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Journal of Experimental Botany, Vol. 66, No. 6 pp. 1661–1671, 2015 doi:10.1093/jxb/erv022 Advance Access publication 19 February 2015 Review Paper Exploring the pleiotropy of hos1 Dana R. MacGregor* and Steven Penfield John Innes Centre, Department of Crop Genetics, Norwich Research Park, Colney Lane, Norwich, Norfolk NR4 7UH, UK * To whom correspondence should be addressed. E-mail: [email protected] Received 14 November 2014; Revised 19 December 2014; Accepted 23 December 2014 Abstract Understanding of the roles that HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1) plays in the plant’s ability to sense and respond to environmental signals has grown dramatically. Mechanisms through which HOS1 affects plant development have been uncovered, and the broader consequences of hos1 on the plant’s ability to perceive and respond to its environment have been investigated. As such, it has been possible to place HOS1 as a key integrator of temperature information in response to both acute signals and cues that indicate time of year into developmental processes that are essential for plant survival. This review summarizes knowledge of HOS1’s form and function, and contextualizes this information so that it is relevant for better understanding the processes of cold signalling, flowering time, and nuclear pore complex function more broadly. Key words: Circadian clock, cold signalling, flowering time, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1), mRNA export, nuclear pore. Introduction Being able to recognize a rapid reduction in temperature and respond appropriately allows a plant to survive in variable environments with freezing temperatures. Likewise, it is particularly important for annual plants to be able to correctly interpret seasonal cues, such as day-length and changing trends in average temperature, so that flowering and seed set occur only at the right time of year. The genes and proteins involved in the response to cold and control of flowering time are under the control of the circadian clock, which ensures that the appropriate timing and level of response occurs. HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1) plays a role in circadian function, but is also a negative regulator of both cold signalling and flowering time, and is therefore a candidate integrator of environmental information into signalling events that are necessary for plant survival. At the molecular level, HOS1 accomplishes these functions by working as part of the Nuclear Pore Complex (NPC), where it is involved in the regulation of protein and mRNA levels through various mechanisms. HOS1 is therefore important for the plant’s ability to adapt to an ever-changing environment. This review investigates the various mechanisms in which HOS1 is able to accomplish these functions while pointing out connections, complexities, and outstanding questions. HOS1 is conserved within plants and is alternately spliced HOS1 is encoded by the Arabidopsis thaliana gene At2g39810, and the HOS1 locus gives rise to two splice isoforms, HOS1-L and HOS1-S (Fig. 1A; Ishitani et al., 1998; Lee et al. 2012b). The alternative splicing of HOS1 means that HOS1-L includes 12 additional amino acid residues, as the HOS1-S start site is within the first intron of HOS1-L (Fig. 1B). This 12 amino acid difference is upstream of the known functional and conserved domains and is upstream of the hos1-1, hos1-2, hos1-3, hos1-5, and hos1-6 mutations (Lee et al., 2001; Lazaro et al., 2012; MacGregor et al., 2013). It is unclear if the location of the hos1-4 mutation (Lazaro et al., 2012) is contained in both © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: [email protected] 1662 | MacGregor and Penfield isoforms, as the splice variant used to describe the position of this point mutation was not defined. When putative HOS1 sequences from multiple plant species are compared, the protein sequence of A. thaliana HOS1-L is more highly conserved compared to HOS1-S, and is nearly identical to the predicted HOS1 protein sequences from A. lyrata and Thellungiella halophila (Lee et al., 2012b). Characterization of the different splice variants showed them to be broadly similar in their expression patterns and their ability to interact with a known ubiquitination target (Lee et al., 2012b). There are two notable differences in these isoforms: (i) HOS1-L is expressed at a higher level than HOS1-S; and (ii) when plants are treated with a transient cold shock, there is a rapid and transient reduction of expression of both splice variants, but the recovery of HOS1-S to pre-cold levels occurs more rapidly than HOS1-L (Lee et al., 2012b). Interestingly, early characterization of HOS1 also described the HOS1-S isoform as well as this rapid, transient reduction in HOS1 mRNA levels in response to a cold shock (Lee et al., 2001). This rapid removal of HOS1 mRNA is suggested to then alleviate the repressive effects of the HOS1 protein, thereby permitting a transient response to the environmental stimuli (see ‘HOS1 Negatively Regulates Cold Signalling’; Lee et al., 2001; Lee et al., 2012b). As these two isoforms appear to be largely similar in expression patterns and function (Lee et al., 2012b), and most studies do not define which isoform they analyse, they are not differentiated in the remainder of this review. However, both isoforms do exist in planta and there is evidence that they play slightly different roles in the regulation of flowering time (see ‘HOS1 Negatively Regulates the Transition to Flowering’, Lee et al, 2012b). It would be interesting to extend this comparative analysis beyond flowering time regulation and re-evaluate the other known functions of HOS1 with the aim of determining where, if at all, the resulting proteins differ. Sequences encoding putative HOS1 from other plant species have also been found and the overall level of conservation is good (Lee et al., 2012b). OsHOS1 from Oryza sativa is 49% identical to AtHOS1 at the amino acid sequence level, and it also performs many of the same functions as AtHOS1 in modulating cold signalling (Lourenço et al., 2013; see ‘HOS1 Negatively Regulates Cold Signalling’). As HOS1 is an important coordinator of several environmental inputs into fundamental developmental processes, it would be interesting to investigate whether this sequence conservation among plants translates into conservation of function across the species beyond just Arabidopsis and rice, as well as extending the analysis to determine if the other roles of HOS1 are conserved. The hos1 mutant is highly pleiotropic hos1 mutants have been shown to disrupt many plant processes. For instance, HOS1 has been identified in screens for altered cold stress responses, early flowering under various different conditions, altered circadian rhythms, and in a proteomic study for components of the nuclear pore (Ishitani et al., 1998; Tamura et al., 2010; Lazaro et al., 2012; MacGregor et al., 2013). One difficulty in unpicking the mechanisms of HOS1 action is that many of these pathways are intricately interconnected. For instance, intermittent cold temperatures delay flowering (Seo et al., 2009), and elements of the chromatin remodelling machinery that act on the flowering time pathway genes are also required for freezing tolerance and cold signalling (To et al., 2011b). The protein modifier SIZ1 acts to regulate protein stability of transcription factors key to both cold signalling and the flowering time pathway (Miura et al., 2007; Jin et al., 2008). The external coincidence model uses the clock to correctly coordinate flowering time with day length (Carré, 2001). Nuclear pore function is required for correct circadian periodicity (MacGregor et al., 2013) and flowering time (Parry, 2014). The circadian clock is not only entrained by temperature fluctuations (Eckardt, 2005), but Fig. 1. The structure of HOS1. (A) Genomic structure of both splice variants of HOS1 (At2g39810) showing exons (grey boxes) and introns (black lines). Figure based on The Arabidopsis Information Resource (TAIR), http://www.arabidopsis.org/servlets/TairObject?id=35204&type=locus. (B) Protein structure of both splice variants of HOS1 showing RING domain (yellow box), homology to Elys (blue dotted box), NLS (green box), and sequence of uncharacterized structure (red boxes). The C-terminal portion of the protein that is important for protein–protein interactions has been identified with a bracket. Figure based on Lee et al., 2012b. (C) Structural comparison between A. thaliana HOS1 from Fig. 1B with Homo sapiens Elys. The Elys structures identified are the region of homology (blue dotted box), the β-propeller domain (orange box), the central α-helical domain (purple box), the DNA-binding domain (pink box), and the C-terminal disordered region (brown box). Figure based on Tamura et al., 2010 and Bilokapic and Schwartz, 2013. Exploring the pleiotropy of hos1 | 1663 the components and correct functioning of the clock itself play an important role in gating temperature signal transduction (Thomashow, 2010; Keily et al., 2013). Therefore, although it is common to compartmentalize phenotypes and functions within one pathway or another, it is important to consider that these signalling pathways do not work in isolation but are tightly connected. Therefore, future research on HOS1 and other nuclear pore proteins would benefit from considering whether the phenotype being studied is a direct consequence of the mutation or an indirect effect acting through one of these other interconnected pathways. To understand what it is that HOS1 does, it is important to consider what HOS1 encodes. The HOS1 sequence encodes for a protein of 927 (HOS1-L) or 915 (HOS1-S) amino acids. As shown in Fig. 1B, both splice isoforms of the protein have a variant C3HC4 Really Interesting New Gene (RING) finger E3 ligase domain, followed by a region of homology to the vertebrate nucleoporin EMBRYONIC LARGE MOLECULE DERIVED FROM YOLK SAC (Elys), and a nuclear localization signal (NLS) (Lee et al., 2001; Dong et al., 2006a; Tamura et al., 2010). Of these, only the RING domain has been demonstrated to be necessary but not sufficient for E3 ligase activity of HOS1, as demonstrated by in vitro ubiquitination assays using HOS1, truncated HOS1 mutants, or point mutations in HOS1 altering conserved histidine or cytosine residues (Dong et al., 2006a); however the E3 ligase activity of these variants or their ability to rescue the loss of HOS1 have not been reported in vivo. Much of the rest of the protein is uncharacterized, does not contain sequences common to domains of known function, and only has sequence similarity to other plant genomes. The final 11 amino acids of the HOS1 sequence are a putative NLS (Fig. 1B), but there are no reports demonstrating that this sequence is required for HOS1 localization. Analysis of HOS1 promoter activity demonstrated that HOS1 is expressed ubiquitously throughout the plant (Lee et al., 2001). Within the cell, Lee et al. (2001) and Lee et al. (2012a) show nucleo-cytoplasmic partitioning of the HOS1 protein: under ambient temperatures, HOS1 is predominantly cytoplasmic, but it concentrates in the nucleus after a shift to cold temperatures. HOS1 has also been localized at the nuclear envelope, specifically as a component of the NPC, at ambient temperature or at all temperatures tested, and in both light and dark conditions (Tamura et al., 2010; Lazaro et al., 2012; MacGregor et al., 2013). The human nucleoporin Nup98 is mobile, dynamically associating with the NPC as well as being localized in both the nucleus and the cytoplasm (Zolotukhin and Felber, 1999; Griffis et al., 2002; Griffis et al., 2004). Therefore, there is a precedent for mobile nuclear pore components, and the movement of HOS1 may be important for its ability to regulate downstream processes. As HOS1 has been characterized as an E3 ligase (Dong et al., 2006a), a component of the nuclear pore (Tamura et al., 2010), and finally a chromatin remodelling factor (Jung et al., 2013), it is clear that the localization of HOS1 is vital for its ability to interact with partners and regulate signalling events as sequestration in one compartment of the cell would limit access to its partners. HOS1 is a component of the nuclear pore and is required for mRNA export Tamura et al. (2010) performed a series of interaction studies to identify components of the Arabidopsis NPC, using the conserved core components of the outer ring of the nuclear pore RNA EXPORT FACTOR 1 (RAE1) and Nup43 as baits (Fig. 2). HOS1 interacted with both RAE1 and Nup43, and has a region of sequence similarity to the vertebrate nucleoporin Elys, lending further support to the association of HOS1 with the NPC (Fig. 1) (Tamura et al., 2010). Elys is a dual nucleoporin/kinetochore protein, and Elys and its homologue MATERNAL EFFECT LETHAL-28 (Mel-28) in Caenorhabditis elegans are required for the correct assembly of the nuclear pores, a functional nuclear envelope, and the formation of subdomains within the nuclear envelope after mitosis (Galy et al., 2006; Rasala et al., 2006; Clever et al., 2012). Analysis of the Elys protein has demonstrated that it is made up of three domains: (i) an N-terminal β-propeller domain, (ii) a central α-helical domain, and (iii) a C-terminal disordered region (Fig. 1C) (Bilokapic and Schwartz, 2013). The first two domains contain the region of homology to HOS1, and synergistically mediate tethering to the NPC, while the disordered region is responsible for the interactions with chromatin (Bilokapic and Schwartz, 2013). However, the region of homology between HOS1 and Elys is a small portion of both proteins (Fig. 1C), and otherwise the protein sequences of Elys and HOS1 are not well conserved. Therefore caution is required when extrapolating protein function from Elys to HOS1. Two groups have independently demonstrated HOS1 is localized to the nuclear envelope using florescent protein fusions (Lazaro et al., 2012; MacGregor et al., 2013); however, the resolution of these images is insufficient to determine whether HOS1 is restricted to the NPC or if it is positioned on the cytoplasmic and/or nucleoplasmic side of the envelope. We cannot predict which side of the pore HOS1 is located because the components with which it interacts are located on both sides of the nuclear envelope (Fig. 2), and Elys has not been positioned on one side or another. However, as HOS1 interacts with chromatin (Jung et al., 2013), it is likely that it is located at least on the nucleoplasmic side of the NPC, if not free in the nucleus. HOS1 is not only located at the NPC, but is also required for the basic NPC function of mRNA export (MacGregor et al., 2013). mRNA is transcribed in the nucleus and then transported to the cytoplasm via the NPC. Mutations to the NPC, including hos1, alter this process leading to an accumulation of polyA-containing mRNA in the nucleus (Dong et al., 2006b; Parry et al., 2006; Xu et al., 2007; Lu et al., 2010; Wiermer et al., 2012; MacGregor et al., 2013; Parry, 2014). Withholding mRNA in the nucleus is likely to have many deleterious effects, and one that has recently been identified is that mRNA retention leads to a long period of the circadian clock (MacGregor et al., 2013). The Arabidopsis circadian clock is composed of three interlocking transcription-translational feedback loops which are responsive to both temperature and light but also compensated against these changes so that the 1664 | MacGregor and Penfield Fig. 2. Components of the nuclear pore complex and their localization within the pore. Rectangles indicate structures that are composed of multiple proteins, and circles, individual proteins; HOS1 is highlighted as a ten-point star. The HOS1-interacting proteins RAE1 and Nup43 are underlined. The tables at the top indicate where loss of function of that gene leads to the indicated phenotype. The colour coding indicates the position within the pore: green, cytoplasmic ring and filaments; orange, central pore proteins; red, transmembrane or membrane-associated proteins; blue, nuclear linker or nuclear basket proteins; yellow, central scaffolding proteins. The pink curved line represents the nuclear membrane. The structure of the nuclear pore is compiled based on figures in Parry (2014) and Tamura et al. (2010) overlaid on Fig. 1 of Raices and D’Angelo (2012). clock consistently runs with a 24-hour period (Pokhilko et al., 2012; Gould et al., 2013). It is known that altering the amounts of any one of the clock components can lead to changes in period length (McClung, 2006); however, analysis of double mutants between hos1 and clock components did not provide evidence for a genetic interaction between HOS1 and these components of the circadian clock (MacGregor et al., 2013). hos1 does result in lowered protein levels of the core circadian component LATE ELONGATED HYPOCOTYL (LHY) but does not alter its rhythm (MacGregor et al., 2013). Therefore, a simplistic model for how hos1 and the other NPC mutants tested change circadian period is that when mRNA export is reduced, it results in changes to the protein amounts of many if not all of the clock components, and these changes collectively lead to a lengthening of the period. The role of HOS1 in the maintenance of circadian periodicity is unique, as unlike the other hos1 phenotypes, the hos1 clock has a long period under all temperature and light regimes tested, and therefore does not appear to have a temperature or light dependency (MacGregor et al., 2013). HOS1 acts as a negative regulator through protein–protein or protein–chromatin interactions The C-terminal portion of the HOS1 protein (Fig. 1B) is important for protein–protein interactions. The region from Exploring the pleiotropy of hos1 | 1665 approximately amino acid 450 either to the end of the protein, or to the NLS, has been used to demonstrate that HOS1 interacts with INDUCER OF CBF EXPRESSION 1 (ICE1, also known as SCREAM), FVE, FLOWERING LOCUS KH DOMAIN (FLK), CONSTANS (CO), and several histone deacetylases such as HISTONE DEACETYLASE 6 (HDA6) and HDA15 (Dong et al., 2006a; Jung et al., 2012; Lee et al., 2012a; Jung et al., 2013) (Fig. 3). Interestingly, for many of these in vitro interactions, such as the interaction with ICE1 or CO, the full-length protein does not appear to interact as strongly with its partners as does the truncated version (Dong et al., 2006a; Jung et al., 2012). There are many possible reasons, both biological and technical, why fulllength HOS1 does not interact as well as the truncated version. For instance, it is possible that expression of full-length HOS1 in the heterologous system results in a protein structure that is less accessible for interactions because it requires additional post-translational modifications or interacting partners not present in yeast. Also, the yeast two-hybrid system may require that the interactions occur at an abnormal subcellular localization for the full-length protein. The region of HOS1 that is deleted to make the truncated HOS1 contains the region of Elys homology, and in Elys this region is contained within the portion of the protein known to mediate tethering to the NPC (Fig. 1) (Bilokapic and Schwartz, 2013). It is also possible that interaction with full-length HOS1 protein results in ubiquitination and degradation of the target, and therefore the interaction cannot be observed because the signal for interaction is not being properly propagated. The interactions identified using the truncated HOS1 are occurring in planta as demonstrated by additional techniques such as in vivo pull-down assays and bimolecular fluorescence complementation (BiFC) approaches. Additional work needs to be done to determine whether the observed differences in the strength of interaction between the full-length and truncated HOS1 are biologically relevant or artefacts of the testing systems. Every protein–protein interaction in which HOS1 participates does not necessarily result in ubiquitination and protein degradation. To regulate cold signalling or flowering time, HOS1 acts to regulate the protein levels of ICE1 or CO, respectively, and is both necessary and sufficient to alter their ubiquitination levels (see below). However, HOS1 also interacts with several other proteins, and for these there is no evidence that the HOS1 interaction leads to ubiquitination or changes in protein levels. For instance, Lee et al. (2012a) were unable to demonstrate ubiquitination of FVE by HOS1 in vitro. Likewise, although HOS1 interacts with HDA6 and the ability of HDA6 to bind to the promoter of FLC is altered by levels of HOS1, amounts of HDA6 protein are not altered with cold and no ubiquitination of HDA6 was observed in the presence of HOS1 (Jung et al., 2013). Therefore, HOS1 is required to serve some additional functions in addition to its role as an E3 ubiquitin ligase. HOS1 negatively regulates cold signalling HOS1 was originally isolated in a screen for mutations that altered the expression of a cold- and osmotic stress-responsive promoter driving LUCIFERASE expression (RD29A:LUC; Ishitani et al., 1998). As its name HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 implies, hos1-1 led to increased RD29A:LUC luminescence when plants were exposed to cold, but there was no significant luminescence in the absence of treatment or between the wild type and hos1-1 treated with abscisic acid (ABA) or salt (NaCl). hos1 led to Fig. 3. Illustration of HOS1 interactions and downstream signalling events important for circadian periodicity, cold signalling, and flowering time. The purple boxes are E3 ubiquitin ligases, the yellow boxes are transcription factors, the orange box is a SUMO modifier, the blue box is an SPA family member, the green boxes are chromatin remodelling factors, and the grey clocks represent the circadian clock. The red rectangles indicate promoters with the known binding sites indicated, and the dark green rectangle indicates the chromatin remodelling that occurs at the promoter of FLC. The linked purple circles represent poly-ubiquitination events of ICE1 and CO by HOS1 or CO by COP1; the 26S proteasome is drawn in cartoon. Linked orange circles represent SUMOylation of ICE1 by SIZ1. Arrows indicate promotion and T-junctions indicate inhibition. Double-lined arrows indicate indirect effects, and single lined arrows indicate direct effects. 1666 | MacGregor and Penfield an increased cold response over a large range of temperatures where, importantly, the wild type shows no luminescence above 10°C yet hos1-1 luminesced up to 19°C (Ishitani et al., 1998). HOS1 is also necessary and sufficient for the normal expression of many additional cold-responsive and coldsignalling genes (Ishitani et al., 1998; Lee et al., 2001; Dong et al., 2006a; Lourenço et al., 2013; MacGregor et al., 2013). There appear to be at least two mechanisms through which HOS1 is altering cold gene expression: through modulation of ICE1 protein levels and by regulating mRNA export. INDUCER OF CBF EXPRESSION 1 (ICE1) is a basic helix-loop-helix transcription factor that interacts with DNA as well as several proteins and is important for cold signalling (Chinnusamy et al., 2003; Lee et al., 2005; Miura et al., 2007; Miura et al., 2011; Hu et al., 2013), stomatal development and patterning (Kanaoka et al., 2008), temperature-regulated pathogen resistance (Zhu et al., 2011), and endosperm breakdown during seed development (Denay et al., 2014). During cold signalling ICE1 acts to control expression of the C-REPEAT/DRE BINDING FACTOR (CBF) family of transcription factors through binding to the CBF3 promoter, initiating a signalling cascade that is necessary for tolerance to cold stress (Fig. 3) (Chinnusamy et al., 2003; Lee et al., 2005; Miura et al., 2007; Miura et al., 2011; Hu et al., 2013). ICE1 is widely conserved among higher plants (Kurbidaeva et al., 2014), and for several ICE1 orthologues there is functional evidence that ICE1 is also necessary and/or sufficient to regulate cold-stress responses (Badawi et al., 2008; Chen et al., 2012; Wang et al., 2012; Chen et al., 2013; Dong et al., 2013; Feng et al., 2013; Zhao et al., 2013; Shan et al., 2014; Xu et al., 2014). The HOS1–ICE1 interaction is necessary for the polyubiquitination of ICE1 and its subsequent degradation by the proteasome; amounts of ICE1 protein are correlated with levels of CBF and other cold-responsive gene expression (Dong et al., 2006a; Lee et al., 2012b; Lourenço et al., 2013). The proposed mechanism for HOS1 regulation of ICE1 is that under ambient temperatures, HOS1 ensures that ICE1 levels are low and the cold signalling pathway is not induced. In response to a short pulse of cold, the level of HOS1 mRNA rapidly and transiently decreases, implying that HOS1 protein levels are reduced; removal of the HOS1 protein alleviates ICE1 degradation, allowing ICE1 to initiate cold responses through CBF3 (Lee et al., 2001; Lee et al., 2012b). Also, in response to a reduction of temperature, HOS1 moves from a predominantly cytoplasmic localization into the nucleus (Lee et al., 2001; Lee et al., 2012a), where it is able to interact with and target ICE1 for degradation, ensuring that the induction of cold responses is transient. To balance HOS1-mediated degradation, ICE1 is also posttranslationally modified by SIZ1, which stabilizes it through the addition of SUMO modifications (Fig. 3) (Miura et al., 2007). Therefore, by regulating ICE1 protein levels through ubiquitination, as balanced by SUMOylation, HOS1 can modulate an appropriate response to transient cold. Further work is required to understand how these transient responses relate to the plant’s ability to survive temperature changes that occur under more natural conditions. Other than this transient response to cold, there is little evidence for transcriptional regulation of HOS1, as shown by analysis of HOS1 in the wild type under various conditions and treatments. HOS1 is ubiquitously expressed throughout the plant and, excluding the rapid response to temperature explained above, its expression is not significantly altered by growing the plants at various temperatures or in response to treatments such as exogenous hormones, salt, or drought (Lee et al. 2001; Lee et al., 2012a). HOS1 does have a diurnal expression pattern showing a peak during the night in both long- and short-day conditions (Lee et al., 2012b), but there is no evidence that its transcription is under circadian control (Lazaro et al., 2012). Under diurnal conditions, the maximal amount of expression is determined by photoperiod and is higher during long days, as confirmed with photoshift experiments (Lee et al., 2012b). Therefore, although HOS1 is required for normal expression of nearly 9% of the Arabidopsis genome (MacGregor et al., 2013) and environmentally responsive changes in expression of many other genes (Ishitani et al., 1998; Lee et al., 2001; Dong et al., 2006a; Lourenço et al., 2013; MacGregor et al., 2013), the regulation of HOS1 itself appears to be largely post-translational rather than at the transcript level, with the exception of the shortterm cold response. Although it is clear that HOS1 is required for wild-type cold signalling and cold-responsive gene expression, it is not clear that these changes then relate back to alterations in a plant’s ability to survive cold stress. Although overexpression of HOS1 leads to decreased ability to survive freezing stress (Dong et al., 2006a), electrolyte leakage tests surprisingly show that plants with the loss-of-function allele hos1-1 or expression of RNAi::OsHOS1 in rice also decrease freezing tolerance in non-acclimated plants, and do not affect freezing tolerance in acclimated plants (Ishitani et al., 1998; Lourenço et al., 2013). These results beg the question as to whether HOS1 has been selected through evolution as a negative regulator of cold signalling or if the role of HOS1 in this pathway is an indirect consequence of some other function. HOS1 negatively regulates the transition to flowering The first report of hos1 noted its early-flowering phenotype under long-day conditions, and suggested that hos1 mutants displayed a constitutively vernalized phenotype (Ishitani et al., 1998). hos1 was also independently identified in a screen for early-flowering mutants in short-day conditions and was called early in short days 6 (esd6) (Lazaro et al., 2012). hos1 shows early flowering under all conditions studied, including different temperature treatments such as intermittent cold, vernalization, or growth at lower ambient temperatures (Ishitani et al., 1998; Lee et al., 2001; Bond et al., 2011; Lee et al., 2012a, b; Jung et al., 2012; MacGregor et al., 2013; Jung et al., 2013). The differentially spliced isoforms appear to have slightly different roles in the control of flowering time, as overexpression of HOS1-L leads to later flowering than does HOS1-S (Lee et al., 2012b), although the underlying mechanism controlling this difference is not known. Exploring the pleiotropy of hos1 | 1667 The flowering control pathways are an excellent example of a highly interconnected environmental response pathway linked to developmental outputs, and FLOWERING LOCUS C (FLC) is instrumental for control over flowering time by temperature signalling (Michaels and Amasino, 1999; Seo et al., 2009). FLC is a MADS-box gene that acts as a key component in the timing control of initiation of flowering in Arabidopsis. FLC is a repressor of flowering which is transcriptionally regulated by vernalization (Sheldon et al., 2000; Michaels and Amasino, 1999; Henderson and Dean, 2004). The regulation of FLC by vernalization requires the cold activation of a long antisense non-coding RNA known as COOLAIR and the recruitment of the PRC2 complex to silence expression through deposition of repressive chromatin marks [for reviews see Ietswaart et al. (2012) or RomeraBranchat et al. (2014)]. In hos1 the expression of FLC is reduced, and in the control region of the FLC promoter the epigenetic marks for active gene expression are reduced and repressive chromatin markers increased (Lee et al., 2001; Lazaro et al., 2012; Lee et al., 2012a; Jung et al., 2013). Chromatin-immunoprecipitation (ChIP) using antibodies directed against HOS1 showed enrichment of the FLC promoter, indicating that HOS1 is present at these locations (Jung et al., 2013). Moreover, HOS1 was shown to interact with various known chromatin regulators including FVE and HDA6, which are known to associate in multi-protein complexes with the regulatory region of the FLC promoter (Pazhouhandeh et al., 2011; To et al., 2011a; Jung et al., 2013). The level of HOS1 in planta is inversely correlated with the enrichment of HDA6 at the promoter of FLC, and binding of HOS1 to FLC is FVE dependent but HDA6 independent (Jung et al., 2013). HOS1’s role in chromatin remodelling is temperature dependent, as cold stress increased the enrichment of HOS1 at the FLC promoter and attenuated enrichment of HDA6 whereas FVE enrichment at the promoter of FLC was not affected by cold treatments (Jung et al., 2013). Therefore, in a temperature-dependent manner, HOS1 and HDA6 compete to bind with FVE at the FLC promoter and the combination that is present determines the state of the chromatin and therefore the level of transcription that occurs. The plants used for the ChIP studies were grown under constant cold or ambient temperature (Jung et al., 2013), indicating that unlike the regulation of ICE1, the role of HOS1 at FLC is not an acute temperature response. As FLC is important for interpreting seasonal temperature cues (Michaels and Amasino, 1999; Sheldon et al., 2000), the role of HOS1 in this pathway means that it plays a role in processing temperature information in response to both intermittent cold treatments (Jung et al., 2012) as would happen during a single day and the longer term changes that signal time of year (Jung et al., 2013). HOS1 affects flowering time through both FLC-dependent and FLC-independent routes. FLC expression is reduced in hos1 compared to the wild type under many conditions, (Lee et al., 2001; Lazaro et al., 2012; Lee et al., 2012a; Jung et al., 2013), although Bond et al. (2011) show that neither FLC nor VIN3 expression is not significantly altered at ambient or low temperatures in hos1. Double mutant analysis demonstrates that the early-flowering phenotype of hos1 is FLC independent. For instance, although the expression of FT and its close homologue TWIN SISTER OF FT (TSF) are increased in hos1, this elevated expression is greater in hos1 than in flc, and the hos1 flc double mutant shows an additive increase in expression (Lee et al., 2012a). The hos1 flc double mutant flowers with the same rosette leaf number as hos1, but the double took fewer days to bolt than hos1 alone under longday conditions (Lazaro et al., 2012). Altogether, these data indicate that there is an FLC-independent route through which HOS1 is required for flowering-time control. A clear mechanism for an FLC-independent role for HOS1 in the control of flowering time under long-day conditions is through its regulation of CONSTANS (CO) protein levels through ubiquitination (Fig. 3). We know that the HOS1-dependent ubiquitination of CO occurs through an FLC-independent pathway because FLC expression is not changed under conditions where CO levels increase (Jung et al., 2012). CO is essential for the photoperiodic flowering pathway activating FLOWERING LOCUS T (FT) in response to long days (Yanovsky and Kay, 2002; Valverde et al., 2004). Expression of CO mRNA is under circadian control, and CO protein is ubiquitinated by another RING motif-containing E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in complex with SUPPRESSOR OF PHYTOCHROME A-105 1 (SPA1), which target CO for degradation specifically in the dark (Fig. 3) (Laubinger et al., 2006; Sawa et al., 2007; Liu et al., 2008; Jang et al., 2008). Under long-day conditions, hos1 co double mutants flowered with the same number of leaves as the co parent, showing that the early flowering of hos1 mutants requires CO. However, under short-day conditions, hos1 is still early flowering, and the hos1 co double mutant flowered as early as hos1 (Lazaro et al., 2012). Therefore, in short days HOS1 negatively regulates flowering through a CO-independent mechanism, whereas in long days HOS1 acts through CO to regulate flowering time. HOS1 is required to degrade CO in the early part of the day (Jung et al., 2012). In hos1 under both short- and longday conditions, the peak of FT expression occurred earlier than in the wild type (Lazaro et al., 2012). HOS1 therefore compliments the activity of COP1-SPA1, which degrades CO during the night (Jang et al., 2008; Liu et al., 2008) to ensure that FT expression is controlled. The synergistic activity of HOS1 and COP1 is supported by the observation that hos1 cop1 double mutants flower earlier than the wild type or either single mutant, and are unable to differentiate between long- and short-day conditions (Lazaro et al., 2012). Coldinduced degradation of CO is COP1 independent as the rate of CO degradation is greater under dark, ambient temperature conditions than it is under cold but well-lit conditions (Jung et al., 2012). In planta, there is also a PhyB-dependent mechanism that degrades CO early in the day (Valverde et al., 2004; Jang et al, 2008); however, the cold-induced degradation of CO occurs independently of PhyB (Jung et al., 2012). Because of the overlap in timing of HOS1 and PhyB degradation of CO, it has been suggested that they act through shared processes to degrade CO (Jung et al., 2012; Lazaro 1668 | MacGregor and Penfield et al., 2012; Endo et al., 2013), but further work is required to demonstrate that HOS1 and PhyB work together rather than simply at the same time of day. Altogether, these data demonstrate that HOS1 activity is required in a time- and temperature-dependent manner to maintain CO protein abundance and prevent precocious flowering. HOS1-mediated degradation of CO is also regulated by temperature and there are many similarities between the regulation of CO and ICE1 by HOS1. HOS1 and CO physically interact at their C-termini, both in vitro and in vivo (Fig. 1B) (Jung et al., 2012; Lazaro et al., 2012). The interaction of HOS1 and CO results in the ubiquitination of CO and its degradation in a mechanism that requires the activity of the 26S proteasome (Fig. 3) (Jung et al., 2012; Lazaro et al., 2012). The level of CO ubiquitination is temperature dependent, where a reduction in temperature results in an increase in ubiquitination (Jung et al., 2012). Finally, there is a correlation between the level and kinetics of FT expression and both CO abundance and proteasome activity (Jung et al., 2012). Altogether, HOS1 appears to be regulating both ICE1 and CO in similar temperature- and ubiquitin-mediated proteasome-dependent mechanisms. Aspects of the hos1 phenotype are also observed in plants lacking other nuclear pore components hos1 mutants are not alone in showing pleiotropic growth phenotypes. Many of the other components of the NPC have been tested for their loss-of-function phenotypes, including screens for phenotypes shared with hos1 as well as several phenotypes not seen in hos1 mutants. It is, of course, possible where mutants have not been compared beside each other, that the phenotypes are present, but have not been observed or recorded; therefore lack of phenotypic evidence cannot be equated to lack of involvement in a given pathway. However, two different reverse genetic approaches demonstrate that members of the same sub-complexes differ in their phenotypes when the loss-of-function mutants are compared (Wiermer et al., 2012; Parry, 2014). These studies demonstrate that a shared localization at the NPC or even within a protein complex does not necessarily indicate a shared syndrome. Despite the different phenotypes that NPC mutants present, there are some phenotypes that recur with loss of NPC function, which suggests that these are core processes regulated by the NPC. In Fig. 2, mutants sharing a common phenotype have been listed in the tables at the top of the figure. HOS1 has been characterized as a negative regulator of cold signalling in response to cold treatment (Ishitani et al., 1998; Lee et al., 2001; Dong et al., 2006a). RNA-sequencing analysis of hos1 at dawn indicates that many cold-responsive and cold-signalling genes are mis-expressed in hos1 when the plants are kept at ambient temperatures (MacGregor et al., 2013). The low expression of osmotically responsive genes 4 (los4/cryophyte) and suppressor of axr1 (sar1 /atnup160) genes were identified through screens for altered cold signalling similar to that which originally identified hos1 (Gong et al., 2005; Dong et al., 2006b). Moreover, hos1, los4, sar1/ nup160, nuclear pore anchor (nua), and the recently identified nucleoporin mutant nup107-1 all showed a long-period circadian rhythm (MacGregor et al., 2013). Therefore correct NPC function, circadian function, and/or cold signalling are all commonly regulated by these genes. Although both LOS4 and SAR1 are known to be involved in cold responses through CBF2 or CBF3 (Gong et al., 2002; Gong et al., 2005; Dong et al., 2006b) there is no evidence that they act through ICE1. In fact, sar1/nup160 is known not to alter the nuclear localization of ICE1 (Dong et al., 2006b). Likewise, neither SAR1/NUP160 nor LOS4 have sequences that would indicate they could act as E3 ligases. As far as has been characterized, the only function that HOS1 shares with this group of genes is their role in NPC function, specifically mRNA export (Cernac et al., 1997; Gong et al., 2002; Gong et al., 2005; Dong et al., 2006b; Parry et al., 2006; MacGregor et al., 2013; Parry, 2014). mRNA export is a crucial function of the NPC, and several NPC genes are essential for the process (Dong et al., 2006b; Parry et al., 2006; Xu et al., 2007; Wiermer et al., 2012; MacGregor et al., 2013; Parry, 2014). Therefore, the simplest explanation for the shared cold and circadian phenotypes between these mutants is that wild-type levels of mRNA export, or another aspect of RNA metabolism, is necessary for correct regulation of both processes. As we have described above, the control of flowering time is a complicated and highly regulated process, and as such mutations that alter flowering time are common. Components of each domain of the NPC, except those at the inner ring or transmembrane ring, are required for wild-type flowering time (Ishitani et al., 1998; Lee et al., 2001; Gong et al., 2005; Dong et al., 2006b; Parry et al., 2006; Xu et al., 2007; Tamura et al., 2010; Bond et al., 2011; Lee et al., 2012a; Jung et al., 2012; MacGregor et al., 2013; Jung et al., 2013; Parry, 2014). Therefore, with this many mutants all sharing altered flowering phenotypes, it is clear that some function served by the NPC is required for flowering-time control. Some but not all of the mutants with flowering-time defects show altered mRNA export, for instance nup62 has early flowering but no alteration in mRNA export (Parry, 2014), and very few of them have been investigated for altered cold responses or circadian period. As we compile more mechanistic detail about the processes regulated by HOS1, it would be interesting to apply what we have learned by studying hos1 to some of these flowering and NPC mutants to determine which of the mechanisms are specific to HOS1 and which are specific to NPC function. Summary and outlook HOS1 is the jack-of-all-trades of the NPC community. It is a large protein with large regions of sequence that have no identified similarity to other known domains and two alternative splice variants. HOS1 functions as a ubiquitin ligase, binds to chromatin, and is an NPC component necessary for mRNA export and genome-wide transcriptional control. It is critical for repressing aberrant cold responses and the transition to flowering, and it does so by being involved in both Exploring the pleiotropy of hos1 | 1669 day-to-day activities, like the control of circadian periodicity, as well as seasonal activities, through its ability to alter the chromatin state of FLC. Therefore, from the molecular level to the whole plant, and from daily to seasonal activities, HOS1 is required for a plant to be able to sense and respond appropriately to its environment and adapt accordingly. HOS1 shares homology with Elys/Mel-28, which is needed for post-mitotic reassembly of the nuclear pores, nuclear envelope, and nuclear envelop subdomains (Galy et al., 2006; Rasala et al., 2006; Tamura et al., 2010; Clever et al., 2012). However, the region of homology overlaps with the Elys region required for tethering to the NPC, not with those needed for chromatin binding or organizational functions. Therefore, although this homology supports the identification of HOS1 as an NPC, it does not give us the ability to assume that they are true functional equivalents or aid in elucidating the function that HOS1 serves at the NPC. As we accumulate information about both proteins independently, it would be beneficial for both communities to determine how much functional overlap there is by investigating hos1 phenotypes in loss of Elys or Mel-28 and vice versa to fully understand the roles that these proteins play, or as importantly, do not play. As the NPC is the gateway between cytoplasm and nucleoplasm, positioning HOS1 at the cytoplasmic or nucleoplasmic side of the complex will greatly inform conclusions made about the role it serves. We know that HOS1 is essential for correct mRNA export (MacGregor et al., 2013), but it is not clear if HOS1 is required to accompany RNA from the site of transcription through the NPC as vertebrate Nup98 is proposed to do (Zolotukhin and Felber, 1999; Köser et al., 2011), functions in mRNA transit through the NPC like Nup58 (Ferrández-Ayela et al., 2013; Bonnet and Palancade, 2014), or if it functions in the exit of the mRNA from the pore like LOS4 (Gong et al., 2005). We can speculate that HOS1 is required at the nucleoplasmic side of the NPC because it binds to chromatin (Jung et al., 2013), but as it has been demonstrated to undergo temperature-dependent nucleo-cytoplasmic partitioning (Lee et al., 2001; Lee et al., 2012a), we cannot assume that it does not serve additional functions away from the NPC. HOS1 is well conserved among plants in primary amino acid sequence, but the only function that has been tested in other species is HOS1’s role as an E3 ligase required for repressing cold signalling (Lourenço et al., 2013). In future translational studies using HOS1, it would be important to avoid analysis of one or a few of HOS1’s functions, but instead to take a broad approach to assessing the translatability of the knowledge gained in A. thaliana to other species. With all of the signalling pathways and molecular events that HOS1 coordinates, it is an ideal candidate for translational studies and real-world applications. 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