PTMs enable an increase in proteomic complexity andfacilitate a quick response to various internal and external stimuli, e.

g. by a covalent addition of afunctional group to the target protein. bHLH proteins can be modified by theaddition of various functional groups, such as N-glycosylation, phosphorylationand 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 suggestsa complex protein regulation, allowing the integration of different signalingpathways.A well-studied example for the transduction ofdifferent stimuli via diverse PTMs is the bHLH protein INDUCER OF CBF EXPRESSION1 (ICE1), which regulates the transcriptionof C-REPEAT/DRE BINDING FACTORS (CBFs). CBFs in turn activate cold-regulatedgenes (CORs), providing freezing tolerance (Stockinger et al.,1997; Chinnusamy et al., 2003; Shi et al.

, 2017). ICE1belongs 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 platformfor many signaling pathways that are also associated with the Fe deficiencyresponse (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 etal., 2006; Dong et al., 2009; Maruta et al., 2012; Kurepin et al.

, 2013).PTMs of ICE1 wereshown to have different effects on protein stability and activity (Figure 4).Upon perceiving a cold signal, ICE1 is antagonistically regulated by a seriesof phosphorylation events (Ding et al., 2015; Liet al., 2017; Zhao et al., 2017).Phosphorylation of ICE1 at position Ser278 is mediated by cold-activated Ser/Thrprotein kinase SnRK2.6 / OPEN STOMATA 1 (OST1), increases its stability andhence promotes downstream gene expression of cold-tolerance genes (Ding et al.

, 2015). This increase in stability results from acompetitive binding between OST1 and RING-type E3 ligase HIGH EXPRESSION OFOSMOTICALLY RESPONSIVE GENE (HOS1) to ICE1 and a disabled interaction betweenphosphorylated ICE1 and HOS1 which, upon interaction with ICE1, facilitatesproteasomal degradation of the protein (Lee et al., 2001; Donget al., 2006; Ding et al., 2015). Smallubiquitin-like modifier E3-ligase SIZ1-mediated sumoylation of ICE1 in additioninterferes 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 bythe cold-activated MITOGEN-ACTIVATED PROTEIN KINASES (MPKs) MPK3/MPK6 cascade wasshown to decrease ICE1 stability and hence to negatively influence freezingtolerance (Zhao et al., 2017). These findings were confirmed and extended byshowing an enhanced transcriptional activity in a transient transactivationassay and reduced proteasomal degradation in a 6x alanine mutant (ICE16A),highlighting the importance of Ser94, Ser203, Ser403, Thr366, Thr382 andThr384.

This suggests a negative effect of MPK3/MPK6 mediated phosphorylation onICE1 activity and stability (Li et al., 2017). This inhibition of a positive regulator in freezingtolerance is proposed to occur under prolonged cold-stress (Dong et al., 2006;Ding et al., 2015).

It is of interest tounderstand how the cold signal is perceived by the cell. Low temperaturesaffect plasma membrane fluidity, which leads to the activation of calcium (Ca2+)channels and hence Ca2+ influx into the cell. The Ca2+-mediatedsignal transduction cascade involves several calcium-responsive kinases, affectingdownstream located expression of cold-responsive genes (Zhu, 2016).


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