In plants, cold stress rapidly induces expression of many transcription factors, including the C-repeat-binding factors (CBFs), which activate transcription of various downstream cold-responsive (COR) genes (Chinnusamy et al., 2007; Zhu, 2016). Meanwhile, transcription of CBF genes is controlled by their own upstream transcription factors, including the bHLH transcription factor ICE1 (INDUCER OF CBF EXPRESSION 1) (Zhu, 2016). In a genetic screen to search for mutations that impair cold-induced expression of the CBF3-LUC reporter gene, AtNUP160 was identified (Dong et al., 2006b). AtNUP160 protein is enriched in the nuclear rim and is critical for nucleo-cytoplasmic transport of mRNAs, as determined by Poly (A)-mRNA in situ hybridization (Dong et al., 2006b). The atnup160-1 mutant plants displayed substantially reduced expression levels of CBF genes, which have been shown to be important for acquired freezing tolerance (Gilmour et al., 2000; Lee et al., 2005). Consistent with the reduced induction of CBFs, expression of cold-responsive genes were altered in the atnup160-1 mutant, accompanied by plant phenotypes of being sensitive to chilling stress and being defective in acquired freezing tolerance (Dong et al., 2006b).

In addition to AtNUP160, Arabidopsis HOS1 (high expression of osmotically responsive genes 1) is another clue for NPC involvement in plant stress responses. HOS1 was reported to be physically associated with the NPC components RAE1 (RNA export factor 1) and NUP43, as shown by immunoprecipitation in a proteomic study of plant NPCs (Tamura et al., 2010). Dysfunction of HOS1 resulted in over-accumulation of polyadenylated RNAs in the nucleus (MacGregor et al., 2013). In addition, HOS1 contains a region with homology to the vertebrate nucleoporin Elys (EMBRYONIC LARGE MOLECULE DERIVED FROM YOLK SAC) (Tamura et al., 2010), which is required for recruiting the Nup107–160 complex to chromatin (Gillespie et al., 2007; Doucet et al., 2010). Therefore HOS1 is considered as an NPC component in plants. Interestingly, Arabidopsis HOS1 was initially identified as a negative regulator in cold stress response from a genetic screening using the RD29A-LUC reporter system (Ishitani et al., 1997, 1998). In response to low temperature, the hos1-1 mutation causes enhanced induction of the CBF transcription factors and their downstream cold-responsive genes (Ishitani et al., 1998), while overexpression of HOS1 represses the expression of CBFs and their downstream genes as well as confers increased plant sensitivity to freezing stress (Dong et al., 2006a). HOS1 encodes an E3 ubiquitin ligase which accumulates in the nucleus in response to low temperatures (Lee et al., 2001). Cold stress also induces protein degradation of ICE1 that physically interacts with HOS1 (Dong et al., 2006a). Further investigation showed that HOS1 was required for the ubiquitination and degradation of ICE1 (Dong et al., 2006a). Since ICE1 is a transcription factor that positively regulates cold stress responses in Arabidopsis (Chinnusamy et al., 2003), it was proposed that HOS1 regulates plant cold stress responses through an ubiquitination proteasome pathway (Dong et al., 2006a).

Genetic screening using the RD29A:LUC reporter system has also identified LOS4 (low expression of osmotically responsive genes 4), which is essential for mRNA export (Gong et al., 2002). LOS4 encodes a DEAD-box RNA helicase, which is most closely related to the NPC-associated Dbp5p/Rat8p in yeast (Gong et al., 2002, 2005). The los4-1 mutant showed reduced gene expression levels of CBFs and their downstream targets, as well as increased sensitivity to chilling stress (Gong et al., 2002). Interestingly, the same genetic screening later also isolated another los4 allele, los4-2, which showed enhanced cold induction of CBF2 and its downstream target genes (Gong et al., 2005). In contrast to los4-1, the los4-2 mutant allele is more tolerant to chilling and freezing stresses, but is sensitive to heat stress (Gong et al., 2005). In situ poly(A) hybridization showed that the export of poly(A) RNAs was blocked in the los4-2 mutant at warm or high temperatures but not at low temperatures, whereas the los4-1 mutation weakened mRNA export at both low and warm temperatures (Gong et al., 2005), indicating that proper mRNA export is important for plant cold stress responses.

Importin β belongs to a large family of Importin β-like nuclear transport receptors that are also known as karyopherins (Gorlich and Kutay, 1999; Merkle, 2003). Proteins containing the importin β domain can function as either importins that mediate nuclear protein import, or exportins that transport proteins out of the nucleus (Merkle, 2003). The Arabidopsis SAD2 (Super sensitive to ABA and drought2) encodes an importin β-domain family protein and was identified in a genetic screening, which was based on alterations in the expression levels of the stress-responsive RD29A-LUC reporter gene (Verslues et al., 2006). The sad2-1 mutant showed increased luminescence after ABA, salt, cold or polyethylene glycol treatments, and exhibited ABA hypersensitivity in seed germination and seedling growth (Verslues et al., 2006). Although the mechanism underlying ABA hypersensitivity in sad2 is not clear, SAD2 may function either in importing a negative regulator of ABA response into the nucleus, or in exporting a positive regulator out of the nucleus, given that importin β proteins can function as nuclear transport receptors. Indeed, the ability of SAD2 in transporting proteins was revealed later on. SAD2 was found to be required for nuclear import of MYB4, an R2R3-type transcription repressor that co-immunoprecipitated with SAD2 (Zhao et al., 2007). As a result of the absence of MYB4 protein in the nucleus, sad2 plants accumulated UV-absorbing pigments and displayed increased tolerance to UV-B radiation (Zhao et al., 2007).

To identify importin genes that function in drought tolerance, Luo et al. (2013) screened T-DNA insertion mutants of Arabidopsis importin β family genes based on the ability to survive after drought treatment. A mutant with a T-DNA insertion in AtKPNB1 was identified to display considerably increased drought tolerance (Luo et al., 2013). AtKPNB1 encodes a homolog of human importin β1, inactivation of which resulted in increased stomatal closure in response to ABA, lower rate of water loss, and substantially enhanced drought tolerance (Luo et al., 2013); while over-expression of AtKPNB1 led to increased sensitivity to drought compared to wild type plants, demonstrating that AtKPNB1 is an important negative effector of drought tolerance (Luo et al., 2013).

In addition to abiotic stress conditions, biotic stress can also trigger plant responses that involve the NPC. The Arabidopsis snc1 (suppressor of npr1-1, constitutive 1) mutant displays constitutive activation of disease resistance response against pathogens, due to a gain-of-function mutation in a TIR-NBS-LRR-type R gene (Zhang et al., 2003). Genetic screening for suppressors of snc1 isolated a series of double mutants named modifier of snc1 (mos), which no longer display constitutive resistance to virulent pathogens as the snc1 single mutant (Palma et al., 2005; Zhang and Li, 2005). Among the MOS genes, MOS3, MOS6, and MOS7 encode proteins associated with the NPC. MOS3 encodes a protein with high sequence similarity with human nucleoporin96 (Zhang and Li, 2005). MOS6 encodes Arabidopsis importin α3 (Palma et al., 2005), while MOS7 is homologous to human and Drosophila melanogaster nucleoporin Nup88 (Cheng et al., 2009). Interestingly, it was found that nuclear accumulation of the defense signaling components EDS1 (Enhanced Disease Susceptibility 1) and NPR1 (Non-expresser of PR genes 1) is significantly reduced in mos7-1 plants (Cheng et al., 2009). Moreover, CPR5 (Constitutive Expresser of Pathogenesis-Related Genes 5), which plays a key inhibitory role in effector-triggered immunity (ETI), was recently found to be a novel transmembrane nucleoporin (Wang et al., 2014; Gu et al., 2016). These findings clearly displayed an important role of nucleo-cytoplasmic trafficking in plant innate immunity.

Nuclear pore complex-dependent plant responses to biotic stress may involve ethylene signaling. In Nicotiana benthamiana, Nup75 (Nucleoporin 75) was identified as essential for plant resistance to Phytophthora infestans, and for the induction of some defense responses, including ethylene-mediated production of phytoalexin (Ohtsu et al., 2014). Arabidopsis HPR1 (HYPER RECOMBINATION1), which is a component of the NPC-associated TREX (Transcription-Export) complex, was isolated through a screening for edr1 (enhanced disease resistance) suppressors. In Arabidopsis, hpr1 mutation not only suppresses the enhanced disease resistance caused by edr1 mutation, but also increases ethylene-induced senescence in the edr1 background, suggesting that HPR1 plays a role in ethylene signaling pathway (Pan et al., 2012). It was also found that hpr1 mutation suppresses plant insensitivity to ethylene as well as RTE1 (REVERSION-TO-ETHYLENE SENSITIVITY1) transcript levels in transgenic Arabidopsis that over-expressed RTE1 (Xu et al., 2015). An enhanced ethylene response was observed in of Arabidopsis eer5-1 mutant, which harbors a mutation in the NPC-associated TREX-2 component THP1 (Christians et al., 2008). Enhanced ethylene response in eer5-1 was correlated with failure to induce appropriately a subset of ethylene-regulated genes (Christians et al., 2008). Therefore, the NPC and its associated RNA export complexes may be involved in ethylene-mediated plant stress responses.

By using in situ hybridization, many studies of nucleoporin mutants have demonstrated accumulation of polyadenylated mRNA in the nucleus (Dong et al., 2006b; Lu et al., 2010; Parry, 2014). Interestingly, stress conditions can also induce bulk mRNA accumulation in the nucleus (Saavedra et al., 1996; Bond, 2006; Muthuswamy and Meier, 2011). It is thus important to understand how certain mRNAs are selectively transported under stress conditions. In yeast, following heat or ethanol stress, poly(A) RNAs accumulates within nuclei, while mRNAs encoding Hsps (heat shock proteins) are efficiently exported from the nucleus (Saavedra et al., 1996; Bond, 2006). Recently, it was revealed that in yeast, cellular stress induces dissociation between regular mRNAs and the export receptor Mex67 as well as its adaptor proteins, thereby preventing general mRNA export; meanwhile, heat-shock mRNAs are efficiently exported in association with Mex67, without the need of adapter proteins (Zander et al., 2016). In fact, adaptor-bound mRNAs, but not free mRNAs, undergo quality control, indicating that at the cost of accuracy, heat-shock mRNAs are exported and translated without delay, allowing cells to survive extreme situations (Zander et al., 2016).

In Arabidopsis under heat or ethanol stress, a correlation seems to exist between altered protein sumoylation levels and bulk mRNA nuclear retention (Muthuswamy and Meier, 2011). Exposing Arabidopsis plants to heat shock and ethanol stress both resulted in elevation in high-molecular-weight SUMO conjugates, accompanied by nuclear mRNA accumulation (Muthuswamy and Meier, 2011). Because mutations in either SUMO E3 ligase SIZ1 or SUMO isopeptidase ESD4 (Early in short days 4) leads to nuclear mRNA retention, it has been suggested that sumoylation acts upstream of mRNA export, likely through the transient sumoylation status of one or more factors involved in mRNA trafficking (Muthuswamy and Meier, 2011).

Trafficking from the cytoplasm to the nucleus is essential for proteins with nucleus-specific functions. Under stress conditions, nuclear import of certain proteins can be critical for the plant to reprogram cellular processes to combat the stress. In Arabidopsis, MYB4 negatively regulate the transcription of cinnamate 4-hydroxylase (C4H) and thereby synthesis of sinapate esters which are UV-absorbing pigments (Zhao et al., 2007). MYB4 was found to co-immunoprecipitate with SAD2, which is an importin β-domain family protein essential for nuclear import of MYB4 (Zhao et al., 2007). Consistently, Arabidopsis sad2 mutant is more tolerant to UV-B radiation compared with wild type plants. Protein interaction between MYB4 and SAD2 requires the conserved GY/FDFLGL motif in the C terminus of MYB4, as demonstrated by the observation that an Asp to Asn mutation in the GY/FDFLGL motif abolishes the interaction between MYB4 and SAD2 (Zhou et al., 2015). Without a functional GY/FDFLGL motif, MYB4 failed to be transported into the nucleus and thus cannot repress their target genes (Zhou et al., 2015).

Besides SAD2, Arabidopsis MOS7 is another plant nucleoporin that has been shown to regulate nuclear accumulation of stress-responsive proteins. MOS7 is homologous to human and Drosophila nucleoporin Nup88 (Cheng et al., 2009). In animals, Nup88 attenuates NES (nuclear export signal)-mediated protein nuclear export (Roth et al., 2003; Xylourgidis et al., 2006). In Arabidopsis, the mos7-1 mutation caused defects in basal and R protein–mediated immunity and in systemic acquired resistance (Cheng et al., 2009). Further investigation showed that nuclear accumulation of the autoactivated R protein snc1 as well as the defense signaling components EDS1 (Enhanced Disease Susceptibility 1) and NPR1 (Non-expresser of PR genes 1) was significantly reduced in mos7-1 plants, whereas nuclear abundance of other tested proteins was unaffected (Cheng et al., 2009), supporting the notion that trafficking of certain stress-responsive proteins can be subject to selective regulation by nucleoporins.

Despite the obvious specificity of the connection between the NPC and plant stress responses, little is known about how such specificity is achieved. Arabidopsis CPR5 (Constitutive Expresser of Pathogenesis-Related Genes 5) was initially identified as a negative regulator of plant Programmed Cell Death (PCD) and Effector-Triggered Immunity (ETI) (Boch et al., 1998; Wang et al., 2014). Loss-of-function mutations in CPR5 resulted in resistance against multiple pathogens, whereas over-expression of CPR5 compromised ETI-associated PCD and pathogen resistance in Arabidopsis (Boch et al., 1998; Wang et al., 2014). CPR5 was later revealed as a transmembrane nucleoporin (Wang et al., 2014; Gu et al., 2016). In addition, transient interference of CPR5 expression yielded in transcriptome patterns that significantly match plant responses to a variety of stress conditions including cold, salt/osmotic stress, abscisic acid, and various pathogens (Gu et al., 2016). It was hypothesized that with compromised CPR5 function, the NPC adopts a structure with significantly increased permeability and/or transport activity that allows deregulated nuclear influx of diverse signaling cargos, which normally undergo nuclear translocation only under stimulus-induced conditions (Gu et al., 2016). Indeed, over-expression of CPR5 caused substantial cytoplasmic retention of NPR1, JAZ1, and ABI5, which are stress- and phytohormone-related nuclear proteins (Wang et al., 2014; Gu et al., 2016). Researchers further revealed that upon activation by immunoreceptors, CPR5 undergoes an oligomer-to-monomer conformational switch, which reconfigures the selective barrier to allow significant influx of nuclear signaling cargos through the NPC (Gu et al., 2016). These findings thus established CPR5 as a converging point in the specific connection between the NPC and the ETI/PCD responses in plants.

In addition to regulating RNA/protein transport, the NPC can also regulate gene expression at the transcriptional level. In as early as 1985, the “gene gating” hypothesis has proposed that certain expanded chromatin regions with transcription units can be attached to the NPC (Blobel, 1985). Subsequently this hypothesis has been supported by many studies in yeast and Drosophila. In yeast, several highly inducible genes are randomly distributed in the nucleoplasm when transcriptionally repressed but are recruited to the nuclear periphery upon activation (Brickner and Walter, 2004; Casolari et al., 2004; Taddei et al., 2006). In Drosophila, the nucleoporins Sec13, Nup98, and a subset of FG-repeat nucleoporins bind to developmentally regulated genes that are undergoing transcription induction (Capelson et al., 2010). Interestingly, certain NPC target genes exhibit transcriptional memory, i.e., after being repressed, these genes remain at the nuclear periphery for several generations and are primed for reactivation (Light et al., 2010). Transcriptional memory of yeast INO1 requires the NPC component Nup100, as well as incorporation of the histone variant H2A.Z after gene repression (Light et al., 2010), indicating that the NPC can directly interact with chromatin and contribute to epigenetic gene regulation in response to developmental and environmental stimuli.

In plants, limited information is available for the mechanism of how the NPC regulates gene transcription. Arabidopsis hos1 mutant exhibits an early flowering phenotype with repressed FLC gene expression. In an effort to explore how HOS1 regulates FLC (FLOWERING LOCUS C) expression, researchers (Jung et al., 2013) found that HOS1 binds to FLC chromatin in an FVE-dependent manner. In addition, HOS1 binding to the FLC locus is dramatically elevated at 4°C. HOS1 also interacts with the histone deacylase HDA6 and inhibits the binding of HDA6 to the FLC locus. Therefore, it was proposed that under short-term cold stress, HOS1 binds to FLC chromatin in an FVE-dependent manner to limit the chromatin accessibility to HDA6, allowing for activation of FLC transcription (Jung et al., 2013). Recently, our work isolated Arabidopsis SAC3B (SUPPRESSOR OF ACTIN3B), a core component of the TREX-2 (transcription-coupled export 2) complex, through a forward genetic screening for anti-silencing factors (Yang et al., 2017). Mutation of SAC3B caused gene silencing of a reporter gene luciferase driven by double 35S (d35S) promoter, accompanied by elevation in the repressive histone mark H3K9me2 and by reduction in RNA polymerase Pol II occupancy (Yang et al., 2017). Moreover, THP1 (Tho2/Hpr1 PHENOTYPE1) and NUA (NUCLEAR PORE ANCHOR) were identified as SAC3B-associated proteins whose mutations also caused d35S::LUC silencing (Yang et al., 2017). The THP1 is another representative component of TREX-2 complex, while NUA is homologous to a component of nuclear pore basket in vertebrate (Krull et al., 2004; Jacob et al., 2007). Importantly, FLC gene expression is decreased in both sac3b and nua mutants (Xu et al., 2007; Yang et al., 2017), while our unpublished IP-MS results also identified HOS1 as a SAC3B-interacting protein. Thus it appears that the Arabidopsis NPC, in association with the TREX-2 complex, controls gene expression through HOS1-dependent chromatin targeting. In the future, it would be interesting to examine whether stress-inducible genes are tethered to the NPC, and whether stress memory can be mediated through chromatin tethering to the NPC.