PTMs enable an increase in proteomic complexity and
facilitate a quick response to various internal and external stimuli, e.g. by a covalent addition of a
functional group to the target protein. bHLH proteins can be modified by the
addition of various functional groups, such as N-glycosylation, phosphorylation
and N-myristoylation events (as suggested for mulberry MabHLH144-like (Sajeevan and Nataraja, 2016)) or acetylation (as suggested for OsbHLH089
(Nallamilli et al., 2014)), to just name a few. This suggests
a complex protein regulation, allowing the integration of different signaling
A well-studied example for the transduction of
different stimuli via diverse PTMs is the bHLH protein INDUCER OF CBF EXPRESSION1 (ICE1), which regulates the transcription
of C-REPEAT/DRE BINDING FACTORS (CBFs). CBFs in turn activate cold-regulated
genes (CORs), providing freezing tolerance (Stockinger et al.,
1997; Chinnusamy et al., 2003; Shi et al., 2017). ICE1
belongs to bHLH subgroup IIIb within the A.
thaliana bHLH family and is therefore closely related to FIT (subgroup IIIa)
(Heim et al., 2003).
Interestingly, the ICE1-CBF-COR cascade presents a central integration platform
for many signaling pathways that are also associated with the Fe deficiency
response (Maurer et al., 2014;
Brumbarova et al., 2015; Le et al., 2016; Shen et al., 2016)
such as ABA, RO-, ethylene or SA signaling (Xiong et al., 1999;
Lee et al., 2002; Chinnusamy et al., 2003; Knight et al., 2004; Christmann et
al., 2006; Dong et al., 2009; Maruta et al., 2012; Kurepin et al., 2013).
PTMs of ICE1 were
shown to have different effects on protein stability and activity (Figure 4).
Upon perceiving a cold signal, ICE1 is antagonistically regulated by a series
of phosphorylation events (Ding et al., 2015; Li
et al., 2017; Zhao et al., 2017).
Phosphorylation of ICE1 at position Ser278 is mediated by cold-activated Ser/Thr
protein kinase SnRK2.6 / OPEN STOMATA 1 (OST1), increases its stability and
hence promotes downstream gene expression of cold-tolerance genes (Ding et al., 2015). This increase in stability results from a
competitive binding between OST1 and RING-type E3 ligase HIGH EXPRESSION OF
OSMOTICALLY RESPONSIVE GENE (HOS1) to ICE1 and a disabled interaction between
phosphorylated ICE1 and HOS1 which, upon interaction with ICE1, facilitates
proteasomal degradation of the protein (Lee et al., 2001; Dong
et al., 2006; Ding et al., 2015). Small
ubiquitin-like modifier E3-ligase SIZ1-mediated sumoylation of ICE1 in addition
interferes with ICE1 degradation and stabilizes the protein (Miura et al., 2005;
Miura et al., 2007; Miura and Hasegawa, 2008).
It was further shown,
that an alanine substitution of Ser403 leads to enhanced protein activity,
decreased ubiquitination and degradation of ICE1, suggesting negatively acting,
additional PTMs (Miura et al., 2011). Indeed, phosphorylation of Ser94, Ser403 and Thr366 by
the cold-activated MITOGEN-ACTIVATED PROTEIN KINASES (MPKs) MPK3/MPK6 cascade was
shown to decrease ICE1 stability and hence to negatively influence freezing
tolerance (Zhao et al., 2017). These findings were confirmed and extended by
showing an enhanced transcriptional activity in a transient transactivation
assay and reduced proteasomal degradation in a 6x alanine mutant (ICE16A),
highlighting the importance of Ser94, Ser203, Ser403, Thr366, Thr382 and
Thr384. This suggests a negative effect of MPK3/MPK6 mediated phosphorylation on
ICE1 activity and stability (Li et al., 2017). This inhibition of a positive regulator in freezing
tolerance is proposed to occur under prolonged cold-stress (Dong et al., 2006;
Ding et al., 2015).
It is of interest to
understand how the cold signal is perceived by the cell. Low temperatures
affect plasma membrane fluidity, which leads to the activation of calcium (Ca2+)
channels and hence Ca2+ influx into the cell. The Ca2+-mediated
signal transduction cascade involves several calcium-responsive kinases, affecting
downstream located expression of cold-responsive genes (Zhu, 2016).