Short and long term low temperature
responses in Arabidopsis thaliana.
Paulina Stachula
Department of Plant Physiology
Umeå 2015
© Paulina Stachula, 2015
Umeå Plant Science Centre
Department of Plant Physiology
Umeå University, 901 87 Sweden
ISBN: 978-91-7601-319-9
Front cover photo by: Katja Gorenc
Electronic version available at http://umu.diva-portal.org/
Printed by: Servicecenter KBC, Umeå University
To my grandmother
Table of Contents
i Table of Contents
Abstract
iii v Sammanfattning
List of papers
vii Preface
1
Introduction
5 1. General effects of chilling temperature (the range between 200C to 00C)
on plant physiology
5 2. General effects of freezing temperature (below 00C) on plant
physiology
6 3. Plasma membrane as a primary site of injury during freeze-thaw cycles 8
3.1. Alterations in the membrane fatty acids composition during cold acclimation 10
3.2. Proteins that stabilize membranes during freeze thaw cycle
11
3.3. Role of sugars in protecting membranes during freezing and dehydration
15
stress
4. Additional compatible solutes that play important roles in cold
acclimation process
17
5. Photosynthesis in cold acclimation
19
5.1. Effects of low temperature on photosynthesis
21
5.2. Acclimation responses of photosynthesis to low temperature
22
5.3. Changes in the environment can be sensed by the chloroplast
24
6. Gene expression during cold acclimation
25
7. Fast responses to low temperature: cold perception
30
8. Role of the circadian clock in cold stress responses in plants
34
9. Long-term cold acclimation-ecological perspective
36
Aim of the study
40
Results and discussion
41 1. Cold signal perception
41
2. Chloroplasts sense changes in environmental conditions
48
3. Plasma membrane signal regulates nuclear gene expression
51
4. Plastid signal: transmitting the status of the chloroplasts to the
nucleus
53
5. Other factors besides plastid signal regulating nuclear gene
i
expression
54
6. Summary of fast responses to changing environment in Arabidopsis
56
7. Long term cold acclimation responses in Arabidopsis
57
7.1. The role of early cold induced pathway in long-term cold treatment
59
Conclusions
61
Acknowlegements
63
References
64
ii
Abstract
A unique aspect of plant biology is their ability to respond very rapidly to shifts
in their ever-changing environment. To do this, plants need to have evolved
mechanisms that enable them to sense their environment and to rapidly
respond to different stresses and different combinations of stress. The main
focus of my thesis has been on plant responses to low temperature. One way
in which the plant cell is thought to sense low temperature is via the
rigidification of the plasma membrane that occurs when the temperature falls
rapidly. I showed that CRMK1, a leucine-rich-repeat-receptor-like kinase,
located in the plasma membrane is a key component of the cold sensing
machinery. I show that a T-DNA insertion mutant of CRMK1 attenuated the
expression of marker cold genes in response to cold stress and decreased the
plants ability to acquire freezing tolerance after 3 days of exposure to low
temperature. I described a protective mechanism in the thylakoid membranes
by which excess electrons, which can accumulate at low temperature and
under high light in cold acclimated plants, can be dissipated. In order to
properly respond to a changing surroundings cells not only have to sense the
fluctuations but they need to communicate this changes to the nucleus and I
showed that organelles such as the chloroplasts can act as a sensor of changing
temperature and communicate this information to the nucleus to change the
expression of cold regulated genes. I presented and identified factors in
addition to low temperature that regulate the expression of genes for cold
tolerance. I showed that a circadian signal, together with plastid and light
signals, influence the expression of nuclear encoded cold tolerance genes
(CBF3 and COR15). The response of plants to low temperature also depends
on the duration of the cold stress. There are plants, such as over-wintering
herbaceous and perennial woody plants, that have to survive and grow for
prolonged periods at low temperature and there are those from extreme polar
or alpine environments that spend entire life in cold. I provided a
comprehensive overview of the biological processes and genes that enable
iii
plants to survive and develop the full cold acclimated state during long-term
over wintering and growth in the low temperature.
iv
Sammanfattning
En unik egenskap hos växter är deras förmåga att känna av och reagera mycket
snabbt när deras miljö förändras. För att klara detta har växter utvecklat
mekanismer som gör det möjligt för dem att snabbt reagera på olika sorters
stress och olika kombinationer av stress. Växter reagerar olika på köld,
beroende på köldstressens varaktighet. Det finns växter som t. ex.
övervintrande örter och förvedade perenner som kan klara långa perioder av
köldstress, det finns det även växter från extrema miljöer (till exempel i
alpina- eller polar trakter) som spenderar hela sina liv i köld. Jag ger i denna
avhandling en översikt av de biologiska processerna och de gener som tillåter
växter att överleva och utveckla det köldacklimatiserade tillstånd som krävs
för att överleva vintern och växa i kyla. Det experimentella arbetet rör hur
växter reagerar på låga temperaturer. Ett sätt på vilket växtcellen tros kunna
känna
av
låga
temperaturer
är
via
den
s.k.
rigidificeringen
av
plasmamembranet, som inträffar när temperaturen sjunker snabbt. Jag har i
mitt arbete visat att CRMK1, ett ”leucine-rich-repeat-receptor-like kinase”,
som sitter i plasmamembranet är en viktig del av växtens maskineri för att
känna av temperaturförändringar. Jag har visat att en växt som saknar
CRMK1 hade minskat uttryck av markörgener för kyla när den utsattes för
köldstress och att denna mutant efter tre dagars köldexponering även hade
minskad förmåga att förvärva köldtolerans. Jag har också beskrivit en skyddsmekanism i tylakoidmembranen genom vilken det elektronöverskott som
ansamlas vid låga temperaturer och intensivt ljus kan ”avledas” hos
köldacklimatiserade växter. För att kunna reagera korrekt på en föränderlig
omgivning måste cellerna inte bara upptäcka fluktuationerna utan även
kunna kommunicera dessa förändringar till cellkärnan och jag har även i mitt
arbete
visat
att
kloroplasterna
kan
fungera
som
sensorer
för
temperaturförändringar och förmedla denna information till cellkärnan, där
uttrycket av köldreglerade gener ändras. Jag diskuterar också andra faktorer
än låg temperatur som reglerar köldtoleransgeners uttrycksnivåer, jag har till
exempel visat att den cirkadiska klockan tillsammans med kloroplast- och
v
ljussignaler påverkar uttrycket av de kärnkodade köldtoleransgenerna CBF3
och COR15.
vi
vii
List of papers
I.
Stachula P, De Dios Barajas Lopez J, Miskolczi P, Vaultier MN,
Zhang B, Ahad A, Nick P, Bakó L, Strand Å, Hurry V.
CRMK1 a receptor-like kinase in the plasma membrane mediates
the cold acclimation response in Arabidopsis.
Manuscript in prep
II.
Norén L, Kindgren P, Stachula P, Rühl M, Eriksson ME, Hurry
V, Strand Å.
HSP90, ZTL, PRR5 and HY5 integrate circadian and plastid
signaling pathways to regulate CBF and COR expression.
Manuscript in prep
III.
Ivanov AG, Rosso D, Savitch LV, Stachula P, Rosembert M,
Oquist G, Hurry V, Hüner NP
Implications of alternative electron sinks in increased resistance
of PSII and PSI photochemistry to high light stress in coldacclimated Arabidopsis thaliana.
Photosynth Res. 113:191-206, 2012
IV.
Stachula P, Vergara A, Delhomme N, Street N, Hurry V.
Essential processes determining life in cold in Arabidopsis
thaliana.
Manuscript in prep
Related publications of Paulina Stachula not included in the thesis
V.
Lundmark M, Stachula P, Hendrickson L, Pesquet E, Külheim
C, Vlčková A, Stenlund H, Moritz T, Strand Å, Hurry V.
The interaction between development and stress in the low
temperature metabolome of Arabidopsis thaliana.
Manuscript in prep
Paper III is printed with kind permission of the publisher.
The papers will be referred to by their Roman numbers in the text.
viii
ix
Preface
Research connected to climate change has shown that anomalies in
temperature have been rising since 1900 (Mann, Bradley et al. 1999) and
recent estimates indicate that in the near future as many as one-third of
European plant species will be threatened by changes in their thermal
environment (Thuiller, Lavorel et al. 2005). Such predictions indicate that we
need to change the way we grow and breed our crops, and also quickly reevaluate our understanding of how our ecosystems will develop in response to
these changes. Despite increasing average temperature across the years, high
temperatures are not the only threat and it is likely that plants, including our
field crops, will also experience an increase in more devastating early spring
frosts events. For example in 2012 large losses were reported in the fruit
industry (50–90% losses of fruit crops such as grapes, peaches, apples,
cherries) because of spring frosts in the Midwest and Northeast of the USA
(Duman and Wisniewski 2014). (Gu, Hanson et al. 2008) reported that losses
to many economically important crops grown in the US, just from freeze
damage in 2007 alone, amounted to 2 billion US dollars. Thus, rather than
focussing only on yield gains and quality traits, our crop plants will need to be
more flexible in response to changes in their environment so that farmers will
get stable yield despite less predictable and more severe weather. To reach this
goal we need first to better understand how the whole plant responds to
changes in temperature in the surrounding environment, beginning with how
temperature is perceived and how the signal is transduced to nucleus, which
in turn activates the transcriptional response resulting in acclimation to
stress.
Levitt (1980) was one of the first scientists to identify a distinction between
mechanical and biological definitions of stress. He proposed that mechanical
stress was the force or action of object A causing the reaction of object B.
Further, any body under stress experiences strain, and strain can be either
1
plastic, which is irreversible (e.g. piece of wood) or elastic, which is reversible
(e.g. rubber band). Levitt’s biological definition of stress was any
environmental factor capable of inducing a potentially injurious strain in
living organisms. Thus, for plants, plastic strain (known also as direct stress
injury) occurs dependent on the length and speed of exposure to stress. For
example, a plant exposed to a rapid and severe drop in temperature can suffer
protoplasmic ice formation resulting into death. Elastic strain (indirect stress
injury), on the other hand, can last from hours to days without causing injury
to the plant and, unlike inanimate objects exposed to mechanical stress, plants
can change in this relatively short time in response to a lower level of a stress.
For example in summer, trees and herbaceous plants in northern latitudes
cannot withstand freezing but exposure to chilling temperatures induces
hardening through a process called cold acclimation and acclimated plants
can survive winter temperatures far below freezing. Thus, many plants can
increase their resistance to various stresses including heat, salt, and drought
conditions in response to an elastic strain.
The principle focus of this thesis is the response of plants to unfavourably low
temperatures, and it is on the responses of plants to cold that I will focus the
bulk of the text below. The definition of chilling injury, as provided by Molisch
(1897) (Lyons 1973), is low-temperature damage in the absence of freezing.
Freezing injury occurs when the external temperature drops below the
freezing point of water. There are many plants, including many of our core
agricultural crops, that are susceptible to chilling injury and can be killed by
their first experience of frost. There are also plants, native to cold climates,
that can survive extremely low temperatures without injury (Levitt 1980).
Plants can be divided into three groups based on their response to low
temperature (Stushnoff, Fowler et al. 1984). The first group consists of frost
tender plants that are sensitive to chilling injury and can be killed by very short
periods of freezing temperature. They cannot tolerate ice in their tissues. This
group of plants include beans, corn, rice, and tomatoes. The second group
2
represents plants that have the ability to cold acclimate and are more frost
resistant than the first group. These plants, while generally sensitive to
freezing during active growth, can acclimate to freezing temperatures and can
survive the presence of extracellular ice in their tissues. The frost resistance of
this second group ranges from the summer annuals, which are killed at
temperatures slightly below freezing, to perennial grasses that can survive
exposure to -40°C. The last group consists of cold hardy plants that are mainly
temperate woody species. Their level of cold tolerance is dependent on the
stage of acclimation, the rate and degree of the drop in temperature, and the
genetic capacity of tissues to stand extracellular freezing and the survival of
dehydration stress. Deep supercooling allows certain tissues in plants from
this group to survive low temperatures without the formation of extracellular
ice.
3
4
Introduction
1. General effects of chilling temperature (the range between
20°C to 0°C) on plant physiology
Photosynthesis is one of the central process in plant physiology and it is
sensitive to chilling temperatures, which is why it is not surprising that
chloroplasts are often first sites of ultrastructural chilling injury in the cell
even before the physical symptoms are visible (Kimball and Salisbury 1973).
These ultrastructural injuries affect other compartments as well and include
swelling and disorganization of the chloroplasts and mitochondria, reduced
size and number of starch granules, dilation of thylakoids and unstacking of
grana, formation of small vesicles of chloroplast peripheral reticulum, lipid
droplet accumulation in chloroplasts, and condensation of chromatin in the
nucleus (Kratsch and Wise 2000). In addition, chilling reduces the fluidity of
the membranes in the cell (Levitt 1980) causing, amongst other things, the
inhibition of mitochondrial and chloroplastic electron transfer reactions
(Huner, Öquist et al. 1998). For example, the chloroplast electron transfer
chain becomes over-reduced under chilling temperatures due to the combined
effects of impaired mobility of plastoquinone within the thylakoid membrane
and reduced utilization of NADPH by the Calvin cycle (Huner, Öquist et al.
1998). Linked to both the inhibited metabolic activity and the impaired
electron transfer reactions, chilling injury is often associated with the
accumulation of reactive oxygen species (ROS) (Prasad and Saradhi 2004).
The activities of the ROS scavenging enzymes will also be attenuated by low
temperature,
inhibiting
the
ability
of
the
scavenging
systems
to
counterbalance the increase in ROS formation. Accumulation of ROS has
negative impacts on membranes (Allen and Ort 2001) presented as electrolyte
leakage. There is no ice formation at chilling temperatures and chilling
conditions can lasts from hours to days. The critical temperature varies
depending on the plant species involved: for bananas it is 15oC, rice 18oC,
5
beans (wilting) 10oC and wheat 4oC (Levitt 1980). Chilling affects the entire
internal environment of the cell and chilling has been found to change the
entire metabolic response of the cell, with some processes recovering quickly
and others slowly. Chilling injury is therefore a result of the combined
consequences of these different metabolic perturbations (Kratsch and Wise
2000).
2. General effects of freezing temperature (below 00C) on
plant physiology
Exposure to freezing temperatures cause more severe effects to cells than
chilling temperatures because freezing can trigger the formation of
intracellular ice, which leads irreversibly to cell death. Freezing injury starts
when ice is formed in the extracellular spaces of plant tissues. Ice formation
is dependent on the presence of ice nucleators (organic or inorganic
substances) and the rate at which the external temperature declines (Mazur
1969, Weiser 1970, Levitt 1980). During freezing, much of the cellular water
moves out from the cell in response to the water potential gradient that is
established between the extracellular ice crystal and the intracellular liquid
water. This water loss causes cell shrinkage and dehydration. If cooling is
sufficiently slow, the cell will dehydrate to the extent required to establish an
equilibrium between the intra- and extracellular aqueous spaces, and
intracellular freezing will not occur (non-lethal effect). If there are no ice
nucleators present (no bacteria, dust or any other particle), and water is very
clean, it remains in a supercooled state above -38°C, the temperature at which
water self nucleates (Thomashow 1998). Plant-produced ice nucleating
agents have been found in the extracellular liquid of a number of species;
including the annual winter rye (Brush, Griffith et al. 1994) and perennial
trees such as Prunus (Gross, Proebsting et al. 1988) and Citrus
(Constantinidou and Menkissoglu 1992). There are also extrinsic nucleators
6
that are usually present on plant surfaces during radiation frost. These
include bacteria (Baca 1987, Lindow 1988), dust particles and wind motion.
As soon as membranes are affected by ice protrusion inside the cell this is
lethal and causes cell death. There are protective strategies that plants can
deploy in order to minimise the action of freezing temperatures and ice
formation. One of such strategy is the synthesis of antifreeze proteins (AFP),
and AFPs have been found in a wide range of overwintering organisms for
example antarctic fish (DeVries and Wohlschlag 1969), plants and insects
(Griffith and Ewart 1995). These proteins have two main functions: the first
is to lower the temperature at which ice is formed, without influencing
melting point of the solution (Duman 1994). The second is to inhibit the
growth of ice crystals by binding to ice nuclei and preventing the small ice
crystals from joining to form larger crystals, a process called recrystallization
(Tursman and Duman 1995). Plant AFPs are secreted into the apoplast and
while they have been found in a number of freezing tolerant monocotyledons
(e.g. winter rye and winter wheat, winter barley), the synthesis of antifreeze
proteins does not appear to be a general response of plants to low
temperature, rather it is specific for particular plants (Antikainen and Griffith
1997). Thus many cold tolerant species are able to mount a defence against
the physical presence of ice in their tissues, but there remain the damaging
effects on plant physiology. The extent that plants experience such damage
will depend on the type of plant and its developmental state. Information on
the responses generated by different cell structures, such as the plasma
membrane and the organelles (main focus in this thesis is related to plastids),
during freezing improve our understanding of how cells can overcome or cope
with extreme stress events such as ice formation in their tissues in order to
survive.
7
3. Plasma membrane as a primary site of injury during
freeze-thaw cycles
As early as 1912, Maximov (Iswari and Palta 1989) stated that irreversible
dysfunction caused by freeze-induced dehydration that occur in the plasma
membrane makes it a primary site of freezing injury. However, there are many
factors in the cytosol and intracellular organelles that can influence the
cryobehaviour of this membrane during cold acclimation and freezing. When
cold tolerant plants are exposed to low non-freezing temperatures they are
able to acclimate to freezing conditions. One of the universal cold acclimation
responses of cold tolerant plants is to increase the concentrations of
compatible solutes present in the cytosol, which results in a decrease in the
extent of freeze-induced osmotic dehydration (Levitt 1980). Amongst these
are specific sugars and proteins that play a protective role for membranes
during freezing (Lineberger and Steponkus 1980), proline that scavenges
active oxygen species (Smirnoff and Cumbes 1989) and glycinebetaine that
promotes recovery of PSII from freezing (Sakamoto and Murata 2002).
Differences in the behaviour of the plasma membrane of non-acclimated and
cold-acclimated protoplasts during freeze-thaw events lead to classification of
three types of freeze induced membrane lesions; expansion-induced lysis,
endocytotic vesiculation together with HII phase, and exocytotic extrusions
and fracture jump lesions (Uemura, Joseph et al. 1995). The first two types of
plasma membrane lesions occur in non-acclimated plants, where dehydration
occurs to a lesser extent than in cold acclimated plants (Uemura, Tominaga et
al. 2006). In the first type of membrane lesion, dehydration results in the
formation of endocytotic vesicles that are not continuous with the plasma
membrane. This type of lesion was first shown in non-acclimated winter rye
and subsequently in Arabidopsis and oat (Steponkus, Uemura et al. 1988,
Uemura and Steponkus 1989), and occurs at temperatures between -2 to -4°C.
Osmotic contractions lead to endocytotic vesiculation just beneath the plasma
8
membrane. Around 40% of the membrane surface area was used to produce
such vesicles in protoplasts of non-acclimated rye subjected to freezing
(Dowgert and Steponkus 1984). This process of endocytotic vesicularization is
irreversible and when thawing occurs cells lyse because of expansion. When
the temperature decreases further, e.g. -5°C, greater dehydration occurs and
the injury is visible as a complete loss of osmotic responsiveness (LOR) while
thawing. LOR occurs in non-acclimated tissue because of phase transition of
the phospholipids within the membrane from a lamellar (bilayer) to a
hexagonal II (HII) phase when the water content of the cell drops to
approximately 20% (Gordon-Kamm and Steponkus 1984, Uemura and
Yoshida 1984). The third type of behaviour of the plasma membrane occurs in
cold acclimated protoplasts and it is connected with more severe dehydration.
In this case, freeze-induced osmotic contraction results in the production of
exocytotic extrusions instead of endocytotic vesicles. During osmotic
expansion upon thawing, exocytotic extrusions are reversibly incorporated
into the plasma membrane and expansion induced lysis does not occur in cold
acclimated protoplasts. In cold acclimated plants there is loss of osmotic
responsiveness but it is not due to HII phase transition (Gordon-Kamm and
Steponkus 1984), but due to fracture jump lesions. Because the plasma
membrane was shown to be the primary site of injury at low temperatures
(both freezing and chilling temperatures) this feature may exert the most
control over plant survival. There are various ways plasma membrane can
react to freeze-thaw cycles depending on severity of dehydration and
alterations in plasma membrane composition in response to exposure to nonlethal cold temperatures. This feature are thought to be of the utmost
importance for plant survival.
9
3.1. Alterations in the membrane fatty acids composition
during cold acclimation
Plasma membranes not only change behaviour in response to a downshift in
the temperature (described in previous section) when it rigidifies (Fig 1), but
cold acclimation results in changes in the lipid composition of the plasma
membrane. The changes that occur in Arabidopsis plasma membranes at low
temperature include an increase of phospholipids and a decrease in
sphingolipids (e.g. glucocerebroside) and free sterols (Uemura, Joseph et al.
1995). Such alterations change the cryobehavior of the plasma membrane and
restore membrane fluidity at low temperatures (Steponkus, Myers et al. 1990).
Similar increases in phospholipid content have been observed in many
different plant species from monocotyledonous to dicotyledonous and from
herbaceous to woody plants (Uemura, Tominaga et al. 2006). Another way to
increase membrane fluidity and to change the cryobehavior of the plasma
membrane is to modify the level of unsaturation of fatty acids. In Arabidopsis,
one week of cold acclimation leads to an increase in di-unsaturated and
decrease
of
mono-unsaturated
species
in
phosphatidylcholine
and
phosphatidylethanolamine (Uemura, Joseph et al. 1995). Changes in
desaturation not only occur in plasma membranes but also in mitochondrial
and plastid membranes (Matos, Hourton-Cabassa et al. 2007). The
availability of Arabidopsis mutants with altered lipid composition lead to the
identification of enzymes responsible for lipid desaturation: the fatty acid
desaturases (FADs) (Wallis and Browse 2002). There are seven classes of
these mutants in Arabidopsis and each one shows a defect in one step of
desaturation. Mutation in loci fad2 and fad3 affect desaturation of extrachloroplast lipids, and the remaining five classes: fad4, fad5, fad6, fad7 and
fad8 affect chloroplast lipid desaturation. Changes in fatty acid composition
in thylakoid membranes of FAD mutants lead to impaired chloroplast
development under low temperature (for example fad2fad6 showed
significant decrease in photosynthesis), which in turn adversely affected the
cold acclimation process (Wallis and Browse 2002). Studies using these
mutants with alerted fatty acid composition in membranes showed that the
10
unsaturation level had significant effects on membrane behaviour and the cold
acclimation capacity of the plant. The mentioned alterations in membranes
are important indicators that its composition adjust to change in temperature
in order to become more stable structure which may lead to improved survival
of the plant.
Figure 1. Schematic presentation of the plasma membrane behaviour at low
temperature (membrane rigidify) and when there is increase in temperature
(membrane fluidize) (modified from (Los, Mironov et al. 2013). PM- plasma
membrane.
3.2. Proteins that stabilize membranes during freeze thaw
cycle
The plasma membrane and thylakoids membranes are believed to be the
primary sites of freeze damage when plants are exposed to temperatures
below zero. Thus, it is important to gain better knowledge of how these
structures can be protected and stabilized during the severe dehydration
events that occur during freezing. A large number of stress-induced proteins
have been identified that may play protective roles and improve membrane
stability at low temperatures. (Guy, Niemi et al. 1985) reported that most
polypeptides induced by low temperature are distinct from heat shock
proteins, suggesting that the observed changes in mRNA abundance induced
by cold was connected with the synthesis and accumulation of specific cold
induced proteins. Around the same time (Uemura and Yoshida 1984)
11
demonstrated that during cold acclimation the plasma membrane is in a
dynamic state and when winter rye seedlings were shifted to cold there was a
significant change in the polypeptides associated with membranes, some
decrease or disappear and some were induced by low temperature. (Volger
and Heber 1975) first presented protein fractions isolated from cold-hardy
spinach and cabbage leaves that showed protective roles against freeze
damage to thylakoid membranes. This line of research was further continued
by (Hincha, Heber et al. 1985, Hincha and Schmitt 1988), where isolated
cryoprotective proteins from cold hardy spinach and cabbage thylakoid
membranes were exposed to freeze-thaw cycles. Such protective proteins were
not present in non-hardy membranes and the absence of these protective
proteins caused membrane damage during freezing stress. It was concluded
from these studies that the function of cryoprotective proteins is to decrease
the permeability of membranes to solutes and to increase their expendability
during thawing (Hincha, Heber et al. 1990).
The first report of a protein with a direct effect on cellular cryoprotection came
from the study of (Sieg, Schroder et al. 1996) who isolated and purified
glycoprotein (cryoprotectin) from leaves of cold-acclimated cabbage. After
sequencing, cryoprotectin was found to be closely related to WAX9, which
belongs to the class of nonspecific lipid transfer proteins (LTP) (Hincha 2001).
Further studies showed that cryoprotectin was structurally and functionally
different from WAX9 as it shows cryoprotective activity but not lipid-transfer
activity (Hincha 2001). In other studies, (Artus, Uemura et al. 1996)
investigated whether well-known cold regulated genes, such as COR15a,
produced proteins with a functional role in freezing tolerance. These studies,
conducted on leaf protoplasts, showed that constitutive expression of COR15a,
which encodes a chloroplast-targeted polypeptide, enhances the in vivo
freezing tolerance of chloroplasts in non-acclimated plants by almost 2°C
(Artus, Uemura et al. 1996). (Steponkus, Uemura et al. 1998) later showed
that this increase in freezing tolerance by constitutive expression of COR15a
12
was a result of a decreased occurrence of freeze-induced lamellar-to-HII phase
transitions. It was further shown by in vitro experiments that purified COR15a
was able to protect L-lactate dehydrogenase against freeze-inactivation
(Nakayama, Okawa et al. 2007) and prevent aggregation of Rubisco
(Nakayama, Okawa et al. 2008). However (Thalhammer and Hincha 2014),
taking a transgenic approach, showed that COR15 had no influence on the
stability of plastid enzymes (such as Rubisco and transketolase) in vivo,
although cold acclimation results in increased enzyme stability. They
concluded that the mechanism through which COR15 enhances freezing
tolerance is through stabilization of the thylakoid membranes, not through
enhanced enzyme stability or refolding after freezing, as suggested in previous
studies.
While COR15 is perhaps the best studied and functionally characterized
cryoprotective protein, a number of additional classes of proteins have been
identified and shown to have protective roles during freezing. One of the
pathogenesis-related (PR) proteins, the osmotin-like proteins isolated from
bittersweet nightshade, was identified by the amino acid sequence similarity
to osmotins and osmotin-like proteins from wild potato, tomato and tobacco,
and were shown to stabilize membranes during freezing (Newton and Duman
2000). Further characterization of this group of PR proteins was facilitated by
screening of a winter cDNA library for the corresponding cDNA sequence,
which gave the nucleotide sequence for the full protein. This group of proteins
have cryoprotective activity in in vitro tests and when added to warm grown
protoplasts of kale (Brassica oleracea) the presence of osmotin was able to
protect protoplasts against freeze-thaw injury down to -8°C (Newton and
Duman 2000). Additionally, a class I β-1,3-glucanase purified in in vitro
thylakoid assay with tobacco, plays a cryoprotective role on thylakoids
membranes by alleviating osmotic stress and preventing vesicle rupture
during thawing because of reduced solute influx across membranes (Hincha,
13
Pfuller et al. 1997). Unlike other cryoprotective proteins, this group is not
induced by 4°C but by lower/freezing temperatures.
A cryoprotective role for galactose-specific lectins was established in in vitro
experiments which demonstrated that lectins bind exclusively to digalactosyl
lipids in membranes (thylakoid membranes are rich in mono- and digalactosyl
lipids) and this hydrophobic interaction with the membrane lipids leads to
reduced fluidity at the membrane surface, resulting in reduced solute
permeability (Hincha, Bakaltcheva et al. 1993). Further studies utilizing
lectins extracted from Ricinus communis concluded that during freeze–thaw
cycles there is one lectin, RCA60, which influences the degree of packing of
lipids, altering the permeability of the thylakoid membranes for solutes
(Hincha, Bratt et al. 1997). Additional groups of lectins isolated from mistletoe
not only bind to the surface of thylakoid membranes but they were also more
abundant during winter when mistletoe is more resistant to cold and are less
abundant during spring when the plant is more susceptible to low temperature
(Hincha, Pfuller et al. 1997), indicating that they are both functional and cold
inducible proteins and likely have cryoprotecitve functions. Cryoprotective
lectins reduce the solute permeability of thylakoids already in the absence of
freezing (Hincha, Bakaltcheva et al. 1993) this feature is specific just for lectins
because other groups such as cryoprotectin and β-1,3-glucanase did not show
any effect on thylakoid rupture in an unfrozen solution (Hincha, Heber et al.
1990).
This diversity of cryoprotective proteins demonstrates first,the complexity of
the protective mechanisms induced by low temperatures and second that they
are present in the different cellular compartments in order to keep the plant
membrane stable during freeze-thaw stress events. The challenge comes (both
for the plants and for the plant scientist) when all these changes have to be
synchronized to maintain improved survival of the plants to low temperature;
14
and it needs to be remembered that proteins are not the only players that alter
membranes functions at low temperature, the other players are sugars and
small molecules.
3.3. Role of sugars in protecting membranes and proteins during
freezing and dehydration stress
Many organisms, such as insects, algae, desiccation tolerant higher plants
(resurrection plants), pollen and seeds of many plant species that are not
desiccation tolerant in their vegetative state, can survive desiccation to various
extents by accumulating disaccharides, mainly sucrose or trehalose. Both of
these sugars are believed to play a major role in cellular desiccation tolerance.
(Crowe, Carpenter et al. 1998) suggested that these sugars replace the water
around polar residues in labile macromolecular assemblages like membranes
and proteins, leading to stabilization of these structures in the absence of
optimal amounts of water. (Buitink 2000) showed that in pea (Pisum
sativum) and cucumber (Cucumis sativa) seeds, vitrification of the cytoplasm
during drying is a crucial aspect of desiccation tolerance. It was shown that
cytoplasmic glasses contain sugars and proteins (Buitink 2000). Uemura and
Steponkus (2003) showed that incubation of Arabidopsis seedlings in sucrose
solutions was connected to the occurrence of various freeze-induced
membrane lesions (see section 3). The appearance of expansion induced lysis
(EIL) between -2 to -4°C was decreased and prevented after incubation in
increasing concentrations of sucrose (10-35mM), while the loss of osmotic
responsiveness (LOR) between -5 and -6°C was reduced by higher
concentration of sucrose (30-200mM). At lower temperatures, between -8 to
-12°C, even higher concentrations of sucrose (up to 400mM) were required to
reduce the occurrence of LOR. Sugars and other compatible solutes replace
water and at the same time increase the spatial separation between
membranes, and push the occurrence of the lamellar-to-gel phase transitions
15
to lower temperatures (Wolfe and Bryant 1999). In cells at low hydration,
vitrification will occur where the sugars are located. If the sugars are between
the membranes, then vitrification should happen there. If the sugars are
excluded from the region between the membranes, then vitrification happens
in the extralamellar volumes near the membranes, but not between them
(Wolfe and Bryant 1999).
In addition to the cryoprotective role played by sucrose in plants, raffinose
also helps to stabilize membranes by replacing the lost water, thus preventing
lipid phase transitions (Hincha, Zuther et al. 2003). Carbohydrate analysis of
drought-, high salinity- and cold-treated Arabidopsis plants showed that
these plants all accumulate a large amount of raffinose and galactinol, which
are not present in plants that didn’t experience abiotic stresses. Raffinose is
synthesised from sucrose by the addition of galactose donated by galactinol.
The synthesis of galactinol is a key regulatory step in the pathway, controlled
by the enzyme galactinol synthase (GolS). Galactinol synthase (GolS) catalyses
the first step in the biosynthesis of RFO from UDP-galactose. Taji et al. (2002)
found three stress-responsive GolS genes (AtGolS1, 2 and 3) among seven
Arabidopsis GolS genes. AtGolS1 and 2 were induced by drought and highsalinity stresses, but not by cold stress. AtGolS3 was induced by cold stress but
not by the other stresses tested. An increase in raffinose has been associated
with an increase in transcript of GolS3 (Liu, Kasuga et al. 1998). The induction
of GolS3 is controlled by the CBF transcription factors (Fowler 2002). The
increase in expression peaks after 12h at 4°C after which the expression
gradually decreases (Kaplan, Kopka et al. 2007). The protective role of sucrose
together with raffinose on membranes was investigated in isolated thylakoid
membranes (Lineberger and Steponkus 1980) and liposomes (Hincha, Zuther
et al. 2003). Sucrose and raffinose act by interaction with protein and lipid
bilayer surfaces (Hoekstra, Golovina et al. 2001). Raffinose was shown to
delay sucrose crystallization (Caffrey, Fonseca et al. 1988), which prevents
membrane
damage.
Raffinose
is
synthesized
extra-chloroplastically
16
(Bachmann and Keller 1995), and it has to be transported into the chloroplasts
by a chloroplast transporter, which has not yet been identified. Zuther et al.
(2004) investigated the role of raffinose in acquiring freezing tolerance and
cold acclimation in different accessions of Arabidopsis Col-0 and Cape Verde
Islands by creating transgenic lines that either constitutively overexpressed a
galactinol synthase (GS) gene from cucumber or a knockout of the endogenous
raffinose synthase (RS) gene. In both cases regardless of whether plants
produced higher amounts of raffinose or no raffinose at all there was no
difference in freezing tolerance in non-acclimated and cold-acclimated plants
(Zuther, Büchel et al. 2004), suggesting that raffinose is not essential for
plants to acquire cold tolerance but it is nevertheless one component of the
cold acclimation response that plants deploy during exposure to stress.
Increased tolerance to freezing and drying appears to result from the sugar’s
ability to lower the temperature of the dry membrane phase transition and
maintain general protein structure in the dry state. It seems reasonable that
any substance which was to increase the tolerance of an organism to freezing
and drying would need to protect both the membranes and the proteins, and
these sugars meet both of these requirements.
4. Additional compatible solutes that play important roles in
cold acclimation process
Natural stress tolerance in any organism cannot be explained by action of just
one factor but it is rather many factors interacting together that enable a plant
to respond and develop tolerance especially to such severe stress like
desiccation and freezing. Certain low molecular weight organic metabolites
act as osmolytes and help plants to survive extreme osmotic stress.
Metabolites that are known to be changed during exposure to abiotic stresses
and enhance stress tolerance, in addition to the sugars discussed above, are:
polyamines, amino acids, lipids and organic acids (Guy 1990). Compatible
17
solutes neutralize the differences in osmotic pressure in the cell and they
protect macromolecules from the destructive action of physical and chemical
factors (e.g. ROS) but when in high concentrations they don’t perturb cellular
functions (Papageorgiou and Murata 1995).
One of the best-studied compatible solutes is glycinebetaine (GB) (Kurepin,
Ivanov et al. 2015). GB is a quaternary ammonium compound that is found in
bacteria, haemophilic archaebacteria, marine invertebrates, plants and
mammals (Rhodes 1993, Chen and Murata 2002, Takabe, Rai et al. 2006).
The biological function of GB in plants has been widely studied, mostly in
herbaceous species such as spinach, sugar beet, maize and barley (Rhodes
1993, Chen and Murata 2002). The major role of GB in improving tolerance
to abiotic stresses is to protect enzymes and lipids that are required to
maintain optimal linear electron flow through thylakoid membranes and
maintain the assimilation of CO2 (Papageorgiou and Murata 1995). However,
although GB has been shown to have protective functions, a number of our
important crops (e.g. rice and potato) are unable to synthesize GB. Studies of
transgenic plants such as A. thaliana, rice and tobacco that were genetically
engineered to synthesise GB have shown that the accumulation of GB
enhances tolerance to various types of environmental stresses at various
stages in the plants life cycle (Sakamoto 2001). Application of exogenous GB
has also been shown to stabilize and protect the photosystem II complex in
the thylakoid membrane from stress-induced inactivation (Murata, Mohanty
et al. 1992, Papageorgiou and Murata 1995). Low levels of GB applied
exogenously also induced expression of stress responsive genes coding for
enzymes that scavenge reactive oxygen species (Chen and Murata 2010),
suggesting that GB successfully balance the negative stress responses induced
by the low temperature and therefore can be used to improve freezing
tolerance of cold stressed plants.
18
Proline is an amino acid but it is also a compatible osmolyte that accumulates
in many organisms, including higher plants, eubacteria, marine invertebrates,
protozoa and algae that have been exposed to environmental stresses such as
drought, high salinity, high temperature, freezing, UV radiation and heavy
metals (Delauney and Verma 1993). It was shown to protect proteins during
freeze-thaw cycles by creating a hydration shell for the protein (Carpenter,
Crowe et al. 1990). Proline also serves as a sink for energy to regulate redox
potentials (Alia, Saradhi et al. 1993), as a hydroxy radical scavenger (Smirnoff
and Cumbes 1989), as a solute that protects macromolecules against
denaturation (Schobert and Tschesche 1978), as a mediator of osmotic
adjustment (Handa, Handa et al. 1986). It lowers the generation of free
radicals and at the same time prevents lipid peroxidation in thylakoid
membranes (Alia, Saradhi et al. 1993). Proline acts to protect developing cells
from osmotic damages, especially during those developmental processes, such
as pollen development and embryogenesis, in which tissues are exposed to
spontaneous dehydration. The importance of proline in improvement of stress
tolerance in plants was shown by manipulation of genes encoding key
enzymes of the proline synthesis or degradation pathway (Nanjo, Kobayashi
et al. 1999, Nanjo, Kobayashi et al. 1999) and it forms part of the large battery
of metabolites and stress-induced proteins that plants utilize to their cellular
membranes and metabolic processes during freezing and freeze-induced
dehydration.
5. Photosynthesis in cold acclimation
Plants routinely experience, and must compensate for, fluctuations in their
light environment. In addition, fluctuations in water availability, nutrient
status and temperature all feedback and impact on the ability of the plant to
maintain a balance between energy capture and utilization. These various
changes occur on daily and seasonal basis and plants need to acclimate to
19
these changes in the surrounding environment. Cold acclimation also does not
progress in the dark or in low CO2, unless the plant has significant stored
carbohydrate reserves (e.g. tubers) (Wanner 1999). Plants therefore need
energy to attain a cold acclimated state and this comes from photosynthesis,
so the photosynthetic system and the entire chloroplast must be able to
acclimate to low growth temperatures and the organelle must be protected
from cold- and freeze-induced injury. Photosynthesis is the process through
which the light energy from the sun is absorbed and utilized to provide the
energy for almost all biological reactions. There are two phases of
photosynthesis. The first one is dependent on the light where primary
photochemistry involves the absorption of sunlight by the pigments of the
light-harvesting protein complex (LHC) and the transfer of this energy to the
reaction centres of photosystem II (PSII) and photosystem I (PSI). There is
transfer of electrons from PSII through the electron-transport chain
(plastoquinone (PQ), the cytochrome b6/f complex (CYT b6/f) and
plastocyanin (PC) to PSI. The chemical energy captured by this process results
in the oxidation of water molecules, the release of oxygen, and the generation
of reductant (reduced ferredoxin and NADPH) and high-energy phosphate
bonds (ATP) (Anderson and Melis 1983). This process takes place in the
thylakoid membranes of chloroplasts. The second phase of photosynthesis is
connected with the carbon-fixing reactions (light-independent reactions) in
the stroma where the ATP and NADPH are used by various enzymes of the
Calvin cycle to fix carbon dioxide and to generate sugars (Waters and Langdale
2009). These sugars can then be metabolized into other carbohydrates and
used for the production of energy, amino acids and lipids. Because of the
central importance of this reaction, the strengths of the various catalysts
generated and the inherently variable environment in which photosynthesis
occurs, mechanism have evolved that enable plants to keep a balance between
absorbed light energy and energy consumed in electron transport and
metabolism (carbon fixation) to protect PSII from over-excitation. The
importance of these processes to cold acclimation is illustrated by the fact that
chloroplasts, especially the thylakoid membranes, are one of the cellular
20
compartments most targeted by the protective mechanisms discussed in the
preceding sections.
5.1. Effects of low temperature on photosynthesis
Low temperatures reduce CO2 fixation, which in turn reduces the availability
of
substrates
for
photosynthetic
electron
transport
(NADP+)
and
photophosporylation (ADP), resulting in feedback inhibition of electron
transport. Low temperatures also directly reduce the rate of photosynthetic
electron transport by increasing thylakoid membrane viscosity, caused by
alteration in biophysical properties of thylakoid lipids (Huner, Öquist et al.
1998), restricting the diffusion of plastoquinone (PQ). Thus, when plants are
exposed to low temperature an imbalance is created when chlorophyll
antenna complexes trap more energy that can be processed biochemically
(Huner, Öquist et al. 1993, Ensminger, Busch et al. 2006, Wilson, Ivanov et
al. 2006). The result of PQ being reduced and of the reduced availability of
electron acceptors (NADP+) at PSI is the synthesis of reactive oxygen species
(Moller 2001). Consequently, cold treatment increases excitation pressure,
induces photoinhibition (Powles and Björkman 1982, Hurry and Huner 1991,
Hurry and Huner 1992, Huner, Öquist et al. 1998) and increases the
production of ROS (Moller 2001). Low temperatures also attenuate the
activity of scavenging enzymes, and this further promotes the overaccumulation of ROS such as superoxide (O2-), hydrogen peroxide (H2O2), and
the hydroxyl radical (.OH) (Moller 2001) and to harmful effects such as lipid
peroxidation, carbonylation of proteins, formation of disulfide and dityrosine
bridges, modification of amino acids in polypeptide chains, DNA damage as
well as modifications of the structure of pigments (Gill and Tuteja 2010). The
other effect of low temperature on photosynthesis is connected with Calvin
cycle, where activity of enzymes involved in carbon fixation slow down. One
of the consequences of thylakoid membranes being in an over-energized state
21
is photodamage, caused initially by the increased formation of ROS.
Superoxide is the major cause of photodamage to PSI in the cold, whereas
hydrogen peroxide and superoxide are the most damaging forms for PSII
(Tjus 2001). The major site of photoinhibition in PSII is the inactivation of the
D1 protein (Aro, Virgin et al. 1993). Recovery from PSII photoinhibition is
strongly temperature dependent and low temperature will decrease the rate of
repair of PSII (Gombos, Wada et al. 1994). Failure to dissipate or avoid
accumulating excess excitation energy leads to photooxidative damage to the
photosynthetic apparatus, which is often manifested as bleaching, chlorosis or
bronzing of leaves (Niyogi 2000).
5.2. Acclimation responses of photosynthesis to low
temperature
Full cold acclimation requires the production of new leaves that have
developed under the new growth condition (Huner, Öquist et al. 1993, Strand,
Hurry et al. 1997). For example, leaves that have developed at 5°C showed
recovery of photosynthesis to the level comparable with control leaves at 23°C
(Hurry, Gardestrom et al. 1993, Strand, Hurry et al. 1997, Strand, Foyer et al.
2003). The recovery of photosynthesis leads to increased tolerance to
photoinhibition (Hurry, Gardestrom et al. 1993) and also to the reestablishment of the balance of energy flow in order to cope with low
temperature stress together with high light stress (Ivanov, Rosso et al. 2012).
In order to maintain the thylakoid energy balance there has to be control of
the flow of energy (electrons) into the electron transport chain and control of
the flow of energy out through the acceptor (NADPH) pool and into
metabolism. The flow of energy into electron transport is modulated during
acclimation to long-term exposure to high light and low temperature by an
increase in xanthophyll-cycle pigments and in a persistent engagement of the
xanthophyll cycle resulting in sustained antenna quenching of excess energy
22
through non photochemical quenching (NPQ). The capacity of NPQ for
maintaining the thylakoid energy balance was elegantly demonstrated by
studies that showed when it fails it can have significant impact on seed
productivity in Arabidopsis under field conditions (e.g. (Kulheim 2002). In
addition, flow of energy into the electron transport chain can be modulated by
alterations in PSII photochemistry, resulting in reaction centre quenching
(Sane 2003, Ivanov, Rosso et al. 2012). The flow of energy out of the electron
transport chain requires modulation of flux through the Calvin cycle. This is
achieved by increased activity of Calvin cycle enzymes related to carbon
reduction cycle (ribulose-1,5-biphosphate carboxylase/oxygenase, (RuBisCO)
stromal fructose-1,6-biphosphatase, (sFru-1,6-BPase) and sedoheptulose-1,7biphosphatase (Holaday, Martindale et al. 1992, Strand 1999). Beyond the
plastid, the flow of captured energy is facilitated through enhanced synthesis
and export sucrose via selective stimulation of sucrose synthesis (Guy, Huber
et al. 1992, Strand, Foyer et al. 2003). Fully acclimated herbaceous annuals
therefore have the ability to not only fix carbon but also to transport this fixed
carbon to sink tissues to support growth and acclimation processes
(Lundmark, Cavaco et al. 2006). Another acclimation response of
photosynthesis is an increase in phosphate availability, which is a result of
adjustments in Pi between different cellular compartments (Hurry, Strand et
al. 2000). Under normal growth conditions the Pi content in the cytosol is
constant and low. When it is in excess, Pi accumulates in vacuole (Foyer and
Spencer 1986). Under cold stress and during cold acclimation, some of this Pi
is released into the cytosol to facilitate the build-up of phosphorylated
intermediates (Strand 1999). Thus, the acclimation of photosynthesis and the
reprogramming of carbon metabolism under growth at low temperature are
linked and are crucial for overwintering herbaceous annuals to survive long
term exposures to cold (Strand, Hurry et al. 1997).
23
5.3. Changes in the environment can be sensed by the
chloroplast
Changes in chloroplast homeostasis are closely associated with changes in
photosynthetic activity. The photosynthetic reactions housed in the
chloroplasts are extremely sensitive to stress (Huner, Öquist et al. 1998).
Therefore chloroplasts could play a critical role as sensors of changes in the
growth environment (Lee, Kim et al. 2007). Changes in the redox status of the
photosynthetic electron transport chain, for example, trigger changes to
nuclear gene expression through a so-called retrograde signalling processes.
Retrograde communication coordinates the expression of nuclear genes
encoding organellar proteins with the metabolic and developmental state of
the plastid and mitochondria through signals emitted from the organelles that
regulate nuclear gene expression (Susek, Ausubel et al. 1993). It is now clear
that several different plastid processes produce these signals and that plastid
to nucleus communication appears to be of particular importance during plant
stress responses (Fernández and Strand 2008). The plastid signals identified
so far can be linked to specific stress conditions. One such signal is connected
to the tetrapyrrole biosynthetic pathway. Under stress conditions the flux
through the tetrapyrrole biosynthetic pathway is inhibited, which coincides
with changes in nuclear gene expression (Strand, Asami et al. 2003, Ankele,
Kindgren et al. 2007). The chlorophyll intermediate Mg-protoporphyrin IX
(Mg-ProtoIX), which transiently accumulates in the chloroplasts under
oxidative stress conditions, is somehow involved in the repression of LHC
transcription in the nucleus in eukaryotes (Kropat, Oster et al. 1997). Another
class of signal molecules is ROS that accumulates under exposure to high light.
Accumulation of ROS serves as a link between excitation pressure in the
chloroplast and nuclear gene expression (Karpinski 1999). Chloroplast signals
have also been shown to play a key role in the cold acclimation process in
barley (Svensson 2006). Barley plants with mutations preventing proper
chloroplast development (albino and xantha) are frost susceptible and
impaired in the expression of several COR genes, suggesting that a functional
chloroplast is required for proper cold acclimation.
24
6. Gene expression during cold acclimation
Weiser was one of the first to hypothesize altered gene expression under low
temperature in woody plants (Weiser 1970). The first study in herbaceous
plants, supporting Weiser’s hypothesis, was Guy et al (1985) whose work with
spinach leaves showed that there was synthesis of a population of new mRNAs
when plants were shifted to 5°C. The extension of this line of research into
other herbaceous plants was carried out in barley (Cattivelli and Bartels 1989)
and Arabidopsis thaliana, where in vitro translation of poly(A)RNA showed
the synthesis of specific translatable mRNA populations under low
temperatures (Gilmour, Hajela et al. 1988). The same technique was used to
show that a unique population of translatable mRNAs was induced by low
temperature in rapeseed (Meza-Basso, Alberdi et al. 1986), and alfalfa
(Mohapatra, Poole et al. 1987). Palva’s group extended this work further to
show a correlation between freezing tolerance and the induction of soluble
proteins in Arabidopsis (Kurkela, Franck et al. 1988).
Since then, the direction in cold acclimation research has been to find and
characterize cold responsive genes and to identify what role they play in
freezing tolerance. An important breakthrough in this field was the
identification of a cis acting regulatory element in the promoter region of
RD29A in Arabidopsis and tobacco plants (Yamaguchi-Shinozaki and
Shinozaki 1994). The dehydration-responsive element (DRE), containing 9 bp
(TACCGACAT), was shown to be involved in the induction of novel genes by
low temperature. The core sequence of this element, which contains CCGAC,
designated the C-repeat (CRT), was found in other COR (cold regulated) genes
and shown to also be required for cold responsiveness in genes such as BN115,
BN19, BN26 from Brassica napus (Weretilnyk 1993, Jiang, Iu et al. 1996), and
wsc120 from wheat (Ouellet, Vazquez-Tello et al. 1998). The CBF (Crepeat/dehydration responsive element binding factor) proteins activate
expression of a set of target effector genes by binding to this, CRT/DRE
25
promoter element (DRE). Stockinger et al. (1997) first isolated the cDNA
sequence of CBF1 from Arabidopsis, which encodes one of the three coldinducible CBFs: CBF1, CBF2 and CBF3 (Gilmour, Zarka et al. 1998, JagloOttosen 1998) or DREB1b, DREB1c, and DREB1a (Liu, Kasuga et al. 1998).
The importance of CBFs for freezing tolerance was demonstrated in
transgenic studies where even at warm temperatures, constitutive expression
of CBF1, 3 and 4 can increase freezing tolerance (Jaglo-Ottosen 1998, Liu,
Kasuga et al. 1998, Gilmour 2000, Haake 2002, Gilmour, Fowler et al. 2004).
Expression of all CBFs responds very rapidly to changes to low temperature,
the transcript levels for all three CBF genes increases within 15 min of cold
treatment, followed by accumulation of COR gene transcripts at about 2 h
(Gilmour, Zarka et al. 1998). CBF induction also takes place during other
abiotic stress conditions such as drought (Shinozaki and YamaguchiShinozaki 2006), high-salinity (Liu, Kasuga et al. 1998) as well as mechanical
agitation (Gilmour, Zarka et al. 1998, Akhtar, Jaiswal et al. 2012). CBF1 and
CBF3 are positive regulators whereas CBF2 has a negative regulatory role
(Novillo, Medina et al. 2007). Since the first discovery of CBFs there have been
many reports showing the expression pattern of these genes and their
regulation in other plant species, for example wheat, rye, and tomato (Ouellet,
Vazquez-Tello et al. 1998, Jaglo, Kleff et al. 2001) suggesting that this family
of genes is not conserved among Brassica class only and is not restricted only
to plants which cold acclimate.
An important breakthrough in efforts to uncover more players in CBF regulon
was provided by Chinnusamy et al. (2003) who demonstrated that “inducer of
CBF expression 1” (ICE1) was a component of the CBF-dependent signalling
pathway and acted upstream of the CBF transcription factors. ICE1 was
identified from a genetic screen where CBF3–LUC plants were chemically
mutagenized and mutants with altered cold-induced CBF3–LUC expression
were isolated. Ice1 inhibits CBF3 expression, together with many of its
downstream genes, leading to a decrease in freezing tolerance. ICE1 induces
26
expression of CBF3 by binding a MYC recognition element present in the
CBF3 promoter (Chinnusamy 2003). Another additional step discovered in
CBF regulon were posttranslational modifiers of ICE1. One of them is the
Small Ubiquitin Related Modifier E3 ligase SIZ1 (SAP and Miz1) (Miura, Jin
et al. 2007) and the ubiquitin E3 ligase High Expression of Osmotically
Responsive Genes1 (HOS1) (Dong, Agarwal et al. 2006). Recently there were
reports adding new components in ICE-CBF-COR pathway that act up-stream
of ICE1 (e.g. OST1, a Ser/Thr protein kinase, was shown to act upstream of
CBFs to positively regulate freezing tolerance. OST1 interacts with ICE1 and
HOS1 by phosphorylation leading to stabilization of ICE1 so that it is not
targeted for degradation by HOS1 (Ding, Li et al. 2015) (Fig 2). A new
component acting in the CBF regulon, JAZ, is a repressor of jasmonate
signalling. There are also components that are not directly connected with the
CBF regulon signalling pathway but revealed influence CBF expression and
the level of freezing tolerance. For example, CRLK1 (calmodulin like receptor
protein kinase) improves freezing tolerance by modifying the CBF regulon
(Yang, Shad Ali et al. 2010). Another example is the presence of negative
regulators of the CBF regulon e.g. the MYB15 transcription factor, which binds
to MYB recognition elements in the promoters of CBF genes. The myb15
mutant shows enhanced cold induction of CBFs and freezing tolerance,
whereas MYB15-overexpressing plants are defective in CBF expression and
hypersensitive to freezing injuries (Fig 2). Thus, research has come a long way
in uncovering many of the components in the CBF response pathways,
showing how this pathway overlaps with other stress-response pathways such
as drought and salt (Stockinger, Gilmour et al. 1997, Shinozaki and
Yamaguchi-Shinozaki 2000). This knowledge has been used to successfully
create transgenic lines with increased freezing tolerance (e.g. overexpression
of CBF1 in RLD-Arabidopsis ecotype, (Jaglo-Ottosen 1998) and these findings
with model plants have been transferred to produce more tolerant crop plants
(e.g. Brassica napus (Jaglo, Kleff et al. 2001) and more tolerant trees (e.g.
(Benedict, Geisler et al. 2006)). The question that remains is how useful is this
knowledge if one wants to shift from controlled environment studies to the
field to develop more tolerant field and woody crops? It is necessary to keep
27
in mind that rarely do plants experience only one stress at a time. It is not only
the combination of various abiotic stresses that plants must be able to cope
with but also the co-occurrence and influence of biotic stresses on the survival
of the plant in the field.
Figure 2. Summary of cold signaling pathway components. Part of the pathway is CBF
dependent with ICE1 which is a MYC-type transcription factor and binds to ciselements in the promoter of CBF3/DREB1A to induce downstream components
expression. There is also CBF independent pathway for example ZAT12 and RAV1
which are induced by cold and influence expression of cold regulated genes and their
targets. (modified from (Chinnusamy, Zhu et al. 2007). OST1 interacts with ICE1 and
HOS1 by phosphorylation leading to stabilization of ICE1 and prevents its degradation
by HOS1. CRLK1 is a Ca 2+/CaM-regulated Receptor-Like-Kinase modulating
expression of CORs. CDPKs, Calcium-dependent protein kinases possibly interacts
with MAPK and activates downstream signalling pathway (Ding, Li et al. 2015).
28
Despite the significant role known for the CBF genes and their downstream
targets in the development of freezing tolerance, further research indicated
that it is not the only pathway required for plants to cold acclimate. One such
example is the Arabidopsis eskimo1 mutant, which is constitutively freezing
tolerant in the absence of cold acclimation. This mutation does not affect
expression of well-known cold regulated genes and this finding lead to a new
hypothesis that plants need to activate different signalling pathways in order
to fully cold acclimate (Xin and Browse 1998, Kurepin, Dahal et al. 2013).
ESK1 was suggested to be a negative regulator of freezing tolerance (Xin,
Mandaokar et al. 2007) and to be responsible for water uptake from roots
(Lugan, Niogret et al. 2010). Furthermore, the transcription factors RAV1 and
ZAT12, for example, are both induced by cold but neither of them are affected
by CBF overexpression, which demonstrates the likely existence of parallel
signalling pathways activated by cold that are independent of CBF expression
(Fowler 2002) (Fig 2). Subsequent research has shown that ZAT12 plays a role
in mediating oxidative and abiotic stress signalling (Davletova, Schlauch et al.
2005). Overexpression of ZAT12 results in a slight increase in plant freezing
tolerance, and to negative regulation of the expression of the CBF
transcription factor family (Vogel, Zarka et al. 2005). Like the CBFs, ZAT12
expression is also regulated by the circadian clock. The strongest induction of
ZAT12 occurs at the end of the subjective day, reverse-phased with the CBFs
(Fowler, Cook et al. 2005). In contrast, RAV1 expression occurs in phase with
CBF expression over the course of a circadian day (Fowler, Cook et al. 2005).
Since these early discoveries by Weiser (1970) and Guy et al (1985), a great
deal of experimental evidence has provided insight into the signalling
pathways that are activated by cold. We also have evidence of how these
signalling pathways interact with other stress response pathways such as
drought (Seki, Narusaka et al. 2002), with light signalling (Catala, Medina et
al. 2011), circadian regulation (Dong, Farré et al. 2011) and with hormone
signalling (Kurepin, Dahal et al. 2013). These findings show that a complex
and integrated signalling network exists in plant cells that impacts on the cold
acclimation responses of all autotrophic organisms.
29
7. Fast responses to low temperature: cold perception
From the text above it is clear that a number of key elements in the cold
signalling responsive pathways have been discovered and their interactions
described but to date we still do not know how plants sense temperature. Early
suggestions were that the plant cell is able to sense cold stress due to a
decrease in membrane fluidity (Levitt 1980), which occurs when the
temperature falls rapidly. However, it is not just a matter of being able to sense
a change in temperature, the cell needs to be able to convert this information
into a signal to generate an appropriate response. This signal must be sent to
the nucleus, via at least some of the elements described in the previous
sections, where a complex transcriptional response is triggered. In
mammalian cells, TRP (Transient Receptor Potential) proteins have been
shown to play a role in signalling pathways under different stimuli e.g. TRPV,
is activated by heat and low pH. ‘thermoTRP’ proteins are involved in
thermosensing and are activated by cool temperatures (10-23°C) as well as
compounds that evoke a sensation of coolness, such as menthol or eucalyptol
(McKemy, Neuhausser et al. 2002). This family of transmembrane ionchannel proteins is cation selective and calcium-permeable, and based on
what we currently know of cold signalling would be reasonable candidates also
for cold sensing in plants. However, to date no proteins related TRPs have
been identified in plants. Turning to the cyanobacterial model, a typical
prokaryotic two-component system consists of a Hik (histidine kinase) and a
Rre (response regulator) (Murata and Los 2006). A pathway for perception
and transduction of low‐temperature signals involves histidine kinase 33
(Hik33) as a membrane‐embedded “cellular thermometer.” In Synechocystis
sp. PCC 6803 when temperature drops, leading to a decrease in membrane
fluidity (Fig 1), Hik33 was found to perceive cold and this sensory kinase
includes a type-P linker, a leucine zipper, and a PAS domain (Los and Murata
2004) (Fig 3A). The type-P linker consists of two helical regions that
transduce stress signals via intramolecular structural changes that result from
30
interactions between the two helical regions and lead to intermolecular
dimerization of membrane proteins (Aravind, Anantharaman et al. 2003).
Later, a histidine residue within the conserved histidine kinase domain is
autophosphorylated, with ATP as the donor of the phosphate group. The
phosphate group is transferred from the Hik to the conserved receiver domain
of the Rre. When phosphorylation occurs, the Rre changes its conformation,
and this change allows the Rre to bind to the promoter regions of genes that
are located further downstream in the signal-transduction pathway.
A reduction in the fluidity of the plasma membrane of Synechocystis appears
to be the primary signal for the low-temperature-induced expression of the
genes for desaturases (Vigh, Los et al. 1993, Murata and Los 1997).
Functionally, a decrease in membrane fluidity at the sites at which Hik33 is
located is thought to alter the structure of Hik33, influencing the spatial
relationship between the monomers of the dimer protein, resulting in altered
activity. The cold sensor Hik33 regulates the expression of a half of the coldinduced genes in Synechocystis (Suzuki, Los et al. 2000, Suzuki, Kanesaki et
al. 2001, Murata and Los 2006). The expression of another group of coldinduced genes, which is not controlled by Hik33, was shown not to be
dependent on changes in membrane fluidity (Murata and Los 2006). The
molecular mechanism of this sensing is still unclear. It was suggested that
lateral diffusion of the membrane spanning domains of the Hik33 may lead to
a drift of its subunits, to dimerization and to autophosphorylation (Los and
Murata 2004). As a member of the two-component regulatory system, Hik33
should operate via its response regulator (Rre) to transduce a signal of
membrane rigidification and to induce the responsive genes. Another example
of temperature sensing comes from histidine kinase DesK of Gram-positive
Bacillus subtilis, which provides more insight into the possible molecular
mechanisms of sensing the changes in membrane properties (Cybulski,
Martín et al. 2010). The DesK sensor kinase includes five TM-spanning
segments that sense an increase in the order of membrane lipids (Fig 3B),
31
promoting
a
kinase
dominant
state
of
DesK,
which
undergoes
autophosphorylation and transfer of the phosphate group to the response
regulator Des (Aguilar, Hernandez-Arriaga et al. 2001). Phospho-DesR
activates the expression of the des gene (Albanesi, Martin et al. 2009) for the
D5-desaturase, the only FAD in B. subtilis that is involved in the maintenance
of membrane fluidity (Mansilla, Cybulski et al. 2004). It is now thought that
the most important parameters of sensing a temperature downshift (37°C to
25°C) is the match between the hydrophobic thickness of the membrane and
the hydrophobic thickness of the transmembrane segments of the integral
membrane protein. Any mismatch between the thicknesses of the lipid bilayer
and the protein may lead to modification in the structure of the protein
(Cybulski, Martín et al. 2010), which may trigger the low temperature
response (Cybulski and de Mendoza 2011). Currently more detailed
information is available about putative action of cold sensor in
cyanobacterium and other bacterial systems. This is becoming a significant
advantage for plant research because description of these mechanisms may
serve as good background for plant scientists to find the mode of action of
plant cold sensor(s). However, the mechanism involved in cold sensing in
higher plants can be more complex because plants consist of a large number
of cells types, where each cell contain subcellular compartments and
membranes that differ in their function and composition.
Figure 3. Two component system, cold sensor in cyanobacterium Synechocystis (A),
Bacillus subtilis (B) modified from (Los, Mironov et al. 2013). Details in text.
32
As with the mammalian TRP proteins, no bacteria-like two-component
system for temperature sensing has yet been identified in plants. One
hypothesis for how cold could be perceived by plants comes from studies of
Medicago sativa (Orvar, Sangwan et al. 2000) where membrane fluidity
influenced rearrangements in the actin cytoskeleton, resulting in Ca2+ influx
into the cytosol from extra-cellular pools. Stabilization of the actin
microfilaments in the cold by the addition of various pharmacological agents
resulted in a loss of cold-induced cas30 expression and a stable level of
cytosolic Ca2+. Conversely, actin destabilization at 25°C led to calcium influx
and subsequent cas30 expression. This suggest that Ca2+ is involved in
primary sensing events and also influence downstream events in signalling
pathway activated by cold and the involvement of calcium in cold signalling
has been presented in many studies (Knight, Campbell et al. 1991, Orvar,
Sangwan et al. 2000). The work of Henriksson and Trewavas, (2003) showed
that cold induced expression of RD29A is connected with the level of calcium
in cytosol. This was proved by using calcium signalling antagonists, which
partially reduces the low temperature induced increase in calcium and at the
same time reduced induction of RD29A expression. Calcium levels were also
shown to modulate expression of other cold responsive genes such as kin10 in
Arabidopsis (Knight, Trewavas et al. 1996). Other possibilities of fast
responses of low temperature signal perception are suggested from work in
Arabidopsis where a plant-specific Ca 2+/CaM-regulated RLK, (CRLK1), was
reported to be involved in cold tolerance (Yang, Shad Ali et al. 2010). The
calcium/calmoduline signal has a positive effect on the kinase activity of
CRLK1, but when calmodulin was inhibited, this inhibition had negative effect
on kinase activity. The crlk1 mutant showed impaired cold-induction of CBF1,
RD29A, COR15A and KIN1, all are well-known cold regulated genes.
The other type of kinases that participate in various signalling cascades and
are very common in the plant kingdom are RLKs (receptor like kinases). The
LRR-RLKs (leucine reach repeat receptor like kinases) form a major RLK
family, and has more than 200 members divided into 15 subfamilies (LRR I–
LRR XV) in Arabidopsis (Shiu, Karlowski et al. 2004). The mode of action is
very different for each subclass of the LRR-RLK family but a common feature
is that most are localized in membranes and mediate abiotic and biotic signals
from the environment to the nucleus, influencing the expression of stress
related genes. Members of the LRR-RLK family play various roles in signalling
events such as: brassinosteroid (BR) perception by the BR INTENSITIVE 1
33
(BRI1) and BR1-ASSOCIATED RECEPTOR KINASE1 (BAK1) (Oh, Wu et al.
2011). CLAVATA1 (CLV1) is responsible for meristem size regulation and
involved in perception of the peptide hormone CLV3 (Deyoung and Clark
2008). The ERECTA family and EPF (EPIDERMAL PATTERNING FACTOR)
play roles in organ growth and stomatal cell differentiation by the peptide
receptors (Kim, Michniewicz et al. 2012). Despite the wealth of information
on how fast responses to cold can be perceived there is still a lack of consensus
for the existence of a true cold sensor in plants, perhaps finding more overlap
between different signalling pathways might be a solution to identify the
sensing mechanism(s) in higher plants.
8. Role of the circadian clock in cold stress responses in
plants
Most living organisms have to adapt to the daily changes between light and
dark cycles, and high and low temperatures. This adaptation has led to the
evolution of an endogenous timekeeper, called the circadian clock. This
biological timing is crucial for organisms to be able to synchronize
developmental and metabolic processes to the most favourable time of the
day. Harmer et al. (2000) hypothesized that circadian clock-controlled genes
are subjected to stress regulation. This was also confirmed in a later study by
Kreps et al. (2002) where it was shown that 68% of the circadian controlled
genes are linked to a stress response pathway and that some of the circadianregulated genes were also cold responsive. For example, CBF3 transcript
levels display a circadian-regulated cycling at warm temperatures, reaching a
peak at Zeitgeber time ZT4 and a minimum at ZT16 (Harmer, Hogenesch et
al. 2000, Bieniawska, Espinoza et al. 2008). Rapid cold induction of all three
CBF genes, together with two of their known targets, was also shown to be
gated by the circadian clock (Fowler, Cook et al. 2005), suggesting that clock
control of the CBF genes may be important for the full expression of cold
34
resistance. In diurnal conditions (light/dark cycles) it was shown that cold
reduces the amplitude of cycles for circadian clock genes and disrupts the
cycling of output genes. When plants are shifted to continuous light, cycling
of circadian genes starts to be arrhythmic (Bieniawska, Espinoza et al. 2008).
These examples demonstrate that low temperature may work in combination
with the circadian clock to influence the cold acclimation state of the plant,
and suggests that the time of the day that the plant is exposed to cold stress
may influence its ability to respond.
The interaction between the circadian clock and plant cold acclimation status
has been investigated by several groups. Central oscillator genes such as CCA1
(Circadian Clock Associated1) and LHY (Late Elongated Hypocotyl) are
directly involved in cold stress responses, because the cca1-11 and the cca111/lhy-21 mutants were more sensitive to freezing and were less able to cold
acclimate in comparison to wild-type (Ws) Arabidopsis plants (Espinoza,
Degenkolbe et al. 2010). The double mutant (cca1-11/lhy-21) also showed
reduced expression of cold regulated genes such as COR15A, COR47, and
COR78 (Dong, Farré et al. 2011), which demonstrate that CCA1/LHYmediated output from the circadian clock participate in attainment of plant
cold tolerance by regulation of the CBF cold-response pathway. Furthermore,
a triple mutant lacking the clock component genes such as PRR9, PRR7 and
PRR5 (Pseudo Response Regulator) was found to have increased freezing
tolerance and to accumulate more compatible solutes such as proline and
raffinose (Nakamichi, Kusano et al. 2009). There is evidence for not only the
CBF signalling pathway being regulated by the circadian clock but also that
the CBF independent pathway can be affected. For example GI (gigantea),
which was suggested to be involved in photoperiodic responses, is involved in
mediating the cold stress response in Arabidopsis (Cao, Ye et al. 2005). GI is
induced by cold, and gi-3 plants showed an increased sensitivity to freezing
stress. It is likely that GI regulates freezing tolerance via a CBF independent
pathway because expression of the “CBF regulon” (CBF1, 2, 3) and some of
35
their targets genes such as RD29A, COR15A, KIN1, and KIN2 were not
different in wild-type control and gi-3 mutated plants (Cao, Ye et al. 2005).
These examples of the interaction between circadian clock and cold
acclimation at the molecular level demonstrate that it is a relevant part of the
cold acclimation process and it shows that for a plant to reach full acclimation
many different signalling pathways have to be integrated in order to correctly
track and fully adjust to the changing environment (Kurepin, Dahal et al.
2013).
9. Long-term cold acclimation-ecological perspective
There are different ways plants can experience low temperature. The first is as
a single frost event, which can happen any time during plant development. It
will appear as stress to the plant and will be seen as sudden change in
temperature. This experience may lead to damage and the extent of that will
depend on many factors such as: severity, duration, whether it is repeated and
whether it occurs in combination with other stressors such as high light. In
addition, the severity of the stress will be influenced by various plant
characteristics, such as the organ/tissue in question, the stage of development
and genotype. The second situation is connected with over-wintering by
winter annuals and how they acclimate to survive winter. We know a good deal
about the physiological changes that occur during prolonged growth in cold
and there are many reports showing that reprograming of photosynthetic
carbon metabolism in favour of cytosolic soluble sugars rather than
chloroplastic starch synthesis occur in response to low temperature (Huner,
Öquist et al. 1993, Strand, Hurry et al. 1997, Strand 1999, Hurry, Strand et al.
2000, Strand, Foyer et al. 2003). These changes in metabolism are also linked
to broader changes in leaf architecture in order not only to support full
recovery of metabolism but also to recover leaf source functions such as
36
carbon export to sink tissues (Lundmark, Cavaco et al. 2006). However, to
date still little is known about the broader transcriptomic reprogramming that
underpins these long-term changes in metabolism and leaf architecture.
The other type of cold experience is when plant grows in a cold environment.
Arctic plants, plants from the sub-Antarctic islands and high alpine species
are examples of plants that spend their entire life-cycle at low temperatures.
The only native flowering plants in the sub-Antarctic islands are Colobanthus
quitensis (Caryophyllaceae) and Deschampsia antarctica (Poaceae) (Xiong,
Mueller et al. 2000). These plants as well as similar extremophiles can be used
as a natural source of resistance genes to abiotic stresses. They can also serve
as ecological markers for adjustment to a changing/warming environment,
because Deschampsia is able to spread in new environment much faster than
other species like bryophytes (Fowbert and Smith 1994). The valuable work of
Edward and Lewis Smith (1988) and Xiong et al (2000) provided an overview
of the natural mechanisms that lead to tolerance to different abiotic stresses
in Deschampsia. It shows adaptation of photosynthesis to low temperature:
with maximal photosynthetic activity occurring at 13°C in D. antarctica, and
30% of its maximal photosynthetic capacity is retained even at 0°C. Moreover,
Deschampsia accumulates proline, sucrose, and fructans, in the leaves during
the growth period, which assures high levels of resistance to cold stress
(Bravo, Ulloa et al. 2001). Furthermore, it produces antifreeze compounds in
the non-acclimated state, which increase after cold acclimation, indicating
that the plant secretes antifreeze proteins into the appoplasts, which increase
their survival in low temperature (Bravo 2005). D. antarctica has high
recrystallization inhibitory activity. This feature prevents the growth of bigger
ice crystals at low temperatures (John, Polotnianka et al. 2009).
Another source of additional information about development of the novel
mechanisms in non-optimal environment are extremophiles which provide
37
new insight into the different mechanisms plants have evolved in order to
survive in extreme environments. One such example is Thellungiella
halophile (Eutrema salsugineum) an extremophile that is able to withstand
different abiotic stresses such as freezing, drought, nutrient-deficiency and
high salinity (Inan 2004, Griffith, Timonin et al. 2007, Guevara, Champigny
et al. 2012). Thellungiella, is a close relative of Arabidopsis, but has much
higher freezing tolerance (from -13.0 to -18.5°C after cold treatment) (Griffith,
Timonin et al. 2007) and it uses different metabolic acclimation strategies
(Bonn, Zinzen et al. 2012). Analysis of Thellungiella expressed sequence tags
provided evidence for the presence of paralogs that have not been annotated
in the Arabidopsis genome as being realted to stress, and for novel genes with
functions relevant to abiotic stress, suggesting that it can be used as a tool for
genetic engineering for traits like “resistance to abiotic stresses” (Inan 2004).
These features of extremophiles, together with newly available transcriptome
sequencing data for Deschampsia (Lee, Noh et al. 2013), can be used to gain
more knowledge into how crops plants can be manipulated in order to produce
higher yields despite unfavourable climate change.
Furthermore, extremophiles can be seen as drivers for evolution. They show a
hysteresis effect that is a memory of the stress. When such plants experience
stress, the next time the stress comes it does not behave as if it is stressed
(Gutschick and BassiriRad 2003). Analysis of gene expression under cold
acclimation, deacclimation and reacclimation provided a view on plant stress
memory (Byun, Koo et al. 2014). It showed that plants with prior exposure to
cold stress respond differently to repeated cold stress, and it is possible for
plants to retain a memory of the previous cold stress at the gene expression
level (Byun, Koo et al. 2014). Modified signal transduction pathways, changed
lipid composition, the presence of defence proteins, and efficient
photosynthesis all contribute to enhanced cold tolerance when plants were
exposed to repeated cold stress (Byun, Koo et al. 2014). Increasing interest
into species naturally living in extreme environments will provide us new
38
insights or development of new strategies which can be introduce to breeding
programs in order to increase crop performance in changing conditions.
39
Aim of the study
The aim of this thesis was primarily to reveal a better understanding of how
cold temperatures are perceived by the plants and how this is converted into
a fast response by the plant cell. Moreover, the secondary goal was to
understand how plants respond over longer periods in the cold so that they
are able to over-winter or even spend their entire life in the cold. The main
questions addressed in this thesis are:
1. What happens during a temperature downshift? I would like to extend
the current knowledge of the very early events of how cold is sensed
and how the cold signal is translated into gene expression leading to
cold acclimation. Where is cold sensed? Is there any signal generated
by the organelles that is transmitted to communicate changes in
temperature to the nucleus? Is there any other signal within the cell
that might contribute to cold acclimation and an increase in freezing
tolerance?
2. What long-term adjustments do plants make to cold? We know that
plants must not only be able to survive short-term frost events but
they must also be able to over-winter, so what is crucial for a plant to
survive/live in low temperature for longer periods?
40
Results and discussion
Plant responses to abiotic stress vary depending on both the duration and the
severity of the stress event. Here I discuss two types of exposure to low
temperature and the characteristic responses for each. The first part of my
discussion will be dedicated to a very short-term exposure to cold (4°C) that
lasts from seconds until a few hours. I will concentrate on the origin(s) of the
various signal(s) that are generated and sent to the nucleus during cold stress
events in order to regulate gene expression and orchestrate a response by the
whole cell. The second type of exposure is when plants are grown in warm
temperature and shifted to low temperature as mature rosettes for a long
period (approximately 40 days), which allows them to develop new leaves in
new conditions and also plants that germinate and complete their life cycle at
4°C. I would like to address the question of what determines plant survival/life
in prolonged exposure to low temperature.
1. Cold signal perception
In cyanobacteria it has been hypothesized that changes to membrane fluidity
that occur in response to a drop in ambient temperature is the primary
temperature sensing mechanism (Los and Murata 2004), and there has been
speculation that the same might be true in higher plants (e.g. (Murata and Los
1997). One example of how cells might sense cold comes from studies with
Synechocystis sp. PCC 6803 where histidine kinase33 (Hik33) forms part of a
two component system shown to be involved in cold perception (Suzuki,
Kanesaki et al. 2001). When the temperature drops, Hik33 autophosporylates
and transfers a phosphate group to receiver domain1 (Rer1), which in turn
triggers expression of desBD genes (Suzuki, Los et al. 2000). This study
further demonstrated that the decrease in membrane viscosity upon cooling
activated Hik33 but the detailed mechanism of signal perception was not
41
shown. Another bacterial model that provides insight into how cold might be
perceived by cells comes from recent studies of Bacillus subtilis. This model is
based on a two-component system, similar to that described for
Synechocystis, but contains additional information. The crystallographic
structure of DesK revealed the conformational changes that occur to the
protein in the transmembrane region during a downshift of temperature
(Albanesi, Martin et al. 2009). Deletion of one out of five transmembrane
domains (TM1) was shown to block the signal from being transduced inside
the cell, suggesting that this particular domain harboured a temperaturesensing motif. This meant that TM1 perceives the change in temperature and
transmits this information to the last transmembrane domain (TM5)
(Cybulski, Martín et al. 2010). Further experiments manipulating the domains
of DesK showed that just TM1, together with TM5, was enough to transmit the
signal through the membrane (Cybulski, Martín et al. 2010). It was further
hypothesized that under low temperature, when the membrane is thicker
because of an increase in the lipid order, this caused the hydrophilic motif to
be trapped inside the hydrophobic membrane environment, and in this
condition DesK shows kinase activity (transfer phosphate group to receiver
regulator). Conversely, when the temperature is higher the membrane lipids
become less organized and the membrane is thinner, which allows DesK to
have phosphatase activity.
While no cold sensing model is available for plants, I show in Paper I that
CRMK1, a leucine-rich-receptor-like kinase (LRR-RLK) acts as one of the
sensors in the Arabidopsis cold signalling network. CRMK1 belongs to large
family of receptor like kinases (RLKs), which are transmembrane proteins
that perceive signals through their extracellular domains and propagate the
signals via their intracellular kinase domain (Shiu, Karlowski et al. 2004).
CRMK1 is localized in plasma membrane (Paper I, Fig 2C) but the mode of
action is not yet known. Perhaps a similar mode of action as that described in
the bacterial component two system might explain the observed function of
42
CRMK1 in Arabidopsis. Supporting this hypothesis I have shown that CRMK1
exhibits autophosporylating activity in protoplasts grown in room
temperature (Paper I, Fig 3) but it is not yet known whether cold can induce
autophosphorylated activity of this protein in vivo. Hik33 was shown to
regulate the expression of only half of the cold induced genes in Synechocystis,
with the remainder being activated by other components not connected with
Hik33 and membrane fluidity (Suzuki, Los et al. 2000, Suzuki, Kanesaki et al.
2001). This suggests that it is not solely membrane viscosity that influences
the activity of a ‘sensor protein’ but that other sensors exist and transmit cold
signals inside the cell. An alternative mode of action for CRMK1 where it may
form sensing complex might be related to brasinosteroid (BR) signalling. In
this case, BR is perceived by the receptor complex containing BRI1 and BAK1,
which are leucine-rich-repeat receptor-like kinases (LRR-RLKs) that interact
with each other (Nam and Li 2002). BR binding stabilizes or promotes the
formation of the BRI1/BAK1 heterodimer, leading to activation of both
receptor kinases via transphosphorylation (Russinova 2004). BR binds
directly to BRI1 or first complexes with an unidentified SBP (Steroid Binding
Protein), which requires processing by a Serine Carboxypeptidase. Whether
any mechanism similar to those outlined above activates CRMK1 is unknown
and will require further studies. Nevertheless, these examples provide credible
posisilities of how such a membrane localized receptor kinase could function
as a cold sensor in plants.
Alternative receptor mechanisms for cold include calcium, and a number of
receptors have been shown to transmit information about the external
environment across the membrane by means of an ion channel that allows
ions into the cell (Cybulski and de Mendoza 2011). For example, it has been
shown that changes in the physical properties of the plasma membrane caused
by low temperature can activate Ca2+ channels in tobacco (Knight, Campbell
et al. 1991), Arabidopsis (Knight, Trewavas et al. 1996), and alfalfa (Orvar,
Sangwan et al. 2000). The increase in cytosolic Ca2+ was able to regulate the
43
activity of many signalling components such as the protein kinase that
phosphorylates ICE1 (Chinnusamy, Zhu et al. 2007) and induces the
expression of CBFs. Knight et al. (1996) also showed that low temperature
increased levels of cytosolic calcium from both extracellular and vacuolar
stores, and is required for cold induction of KIN1, a member of the CBF
regulon. Furthermore, Doherty et al. (2009) provided evidence of a direct link
between calcium signalling and cold induction of the CBF pathway with the
discovery that CAMTA3 factors bind to a regulatory element present in the
CBF2 promoter and the camta1camta3 double mutant showed impaired
freezing tolerance compared to wild type plants. This link shows that CAMTA
function as important components of the fast response, or early warning
system, thanks to their ability to transduce calcium signatures (Doherty, Van
Buskirk et al. 2009). The other components that are regulated by the elevated
level of cytosolic calcium during a drop in temperature are the phospholipases
(Krinke, Flemr et al. 2009, Ruelland, Kravets et al. 2015). For example
phospholipase C (PLC) activation by Ca2+ results in an increase in
diacylglycerol kinase (DAGK)-produced phosphatidic acid (PtdOH) (Ruelland
2002). The activation of PLC is one of the very early steps in the cold induced
signalling pathway that leads to the induction of cold responsive genes such
as MYB73, CZF1 and TCP transcription factors that belong to the CBF
independent pathway (Vergnolle, Vaultier et al. 2005). These examples
demonstrate the importance of calcium as a signalling component of the low
temperature response, transducing the signal to the cytosol in order to initiate
the expression of cold regulated genes.
From the data briefly outlined above, there is no doubt that calcium is
important in cold signalling and it is an early indicator of stress but the main
question remains how is the stress perceived. Monroy et al. (1993) presented
calcium permeable channels as potential cold sensors, because calcium
channel blockers inhibit cold acclimation. More recently the Arabidopsis
plasma membrane calcium transporters, ACA8 and ACA10 were shown to be
44
cold induced. ACA10 contains a DRE/CRT-motif in its promoter region and
its expression is induced by cold stress in guard cells and vascular tissue
(Schiott and Palmgren 2005). It was suggested that both calcium transporters
participate in generating cold-induced calcium oscillations by mediating
calcium efflux from the cytoplasm (Monroy, Sarhan et al. 1993). Similarly the
tonoplast Ca2+/H+ antiporter Calcium Exchanger1 (CAX1) is cold induced
(Catala, Santos et al. 2003) and in Arabidopsis CAX1 was shown to acts as a
negative regulator of cold acclimation because cax1 mutant showed higher
expression of CBFs and enhanced freezing tolerance after cold acclimation.
The evidence of involvement of calcium channels in the low temperature
response is strong, as is the evidence for calcium transporters being involved
in modulating the signalling response, but there is still the unanswered
question(s) of how and where cold is perceived. As mentioned earlier,
perceiving cold may be complex and may not rely on only one sensor. For that
reason it will be interesting to investigate whether there is a connection
between CRMK1, which I demonstrate in Paper I that is involved in cold
sensing, with some mentioned above calcium transporters (Fig 4).
45
Figure 4. Possible mode of action of CRMK1. CRMK1 may interact with other kinase
or with different protein. Activity of CRMK1 migth be modified by membrane fluidity
changed at low temperature. Downstream events induced by cold such as CBFs and
CORs expression and ZAT12 expression as marker for parallel pathway activated under
cold stress independent of CBFs regulon.
As I presented above, sensing low temperature in higher plants probably
requires action of not one single receptor but rather a sensor complex. If we
assume that there is a complex of sensors, perhaps this group of proteins
might sense low or high temperatures. There are many steps in an initial
signalling cascade that are similar between low and high temperatures; such
as changes in plasma membrane fluidity, with Ca2+ flux across the plasma
membrane being a possible secondary messenger (Sangwan, Orvar et al.
2002, Sung, Kaplan et al. 2003). The inward calcium flux can influence many
different pathways, for example calmodulin AtCaM3 was shown to be required
for the heat stress response (HSR) and the expression of heat shock proteins
46
(Liu, Sun et al. 2005). The calmodulin binding transcription factors (CAMTA)
were also shown to be involved in cold signalling (Doherty, Van Buskirk et al.
2009). Based on these results and the fact that CRMK1 is localized in plasma
membrane and that there are also some common downstream elements in
both pathways, such as the transcription factor families including the DREB
family, I tested whether CRMK1 could also be involved in the hightemperature response pathway. I investigated the expression of marker-genes
that are key players in the heat-shock response such as transcription factors
MBF1c, ZAT12 and DREB2a (Rizhsky 2004, Suzuki 2005, Sakuma,
Maruyama et al. 2006). The results obtained did not support the conclusion
that CRMK1 was involved in sensing high temperature (Paper I, Fig 6), which
suggests that CRMK1 is low temperature sensor rather than general
thermometer and that there are likely to be distinct mechanism that respond
to high temperatures despite common steps of both heat and cold signalling.
This apparent specificity is perhaps not surprising when you consider that
other abiotic stresses; for example drought, salt or cold stress, share many
overlapping steps in their signalling cascade but none share the same sensing
mechanism, at least so far (Ishitani, Xiong et al. 1997, Liu, Kasuga et al. 1998,
Nakashima, Yamaguchi-Shinozaki et al. 2014). My findings support the
conclusion that CRMK1 does not play a role in the heat-shock response (Paper
I, Fig 6) and I concluded that CRMK1 is not a biological thermometer but is
restricted to sensing shifts to low temperature.
In the field it is common for plants to experience more than one abiotic stress
at the time. Low temperatures often co-occur with high light and osmotic or
water stress, for example. This is why identifying one sensor is very
challenging and discovering one such sensing component is just the first step
on the way to improve survival of plants to unfavourable environments.
Further research is necessary to identify interacting partners of CRMK1, and
this will enable us to more firmly anchor cold sensing by the plasma
membrane and will provide valuable insight into cold sensing and
47
downstream signalling in higher plants. Furthermore, while the plasma
membrane may be the primary source of receiving information about low
temperature from changing environment, it is not the only structure in the cell
able to perceive a temperature downshift. As I describe in the introduction
(see section 5.3) the chloroplast is the target for many cold acclimation
mechanisms, illustrating both its importance to acclimation and its sensitivity
to stress. In the following section I discuss my results that show the
involvement of the plastids in sensing and acclimation responses.
2. Chloroplasts sense changes in environmental conditions
Many crucial processes for plant growth and development occur in the
plastids, not only related to photosynthetic carbon fixation but also critical
steps in nitrogen and sulphur assimilation, and also biosynthesis of fatty acids,
tetrapyrroles, amino acids and many secondary metabolites. This
demonstrates the importance of the organelle in general and what is
important as well is that chloroplasts also play a role as a stress sensor,
including sensing low temperatures (Wilson, Sieger et al. 2003) and excess
light (see section 5.1, 5.2 introduction). Recent studies with barley mutants
affected in chloroplast development showed that these mutants were also frost
susceptible (Zhu, Dong et al. 2007), suggesting that full development of cold
acclimation requires fully functional chloroplasts and indicating that the
plastids can be the rate limiting factor in the responses to low temperatures
(Crosatti, Rizza et al. 2012).
It is well known that plants that actively grow during the winter (under low
temperature) are more resistant to photoinhibition (Huner, Öquist et al.
1998). This happens in winter cereals such as winter wheat where there is a
48
better performance of photosynthesis (light-saturated rates of CO2 exchange
and photon yields for CO2 exchange and O2 evolution) in low temperature in
contrast to wheat cultivars grown in 20°C (Hurry and Huner 1991).
Furthermore, winter rye grown in 5°C exhibit increased tolerance to
photoinhibition (Huner, Oquist et al. 1993). In addition, it is also well known
that exposure of plants to excess light can damage both photosystems.
Photosynthetic organisms have to control their photosynthetic energy
balance, the difference between energy absorbed by the photochemical
reactions (temperature independent) and energy utilized by metabolism,
growth and development (temperature dependent). Whenever this balance is
disturbed by environmental cues such as low temperatures or excess light,
high excitation pressure it can cause changes within the photosynthetic
electron transport chain (Oquist 2003, Ivanov, Sane et al. 2008). When there
is an increase in excitation pressure during cold acclimation, cereals increase
their capacity to regenerate RuBP by increasing the amount of RuBisCo and
other enzymes in the regenerative phase of the Calvin-Benson cycle (Hurry,
Tobiaeson et al. 1995). This upregulation of Calvin cycle capacity results in an
increase in electron flux through carbon fixation, protecting the components
of the electron transport chain from damage (Hurry and Huner 1991, Hurry
and Huner 1992, Huner, Öquist et al. 1993, Huner, Öquist et al. 1998).
Similarly, when Arabidopsis plants are exposed to low temperature for a short
period of time (3 days) there is a significant decrease in CO2 assimilation
compared to warm grown plants (Paper III, Fig 1a, (Strand, Foyer et al.
2003)). Like most plants, Arabidopsis also show a decrease in photochemical
efficiency of PSII (Paper III, Fig 2a) and decrease in PSI photooxidation state
(P700 to P700+) (Paper III, Fig 3a) when transferred to low temperatures.
When control and 3-day cold stressed plants are exposed to excess light, this
results in photoinhibition. However, when plants develop in low temperature
and have fully cold acclimated leaves (around 40 days after shifting to low
temperature) they show almost full recovery in CO2 assimilation at low
temperature, demonstrating that readjustment of the photosynthetic
machinery is required for proper growth and development in the new
temperature regime (Strand, Foyer et al. 2003). This recovery in CO2
49
assimilation at low temperature also renders the cold developed leaves more
resistant to photoinhibition (Oquist 2003). However, in Arabidopsis the
recovery of photosynthesis is not complete and this suggests additional
mechanism may exist that assures photoprotection at the low growth
temperature (Ivanov, Rosso et al. 2012). One such mechanism is the
dissipation of excess excitation energy connected with formation of the
xanthophyll pigment zeaxanthin, which has been shown to be an important
photoprotective mechanism against photoinhibition of PSII (Horton, Ruban
et al. 1996). A related mechanism is thermal release of excess energy within
PSII reaction centres, which is enhanced in cold acclimated plants (Sane
2003, Ivanov, Sane et al. 2008). Both mechanisms release excitation energy
safely as heat. As an alternate strategy to enhanced thermal dissipation, in
Paper III I show that the plastid terminal oxidase (PTOX) has a role as an
alternative pathway for electrons arriving downstream of PSI. In addition,
PTOX may be also involved in modulation of the cyclic electron transport
(based on differential responses to inhibitors specific for PTOX CET in control
and cold stressed Arabidopsis) in order to enhance tolerance to
photoinhibition and prevent or minimise ROS production (as a consequence
of imbalance in excitation pressure). I show that in cold acclimated plants
there is a strong up-regulation of PTOX protein abundance and gene
expression (Paper III, Fig 7a, b, c). Paper III clearly demonstrates changes that
are taking place in chloroplasts under high light and low temperature. I
describe here a protective mechanism that Arabidopsis developed in
particular conditions that provides additional possibility to remove excess
electrons from the stroma by the use of alternative sink such as plastid
terminal oxidase located on the chloroplasts thylakoid membrane. Coldinduced enhancement of the PTOX-dependent alternative electron transport
pathway may reduce the formation of ROS, increasing tolerance to
photoinhibition in cold acclimated plants and serves as proton pump into the
lumen to enhance NPQ.
50
3.
Plasma
membrane
signal
regulates
nuclear
gene
expression
The ‘ICE1-CBF-COR’ regulon is the best-characterized signalling pathway
induced by low temperature (Fig 2). Expression of CBFs responds very rapidly
to changes to low temperature, the transcript levels for all three CBF genes
increases within 15 min of cold treatment, followed by the accumulation of
COR gene transcripts at about 2 h (Gilmour, Zarka et al. 1998). I demonstrate
that CRMK1 is localized to the plasma membrane and transduces a cold signal
to the nucleus, which in turn influences expression of the CBF pathway. To
support this statement I show that the CBF pathway is disrupted in a T-DNA
knockout of CRMK1 (Paper I, Fig 4). Following cold treatment a strong
reduction in CBF1 and CBF3 expression in response to cold was observed in
crmk1 after 1 hr of cold stress (Paper I, Fig. 4A, B). In addition, target genes
such as KIN1 also show reduced expression in crmk1 after 6h of cold treatment
(Fig 5). I don’t know what the mode of action of CRMK1 is or how the cold
signal is sent to nucleus, however, evidence from other LRR-RLK members,
such as BRII/BAK1 (Russinova 2004), suggest that a search for a receiver
protein or studies to see whether it creates heterodimer/homodimer with
other components might be fruitful, as might studies of the proteins function
under conditions that alter membrane thickness (Cybulski, Martín et al.
2010). In Bacillus subtilis there is a special motif called SB-motif found in
DesK, which is responsive to membrane thickness that is altered during
temperature fluctuations (Cybulski, Martín et al. 2010). When the growing
temperature decrease there is an increase in lipid ordering which results in
membrane being thicker, the SB-motif is trapped inside the anhydrous
environment, this destabilization promotes kinase activity. In opposite
situation when temperature rise lipids become more disordered and it results
in membrane being thinner, SB-motif is able to reach aqueous environment
and stabilize the kinase-repressed state of DesK, which is then possible to
transmit phosphate group to receiver domain, DesR. This is in turn the switch
to activate transcription of des genes coding for the acyl lipid desaturase ∆5Des (Cybulski, del Solar et al. 2004) and unsaturated fatty acid products of
51
acyl lipid desaturase ∆5-Des increase fluidity of the membrane (Vigh, Maresca
et al. 1998). This mechanism of a tight interaction between membrane
thickness and a two component system was suggested to be common to many
organisms (Cybulski, Martín et al. 2010), and may be similar to the
mechanism working in higher plants, and further research to identify the
interacting partners for CRMK1 could provide a valuable breakthrough in our
understanding of cold perception.
Figure 5. Relative expression of KIN1 under 1, 3 and 6h of cold treatment in wt (Col0), and two T-DNA insertion lines of CRMK1. Wisc line usesd for further analysis due
to more pronounced and reproducible response to low temperature.
52
4. Plastid signal: transmitting the status of the chloroplasts
to the nucleus
Different fluctuations in the environment connected to temperature shifts or
light conditions are sensed by the chloroplasts enabling photosynthesizing
organisms to adjust to changes in their environment, either by increasing their
ability to consume the products of photosynthetic electron transport,
diminishing the input of energy (reduced light capture efficiency) or
developing protective mechanisms. Thus, in order to properly respond to a
changing environment plants not only have to sense the fluctuations but they
need to communicate this change to the nucleus. Many studies have identified
components localized within the plastid (plastid signals) and the transcription
factors within the nucleus that respond to changes in temperature.
Accumulation of Mg-ProtoIX (plastid signal) and its methylester Mg-ProtoIXME (one of the intermediates in chlorophyll biosynthesis (‘chlorophyll
branch’) was demonstrated to occur in parallel with changes in nuclear gene
expression (Strand, Asami et al. 2003), Paper II, Fig S1A, B). These results
indicate that a plastid signal has to communicate information to the nucleus
about the chloroplasts status in order to orchestrate a coordinated response
to unfavourable conditions. For example, when plants are stressed there is
increase in Mg-ProtoIX levels and a strong repression of nuclear encoded
photosynthetic genes such as LHCB1.1, and RBCS expression observed in
wild-type plants (Ankele, Kindgren et al. 2007). Retrograde communication
(from organelles to nucleus), controls the expression of nuclear genes
encoding organellar proteins with the metabolic and developmental state of
the plastids (Fernández and Strand 2008). The chloroplast protein complexes,
mainly photosynthetic complexes, contain components that are encoded by
both chloroplast and nuclear genomes. This suggests that there has to be
communication between these compartments and this also demonstrate
53
importance of proper coordination of sensing and signalling cascade in order
to maintain proper cellular functions.
5. Other factors beside plastid signal regulate nuclear gene
expression
Plants need to process and integrate various signals to properly respond to
changes in their environmental conditions. It has been suggested that
expression of cold regulated genes is modified by components of light
signalling pathway and that the correct integration of low temperature and
light signals is important for the plant to develop cold acclimation status
(Catala, Medina et al. 2011). Elongated Hypocotyl 5 (HY5) is a bZIP
transcription factor that binds to the promoters of light-inducible genes. It
was also shown that HY5 responds not only to different light signals but to
plastid signals as well (Kindgren, Norén et al. 2012). HY5 was shown to
positively regulate CBF-independent cold-induced gene expression through
the Z-box and other cis acting elements, ensuring the complete development
of cold acclimation (Catalá, Medina et al. 2011). However, the link between
HY5 and the regulation of the CBF genes in response a plastid signal has never
been shown. Inducer of CBF Expression 1 (ICE1) is a MYC basic helix-loop
helix (bHLH) transcription factor that binds to the MYC elements in the CBF3
promoter and activates the expression of CBF3 in response to low temperature
(Chinnusamy 2003). This regulation does not occur in warm conditions
because ICE1 is inactive (Medina, Catalá et al. 2011). At warm temperatures
the expression level of CBF1, CBF2, and CBF3 oscillate with a peak at about 8
h after dawn (zeitgeber time 8; ZT8) and the lowest expression at ZT20
(Harmer, Hogenesch et al. 2000, Bieniawska, Espinoza et al. 2008). This
suggests the existence of additional pathways by which CBFs expression is
regulated, and that it is not only temperature that influences expression of the
CBF regulon. One example is the regulation of CBF expression by Late
54
Elongated Hypocotyl (LHY) and Circadian Clock-Associated 1 (CCA), key
components of the circadian clock machinery (Somers 1999). During the day,
CCA1 and LHY bind the CBFs and promote CBFs transcription. In the evening,
CCA1 and LHY are at low levels and have little effect on CBF expression (Dong,
Farré et al. 2011). The negative regulation of CBF expression occurs via
Phytocrome Interacting Factor 7 (PIF7), which is a bHLH transcription factor
that binds to a Gbox present in the promoter of CBF2. In the pif7 mutant, the
expression levels of CBF1 and CBF2 are not affected under diurnal cycles but
only in the subjective night and under continuous light. This indicates that
PIF7 acts as a repressor of CBF1 and CBF2 expression under circadian control
(Kidokoro, Maruyama et al. 2009).
In Paper II I present evidence outlining how the circadian clock, the light
signal (HY5) and the plastid signal (Mg-ProtoIX) might be integrated to
regulate CBF3 and COR15 expression under warm growth conditions. In the
light/night cycles Mg-protoIX was shown to oscillate diurnally (Paper II, Fig
2B) and CBF3 expression is also under diurnal regulation (Paper II, Fig 2A).
When photoperiod is constant, there are no fluctuations of the plastid signal
and no contribution to the regulation of CBF3 expression by Mg-ProtoIX.
There is only circadian cycling input to the regulation of CBF3 expression in
constant light (Paper II, Fig 2A). HY5 was shown already to be responsive to
a plastid signal (Kindgren, Norén et al. 2012) triggered by diurnal changes in
tetrapyrrole levels (Paper II, Fig 4). Here we show that HY5 binds to promoter
regions of CBF3 and COR15 to repress their expression (Paper II, Fig 3). These
results demonstrate a novel mechanism for controlling CBF and COR gene
expression, and we wanted to check whether mutants related to the light
signal (hy5), the plastid signal (crd) and the circadian signal (ztl) showed
altered susceptibility to freezing under warm growth temperatures. We
expected that crd and ztl mutants might be less freezing tolerant than wild
type because both of them shows reduced levels of CBFs and CORs, and hy5
was expected to be more freezing tolerant because of higher expression level
55
of CBF3 and COR15 (Paper II, Fig 6C, Fig 7A). However, our results (Fig 6)
did not show significant changes in freezing tolerance between control and the
different mutants suggesting that combination of those three signals require
additional factors before they contribute to cold acclimation.
Figure 6. Electrolyte leakage from warm grown wild type (Col-0), crd, ztl, and hy5
insertional lines after exposure to the temperature indicated (programmed to cool at
2°C /h). Data are means ±SD (n=4 leaves discs, each from different plant).
6. Summary of fast responses to changing environment in
Arabidopsis thaliana
In the first part of result and discussion section of my thesis I have discussed
early responses to changes in temperature. I put together what is known about
cold sensing on the plasma membrane level in other organisms and in higher
plants. I have presented and discussed various possibilities of cold sensor in
plants in relation to brasinosteroid signaling as example of RLK signalling
pathway. I also show that plants can sense different ranges of temperature
from low temperature what was mentioned in Paper I. I discussed similarities
of heat shock responses and low temperature sensing possibilities thus to
56
emphasize importance of plasma membrane as the primary site of
temperature change injury. Another aspect of sensing is how change in
temperature is sensed by organelles as example I showed Paper III where
protective mechanism was described in thylakoid membranes of alternative
way for excess electrons occurring in high light in cold acclimated
Arabidopsis. Coordination of cold stress sensing and signalling to nucleus as
well as communication between chloroplasts and nucleus function in
integrated and complex network leading to proper responses in order to grow
and develop in ever-changing environment. I presented and discussed various
factors apart from low temperature which regulate CBFs and CORs
expression. This was the subject of Paper II where I show that circadian signal
together with plastid and light signal influence nuclear encoded gene
expression (CBF3 and COR15). However, none of the mutants indicated
significant changes in freezing tolerance, suggesting additional factors that
contribute to cold acclimation in combination of these signals.
7. Long term cold acclimation responses in Arabidopsis
The next part of my results and discussion section is connected with long-term
cold responses. The main question is how the plant cell reacts to very long
exposure to low temperatures in comparison with short-term responses. The
goal of the Paper IV is to discover the processes that are activated in
overwintering plants and in those that spend their life in low temperature. Our
analysis showed that there are more than 9000 differentially expressed genes
(degs) in these long-term treatments compared to around 3000 degs (Paper
IV, Fig. 3) in the short-term responses. This suggests that many changes occur
in plants as they develop new leaves or grow from seed in low temperature.
We obtained a global view of the biological processes changing between shortand long-term cold response genes. Analysing the GO profiles of up-regulated
57
genes comparing short-term cold treated group v/s long-term cold treated
group (Paper IV, Fig. 4A), we found that the GO-BP terms “nucleobasecontaining compound metabolic process”, “gene expression”, “nucleic acid
metabolic process” and “RNA metabolic process” correspond to GOs that are
more abundant in the short-term group. These results demonstrate that gene
expression changes globally between short (up to 24h) and long (cold
developed leaves and gold grown plants) exposures to low temperature. The
processes that were up-regulated and more abundant in long-term cold
treatment are: “response to other organism”, “defence response to other
organism”, “innate immune response”, “signal transduction”, “defence
response, incompatible interaction”, “protein modification process” and
“single-organism transport”. These results demonstrate that the long-term
cold response genes include a large set of genes annotated as belonging to
biotic stress responses that, to date, have not been identified as playing role in
abiotic stress acclimation and cold tolerance. In the down-regulated groups
we found that “system development” and “organ development” are the
biological functions with the largest changes between short- and long-term
responses (Paper IV, Fig. 4B). The strong representation of these two GO
terms in the down-regulated genes indicates that for both short- and longterm responses, but especially for long-term cold response genes, there is a
down-regulation of processes involved in modulating plant growth and organ
development during acclimation to cold.
My results provide new insights into the role of cation homeostasis, signalling,
protein modifications, protein sorting and the vacuole that were shown to be
specifically enriched in up-regulated long-term group of plants (Paper IV, Fig.
6A). In the down-regulated group of long-term treatment, processes related
to development and growth were the most specifically enriched (Paper IV, Fig.
6B). Further analysis demonstrated the strong representation of genes related
to “chromatin silencing by small RNA” and there are many genes related with
these process being down-regulated in long-term cold response genes,
58
supporting the conclusion that controlling growth related processes is a key
feature of long-term acclimation and maintenance in the cold.
7.1 The role of early cold induced pathway in long-term cold
treatment.
To gain insight into the role of the “CBF regulon” during extended exposure
to low temperature we used a recent conservative CBF regulon gene list (Park,
Lee et al. 2015) and investigated the expression profiles of this regulon in our
experiment. I provided a first attempt to show the role of CBF regulon in longterm cold acclimation responses in Arabidopsis. I did not observe expression
of CBF1, 2, 3 in cold grown plants (Paper IV, Fig. 7A) but other transcription
factors that were induced by short cold treatment stayed active in long-term
cold treatment. Other members returned to warm levels of expression over the
longer term, demonstrating that not all the members of the CBF regulon are
required for a plant to reach full cold acclimation.
In addition to the short-term cold response, CBF1 and CBF2, but not CBF3,
showed very strong induction in cold developed leaves, suggesting that
pathways regulated by these CBFs are reactivated when plants reach full cold
acclimation state that represents development in low temperature and some
of the CBF targets do not respond to prolonged growth in low temperature,
suggesting that CBFs can regulate additional targets which are different than
CORs. The cold grown (CG) plants show normalization of growth and
metabolism not an “intermediate’’ response to low temperature. Our findings
demonstrate that the CBF regulon still plays a role in long-term cold
acclimation but not so significant as much as in short-term cold responses,
because there are members which are induced by prolonged growth (23 genes
59
out of 114 were re-induced by prolonged growth in low temperature) in low
temperature but still the majority of them came back to control expression
levels. This clearly demonstrates that there must be additional pathways in
order to reach full acclimation. This is not surprising because in short-term
cold responses there are existing other pathways induced by this abiotic stress
(Park, Lee et al. 2015) so the overlap and cross-talk exists in long-term cold
acclimation responses as well.
60
Conclusions
The work presented in this thesis is an important step towards understanding
how plants perceive low temperature. I described a plasma membrane
localized cold sensor that modulates the response of the key cold-responsive
transcription factors CBF1 and CBF3 in response to cold shock.
Furthermore, in this thesis I shed light on the factors such as light, plastid and
circadian signals that participate in the regulation of transcription of the best
known cold induced genes (CBF3 and COR15). However, none of the mutants
related to the light signal (hy5), the plastid signal (crd) or the circadian signal
(ztl) showed altered susceptibility to freezing under warm growth
temperatures, suggesting there must be additional factors acting together with
these signals in order to contribute to cold acclimation.
The aspect that I highlighted in this thesis is that chloroplasts are sensitive to
any fluctuations in environment and one way thylakoid membranes can
remove disturbance caused by excess light is by activating alternative electron
transport pathways in inducing greater resistance of both PSII and PSI to high
light stress which is one of the outcome of a stress response.
In this thesis I showed that, in addition to short-term responses to cold, there
are comprehensive long-term responses to low temperature that are needed
in order for plants to acclimate to prolonged changes in their thermal
environment, such as occurs during winter. The transcriptome of
overwintering and cold grown plants changes to a greater degree than shortterm treated plants. There is major change in how DNA is processed during
long-term cold treatments, which does not happen in short-term acclimation
responses. This may provide a mechanism for the up-regulation of many genes
specific to prolonged growth in the cold. Processes that are related to
signalling pathways involving Ca2+, ethylene, jasmonic acid, and kinase
pathways such as MAPK cascades already known to be involved in very early
responses to cold are up-regulated in long-term cold acclimation showing that
61
reactivation of particular pathways has to occur in order to activate proper
cold response and acclimation. I showed that the CBF regulon, which is
induced by early exposures to cold, is not as important in long-term
acclimation. I found biotic factors that were specifically enriched in the upregulated long-term cold response group, demonstrating a novel link between
biotic and abiotic factors in prolonged growth in low temperature. The growth
and developmental processes were down-regulated in long-term cold
treatment that might explain why overwintering and cold grown plants show
growth retardation phenotype.
62
Acknowledgements
First of all, I am deeply grateful to all people from UPSC who has been
everyday with me during PhD period and have helped to grow scientifically
and personally as well.
I would like to thank Vaughan who gave a lot of financial support for whatever
course/conference I wanted to take part does not matter where (Finland,
Norway, Luxembourg, Brazil, Uppsala, Denmark) and also for the help in the
last months of writing although very demanding but very fruitful at the end I
hope.
I would like to thank to Alexander who brought new ideas in the last moment
to the manuscript. I only regret that so late I had a chance to experience having
postdoc in the group, but the feeling was great.
Sorry that some people might feel left out but I am terrible in doing such
things, it was not done on purpose. I will always remember all those who were
with me with good word and just simple interest in my work. This was really
helpful especially I would like to thank Pali for his professional guidance with
kinase assay and cloning.
Na specjalne podziękowania zasłużyli moi dwaj chłopcy (Marcin i Kacper), a
szczególnie Marcin, który mnie wspierał w najtrudniejszych momentach tego
doktoratu. On chyba jako jedyny najlepiej wie ile to kosztowało samozaparcia,
aby dociągnąć do mety. Dzięki Marcinkowi stałam się bardziej odważnym
naukowcem, a co ważniejsze człowiekem. I oczywiście jest współautorem tego
”dzieła” . Kacper od momentu gdy sie pojawiłeś na świecie życie moje
nabrało więcej barw i radości, których nikt inny mi nie dał tak bardzo jak Ty i
które dawały i dają siłę do walki z codziennością.
Podziękowania również należą się dla mojego znajomego i kogoś więcej niż
przyjaciela, który od pierwszych dni towarzyszył mi w Umeå w blaskach i
cieniach wszytkich doświadczeń, których doznałam tu na Północy. I serdeczne
dzięki za dach nad głową kiedy go nie bylo. Także współautor tego ”dzieła”
Dziękuję serdecznie wszystkim którzy w mniejszy bądź większy sposób
przyczynili się do tego, żebym mogła być kim teraz jestem.
63
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