In their natural habitat, plants are continuously challenged by adverse environmental
conditions. Among them, cold stress (CS) is a major environmental factor limiting
agricultural productivity and geographic distribution (Chinnusamy et al., 2007). In
this respect, chilling (15-0°C) and freezing (<0°C) stress should be distinguished
(Thomashow, 2010). CS responses at the cellular level are characterized by an extensive
reprogramming of gene expression and metabolic fluxes (Stitt and Hurry, 2002; Miura
and Furumoto, 2013). Clearly, these modifications are mainly linked to the onset of
tolerance mechanisms, which ultimately lead to acclimation. Several metabolites are
known to contribute to this process, including amino acids, polyamines, polyols, and
soluble sugars (Krasensky and Jonak, 2012 and references therein). Among them, particular
focus was recently given to understand the multifunctional role of soluble sugars
in enhancing cold tolerance (Nägele and Heyer, 2013).
Accumulation of soluble sugars following CS is known since long (Levitt, 1958), including
studies on their potential roles in stabilizing biological components, particularly
for Raffinose Family Oligosaccharides (RFO) (Santarius, 1973). Despite this well-known
correlation, more recent investigations shed light on the potential underlying biological
mechanisms involved (Valluru et al., 2008; Sicher, 2011; Peng et al., 2014). One of
the major factors affecting overall cellular stability under CS is membrane phospholipid
composition regulating membrane fluidity (Ruelland and Collin, 2011 and references
therein) associated with cold stimulus perception, as suggested by the protein kinases
cascade activation triggered by dimethyl sulfoxide (DMSO)-mediated membrane rigidification
(Furuya et al., 2014). Different saccharides are capable to directly stabilize biological
membranes under stress conditions. Sucrose (Suc) can directly protect cell membranes
by interacting with the phosphate in their lipid headgroups, decreasing membrane permeability
(Strauss and Hauser, 1986). Fructans, fructose-based oligo- and polysaccharides, and
RFO can increase stability of phospholipidic mono- and bilayers by direct insertion
between polar headgroups (Vereyken et al., 2001; Hincha et al., 2003). Fructans are
localized in the vacuole, suggesting that their contribution to membrane stabilization
may be restricted to the tonoplast. However, their detection in the apoplast of cold-stressed
plants also suggests a role in the protection of the plasma membrane, where they can
be delivered by a vesicle-mediated transport (Valluru et al., 2008). This scenario
seems to be different for RFO. Despite their cytosolic biosynthesis, their protective
action may be restricted to chloroplast inner membranes, as suggested by research
on Arabidopsis thaliana (Nägele and Heyer, 2013 and references therein). Thus, specific
changes in subcellular concentrations of potential stress protectants may greatly
influence successful responses (Lunn, 2007) (Figure 1). The regulation of the activity
and/or expression of soluble sugar transporters, especially those involved in chloroplast
and Tonoplast Monosaccharide Transporters (TMTs) (Wormit et al., 2006) and Sugars
Will Eventually Be Exported Transporters (SWEETs) (Klemens et al., 2013), may play
a central role in such processes. Cold-stressed AtSWEET16 overexpression lines showed
increased freezing tolerance and increased glucose (Glc) and Suc levels (Klemens et
al., 2013). The fructose (Fru)-specific transporter AtSWEET17 plays a primary role
in Fru homeostasis following 1-week 4°C treatment (Guo et al., 2014b). These authors
suggested that the Fru-specific transport features of this carrier may be mediated
by a Fru-specific signaling pathway. Taken together, these works indicate that the
activity and/or expression of sugars transporters may be regulated by sugar signaling,
affecting the subcellular distribution of sugars and overall cellular sugar homeostasis,
which may be tightly linked to the cellular redox homeostasis (see next paragraph).
In that respect, it will be particularly interesting to characterize the nature of
the Raffinose importer in the chloroplast (Schneider and Keller, 2009) and to decipher
its activation by sugar- and hormone signaling under CS in tolerant accessions.
Figure 1
Protective effects of cold-induced saccharides at the subcellular level. The figure
highlights the (putative) action sites of sugars accumulating during CS responses
in higher plants cells. The grey dotted line refers to the proposed vesicular transport
mechanism of fructans from the vacuole to the plasma membrane in fructan accumulating
species (Valluru et al., 2008). The green dotted line refers to the possible roles
of anthocyanins in CS protection. Anthocyanins are also imported in the vacuole through
ABC class transporters (Francisco et al., 2013), where they can contribute in alleviating
CS. The blue arrow represents the signaling pathway leading to the activation of CBFs.
The biosynthesis and metabolic conversions of the sugars involved is oversimplified
and represented by grey arrows. CBFs, C-repeat binding factors; GAs, gibberellins;
GAox, GA oxidase; GolS, galactinol synthase; βAM, β-amylase; Suc, sucrose. Specific
effects of different sugars/anthocyanins are highlighted in italic. Readers are referred
to the figure legend and the text for further details.
Reactive oxygen species (ROS) production partially contributes to chilling and freezing
damage (Nishizawa et al., 2008 and references therein). Recently, several carbohydrates
were proposed as important components of the cellular ROS scavenging system, perhaps
in synergism with other components such as phenylpropanoids (Couée et al., 2006; Nishizawa
et al., 2008; Van den Ende and Valluru, 2009). Living cells do not possess an efficient
enzymatic system to scavenge the highly deleterious hydroxyl radical (·OH) (Gechev
et al., 2006). Furthermore, carbohydrates have generally higher scavenging ability
against ·OH as compared with other radicals, such as superoxide (O·−
2) (Stoyanova et al., 2011). The recent works of Peshev et al. (2013) and Peukert
et al. (2014) provided new mechanistic insights into this process. They observed that
Fenton reaction-derived ·OH scavenging by fructans in vitro lead to the formation
of new oligosaccharides and oxidized sugars. Such oxidized sugars can also be found
in vivo, suggesting that fructans function as scavengers in planta (Peukert et al.,
2014). Alternatively or additionally, fructans have been proposed as stress signals,
further amplifying stress responses that may be initiated by Suc-specific signaling
pathways (Van den Ende, 2013).
Contrary to fructans, the signaling capacity of small metabolic sugars is widely recognized
(Ramon et al., 2008; Ruan, 2014) and several lines of evidence indicates their involvement
in regulating various stress responses (Van den Ende and El-Esawe, 2014). Recently,
a possible mechanistic link between sugars and CS tolerance was proposed by Peng et
al. (2014). They successfully expressed PtrBAM1, a stress-responsive chloroplastic
β-amylase-coding gene from Poncirus trifoliata in tobacco, under the constitutive
promoter CaMV35S. They found that β-amylase activity was strongly enhanced by CS accompanied
with a massive accumulation of maltose and other soluble sugars (Figure 1). Importantly,
this breakthrough paper provides the first evidence that PtrCBF1 (C-repeat-binding
factor 1), a transcription factor belonging to a family of central regulators of CS
responses highly conserved throughout plants kingdom (Chinnusamy et al., 2007), can
bind directly to the promoter of PtrBAM1, providing a unique link between CBF-mediated
cold responses and sugar dynamics. Thus, cold-dependent sugar accumulation may, at
least partially, depend on the CBF transcriptional cascade, as previously suggested
by the CBF-dependent metabolic changes observed during CS (Cook et al., 2004).
It is noteworthy that Suc can trigger fructan synthesis and accumulation in fructan
accumulators such as wheat, by activating the transcriptional factor TaMYB13, which
directly controls gene expression of enzymes involved in fructan synthesis (Kooiker
et al., 2013). A possible scenario for future research could be that CBF-dependent
increases of Suc under CS may trigger fructan synthesis and accumulation in wheat
and other fructan accumulators, allowing a highly coordinated metabolic countermeasure
onset, via an orchestration of direct and indirect signaling and scavenging mechanisms,
as proposed above (Figure 1). Accordingly, in winter wheat, fructans accumulate in
young plants during cold acclimation in the autumn, and this process is also associated
with increased snow-mold resistance (Yoshida et al., 1998). The high correlation between
fructan accumulation and cold tolerance in the wheat family was recently confirmed
by studies on artificially obtained wheat hexaploid lines characterized by different
degrees of freezing tolerance (Yokota et al., 2015). In line with these views, it
has been shown that transgenic rice plants carrying wheat fructosyltransferase (FT)
genes showed an increased CS tolerance (Kawakami et al., 2008).
Galactinol synthase (GolS), the enzyme catalyzing the first step in RFO biosynthesis,
is considered as a target gene of the CBF regulon (Taji et al., 2002), leading to
RFO accumulation under CS. Notably, galactinol, and raffinose have also been proposed
as important signals during biotic interactions (Kim et al., 2008). Another emerging
point of convergence between CBFs and sugars is that AtCBF1 enhances accumulation
of DELLA proteins, fundamental repressors of gibberellin (GA) signaling and positive
regulators of stress responses (Claeys et al., 2014), by stimulating GA catabolism
through increased expression of GA2-oxidase genes (Achard et al., 2008). Furthermore,
it has been recently demonstrated that DELLAs can be specifically stabilized by Suc,
but not by Glc (Li et al., 2014). DELLA proteins stimulate anthocyanin synthesis through
activation of the PAP1/MYB75 transcription factor (Li et al., 2014). In general anthocyanin
levels positively correlate with cold tolerance (Janska et al., 2010), probably by
protecting chlorophyll from over-excitement under freezing conditions (Hannah et al.,
2006).
Soluble sugars levels are strictly connected with starch synthesis and breakdown dynamics,
which are on their turn tightly regulated by the circadian clock (Graf et al., 2010).
In turn, sugar levels have fundamental roles in entraining the clock (Haydon et al.,
2013). Cold-responsive genes such as AtCBF1 show diurnal oscillations in their expression
(Nakamichi et al., 2009). Moreover, expression of central clock components and diurnal
regulated genes is largely influenced by CS (Miura and Furumoto, 2013), providing
tight connections between the clock, metabolic adjustments and CS responses. Recently,
Sicher (2011) shed light on the importance of starch dynamics during chilling responses
in Arabidopsis, by comparing starch and different sugar profiles during chilling stress
in light/dark conditions in wild-type and pgm1 starchless mutants. This author demonstrates
that synthesis and accumulation of the two most highly induced sugars during chilling
stress, maltose and raffinose, strictly depend on the presence of starch, demonstrating
the intimate interconnection between RFO, sucrose, and starch metabolisms (Figure
2). It is known that both target of rapamycin (TOR) and SnRK1 kinases influence such
processes (Dobrenel et al., 2013), but the exact underlying mechanisms need further
exploration. Future research on crop species under CS should focus on the dynamics
of all carbohydrate pools in a diurnal context, to be able to better understand the
complete picture.
Figure 2
Overview of soluble sugars, RFO, fructans and starch metabolic pathways. The scheme
illustrates connections and divergences between the above mentioned pathways, taking
in account their different subcellular environments. Enzymes involved in catabolic
steps are written in italic. Note that only the metabolism of linear fructans is illustrated,
due to space constraints. For further details, readers are referred to http://plantsinaction.science.uq.edu.au/book/export/html/121.
for starch and sucrose metabolism; and to Vijn and Smeekens (1999) and Nishizawa et
al. (2008) for more details on fructan and raffinose biosynthesis, respectively.
Besides the CRB signaling pathway, which is necessary but not sufficient to trigger
cold acclimation, chilling, and freezing tolerance, several phytohormones also play
critical roles by positively or negatively influencing cold resistance and acclimation
(Thomashow, 2010; Miura and Furumoto, 2013). Among them, ethylene and abscisic acid
(ABA) stand out for their well-known crosstalk with sugar signaling pathways (Gazzarrini
and McCourt, 2001). Sugar signaling was also shown to take part in stress responses,
and this is particularly evident when Suc-specific responses are involved (Van den
Ende and El-Esawe, 2014), including the upregulation of the phenylpropanoid biosynthetic
pathway (Serrano et al., 2012; Li et al., 2014), with a strong impact on the anthocyanin
biosynthetic branch (Teng et al., 2005). As in development, during CS response the
ABA-ethylene dynamics conserve an antagonistic nature, with CBF1 as major crosstalk
point (Thomashow, 2010; Shi et al., 2012). Both ABA and Suc promote the accumulation
of DELLA proteins (Guo et al., 2014a; Li et al., 2014), urging further research on
possible ABA-sugar signaling synergisms under CS. Intriguingly, the accumulation of
DELLA proteins is also mediated by CBF1 through posttranslational mechanisms, which
seems to be required for the full activation of freezing tolerance in A. thaliana
(Achard et al., 2008). Thus, it can be speculated that DELLA proteins play an important
role in orchestrating ABA and sugar-induced CS responses, but this requires further
research. This idea was proposed even in a much broader context by De Bruyne et al.
(2014), considering DELLA proteins as pivotal modulators of the physiological balance
between growth and overall (also biotic) stress responses by integrating sugar and
hormonal inputs.
Thanks to their biochemical properties and availability, sugars are likely to be used
by plants in counteracting the most commonly occurring adversities in their natural
environment. Overall, modulation of compatible solutes, among which sugars typically
represent a vast majority, may represent one of the basic mechanisms involved in multistress
tolerance (Puniran-Hartley et al., 2014). Plant responses to evolutionary pressures
in stressful environments led to the diversification of sugar structures and functions,
as well represented by the RFO and fructan cases, among other oligosaccharides (Van
den Ende, 2013). Furthermore, the ability of sugars to modulate expression of stress-related
genes involved in both abiotic and biotic stress responses, such as phenylalanine
ammonia lyase and pathogenesis related proteins (Herbers et al., 1996; Barau et al.,
2014), testifies the high integration level of carbohydrates in cellular defensive
strategies. Moreover, it has been demonstrated that sugar dynamics in the apoplastic
environment need to be dissected from those occurring within the cells, a very important
notion for future research (Barau et al., 2014). Recent data strongly support the
involvement of invertases, key controllers of compartment-specific Suc/hexose ratios,
in response to both biotic and abiotic stresses (Albacete et al., 2014; Sun et al.,
2014). An important goal for future research will be to unravel how invertases and
other Suc metabolizing enzymes are precisely connected to the main stress signaling
pathways, and in which way they influence growth/defense balances, intimately connected
to TOR and SnRK1 activities. In the coming years, dissection of stress-specific signaling
pathways initiated by sugar signaling will likely become one of the most exciting
topics in plant physiology, disclosing new possibilities to increase multistress tolerance
in crops.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.