chronic Winter Photoinhibition; WPI
0.5h
: Dynamic Winter
Photoinhibition; WPI: Winter photoinhibition; Z: Zeaxanthin.
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1. INTRODUCTION
High mountain climates are characterized by low temperatures, low atmospheric pressure and high
proportion of short wavelength radiation (Körner 1999). Photosynthetic organisms acclimated to these
extreme conditions need to complete their life cycle within a short vegetative period and to accumulate
sufficient reserves for a long-lasting winter (Streb and Cornic 2012). Among perennial alpine plants,
there are some species which require snow cover to overwinter successfully because the conditions
below the snow are milder, mainly due to the amelioration of extreme temperatures and to the
decrease of light intensity (e.g. (Strand and Öquist 1985)). On the contrary, other species are exposed,
at least periodically, to the adverse conditions out of snow banks. The major stresses that these plants
with evergreen foliage have to cope with are the freezing of apoplastic water (Sutinen et al. 2001) and
the combined effect of high light and low temperature, known as “photochilling” (Huner et al. 2003;
Ivanov et al. 2003).
Photochilling stress is due to the fact that low temperature slow down enzymatic carbon assimilation
(Falk et al. 1996), whereas the absorption of light by the photosynthetic apparatus remains constant
because it is temperature independent. As a consequence, light energy absorption by antennae is much
higher than its potential use by the photosynthetic machinery, so the photosynthetic apparatus remains
overexcited. This situation greatly increases the risk of photooxidative damage, and plants must up-
regulate photoprotection mechanisms to counteract these effects. Apart from the reduction of light
absorption through morphological modifications or the adjustments in photosystems (PS) antenna size,
plants employ other physiological photoprotection mechanisms that can be grouped in three main
strategies: (i) the up-regulation of alternative energy emission pathways such as the dissipation of
exceeded light energy as heat (thermal dissipation) (Öquist and Huner 2003; Demmig-Adams and
Adams WW III 2006) (ii) the increase of metabolic activity of energy sinks (Asada 1999; Niyogi
2000) and (iii) the deactivation of reactive oxygen species (ROS) through the antioxidant metabolism
and/or the repair of oxidative damage (Noctor and Foyer 1998; Mullineaux and Rausch 2005).
Regulated thermal dissipation is associated with a decrease in fluorescence yield, which is estimated
by the fluorescence parameter called non-photochemical quenching (NPQ). For its activation, NPQ
requires three different components: transthylakoidal proton gradient (ΔpH), PsbS protein (Li et al.
2002) and activation of violaxanthin cycle (V-cycle) (Niyogi et al. 1997; Niyogi et al. 1998).
Depending on the maintenance or not of the activation this mechanism in darkness, NPQ can be
considered dynamic, when is completely reversed after one winter night (12 hours), or sustained, when
it needs more time, even several days of low light and optimal temperature for a complete recovery.
The consequence of sustained (also refereed to as chronic) thermal dissipation is a concomitant
reduction of photochemical efficiency. Hence, this process results in a depression of maximal
photochemical efficiency and as a consequence can be considered as a type of photoinhibition
(Demmig-Adams and Adams WW III 2006). Contrasting with other processes that generate an
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uncontrolled damage in photosynthetic machinery, particularly of reaction centers (photodamage),
chronic thermal dissipation is a highly regulated protective mechanism.
Severe winter stress activates sustained thermal dissipation, which involves the slow reversion from a
dissipative state in the photosynthetic apparatus of evergreen plants (Verhoeven 2014). This is also
termed as a sustained/chronic winter photoinhibition (WPI) when recovery is extremely slow (more
than one night) even after incubation under optimal conditions. WPI is apparently independent on ΔpH
(Verhoeven et al. 1998; Gilmore and Ball 2000; Demmig-Adams et al. 2006) and PsbS protein (Öquist
and Huner 2003; Adams WW III et al. 2004), but it has been demonstrated that it requires the presence
of zeaxanthin (Z). Thus, when WPI is activated, Z is retained and persistently engaged in thermal
dissipation (Demmig-Adams and Adams WW III 2006). A unified view of winter downregulation of
photosynthesis in woody species, integrating the roles of pigments and proteins, and different types of
“quenching” that occur simultaneously, has been recently proposed by (Verhoeven 2014).
In temperate alpine ecosystems, the mechanism of WPI was well characterized in woody plants e.g.
(Demmig-Adams and Adams WW III 2006; Zarter et al. 2006; Verhoeven 2014) and some herbs
(Streb et al. 2003; Østrem et al. 2011; Sanchez and Smith 2015; Sui 2015). These studies showed that
a wide range of species use the downregulation of photosynthesis as a photoprotective mechanism
under wintry conditions. Although metabolic and protein changes involved on WPI have been well
characterized in a few woody species e.g. (Demmig-adams and Adams III 2014), it is still unknown
how widespread this character is among other evergreen species, specially mosses and lichens. What is
more, a recent literature compilation (Míguez et al. 2015) has revealed that very scarce number of
works have studied WPI in lichens, bryophytes, terrestrial algae or ferns, even though these groups are
dominant in many boreal and alpine ecosystems. Hence, in the present work we aimed to fulfill these
gaps by comparing the well-known response of woody species with the rest of alpine flora (herbs,
lichens and mosses) at three different levels: (i) performing a survey on the frequence of this character
under field winter conditions in mosses, lichens and herbs; (ii) analyzing the potential for
photosynthetic recovery under the simulation of a period of warm temperatures in winter, in selected
species from the different functional groups (iii) elucidating the role of pigment and thylakoid proteins
in photoprotection and along recovery process.
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2. MATERIALS AND METHODS
2.1. Site description, plant material and experimental design
Field experiments were carried out (i) during late spring (June), after snow melt but before the
occurrence of any summer drought and (ii) in late winter (March) due to the complete coverage by
snow on previous months. Besides, according to a recent study of (Verhoeven 2013), the slowest rates
of photosynthesis recovery are observed in late winter, indicating that in this period, at least conifers,
present the deepest downregulation of photosynthesis. In both seasons, samples were collected at
noon. The temperatures at that time in the field oscillated between 3 and 7
ο
C in winter and between 19
and 25
ο
C in spring. Photosynthetic organisms collected in winter were not covered by snow. The
altitude of sampling sites was between 1750 and 1850m corresponding to the subalpine bioclimatic
level. Two approaches, the first observational (experiment 1) and the second manipulative (experiment
2), were carried out:
Experiment 1: In order to encompass a wide range of different species, a screening, comprising 50
subalpine species representative of the main functional groups (woody plants, herbaceous species,
mosses and lichens) was carried out in 2012. The samplings were performed in winter and spring in
three different mountainous areas in the north of Spain (Table 1). Immediately after collection,
samples were incubated under darkness at 100% relative humidity (in plastic bags with wet paper) and
at room temperature (20ºC) during 14h to allow their recovery from any kind of dynamic WPI (here
termed WPI
0.5h
). Chl fluorescence measurements were taken after 30min and after 14h under those
optimal conditions in 5 individuals of each species. After the second measurement, 5 replicates per
species (100 mg approximately) were sampled and immediately frozen into liquid nitrogen and
thereafter preserved at -80ºC until pigment and protein analysis.
To calculate WPI, it has been considered that the photochemical efficiency of PSII (F
v
/F
m
)
measured in
late spring after 14h of recovery in darkness is the highest value that each species can reach. Taking
this assumption into consideration, the percentages of dynamic winter photoinhibition (WPI
0.5h
) and
chronic winter photoinhibition (here termed WPI
>12h
) were calculated for each species as follows:
WPI
0.5h
=(F
v
/F
m 14h winter
– F
v
/F
m 30min winter
)/(F
v
/F
m 14h spring
) x 100
WPI
>12h
=(F
v
/F
m 14h spring
– F
v
/F
m
14h winter
)/(F
v
/F
m
14h spring
) x 100.
WPI
all
=WPI
>12h
+
WPI
0.5h
Where: 14h and 30min indicate the time that plants were incubated in darkness and 20°C before the
fluorescence measurement. This approach is in agreement with the kinetics of F
v
/F
m
recovery
previously described by other authors (Verhoeven 2013), where a rapid component lasts less than 2h.
