The nucleoporin NUP160 and NUP96 regulate nucleocytoplasmic export of mRNAs and participate in ethylene signaling and response in Arabidopsis

Arabidopsis nucleoporin involved in the regulation of ethylene signaling via controlling of nucleocytoplasmic transport of mRNAs. The two-way transport of mRNAs between the nucleus and cytoplasm are controlled by the nuclear pore complex (NPC). In higher plants, the NPC contains at least 30 nucleoporins. The Arabidopsis nucleoporins are involved in various biological processes such as pathogen interaction, nodulation, cold response, flowering, and hormone signaling. However, little is known about the regulatory functions of the nucleoporin NUP160 and NUP96 in ethylene signaling pathway. In the present study, we provided data showing that the Arabidopsis nucleoporin NUP160 and NUP96 participate in ethylene signaling-related mRNAs nucleocytoplasmic transport. The Arabidopsis nucleoporin mutants (nup160, nup96-1, nup96-2) exhibited enhanced ethylene sensitivity. Nuclear qRT-PCR analysis and poly(A)-mRNA in situ hybridization showed that the nucleoporin mutants affected the nucleocytoplasmic transport of all the examined mRNAs, including the ethylene signaling-related mRNAs such as ETR2, ERS1, ERS2, EIN4, CTR1, EIN2, and EIN3. Transcriptome analysis of the nucleoporin mutants provided clues suggesting that the nucleoporin NUP160 and NUP96 may participate in ethylene signaling via various molecular mechanisms. These observations significantly advance our understanding of the regulatory mechanisms of nucleoporin proteins in ethylene signaling and ethylene response.


Introduction
Ethylene is involved in regulation of various processes during plant growth and development, including seed germination, flowering, sex determination, fruit maturity, senescence, and stress responses (Dubois et al. 2018). Arabidopsis thaliana is a good model plant for studying ethylene signaling. When ethylene or ethylene precursor ACC (1-aminocyclopropane-1-carboxylic acid) was added to the medium, the etiolated Arabidopsis seedlings exhibited a typical "triple response" with exaggerated apical hook, swelled hypocotyl, and shortened hypocotyl and root (Bleecker et al. 1988;Guzmán and Ecker 1990). Ethylene signaling starts with the binding of ethylene to receptors. In Arabidopsis, there are five ethylene receptors (ETR1, ETR2, ERS1, ERS2, EIN4) (Chang et al. 1993;Hua et al. 1998;Hua and Meyerowitz 1998;Sakai et al. 1998). Ethylene receptors mainly localize to the ER and Golgi organelles (Chen et al. 2002;Schaller and Bleecker 1995;Dong et al. 2008). Among the five ethylene receptors, the subfamily I receptors (ETR1, ERS1) are believed to play a predominant role in ethylene receptor signaling (Hua et al. 1995;Hua and Meyerowitz 1998;Hall and Bleecker 2003;Qu et al. 2007;Liu et al. 2010).
To explore the regulation mechanism of the ethylene receptor ETR1, we and colleagues previously reported isolation and identification of the ETR1 receptor-associated protein RTE1, RTH and CPR5 based on their regulatory functions in the ETR1 receptor signaling (Resnick et al.  Zhou et al. 2007;Dong et al. 2008Dong et al. , 2010Wang et al. 2017;Zheng et al. 2017;Chen et al. 2022). The Arabidopsis RTE1 is an activator of the ETR1 receptor, and they can physically interact (Resnick et al. 2006;Dong et al. 2010). Genetic analyses indicated that RTE1 is essential for the ETR1 receptor to function, but not for the other receptors (Resnick et al. 2006(Resnick et al. , 2008. In Arabidopsis, the homologue of RTE1 (RTH) regulates ethylene signaling via a physical interaction with RTE1 (Zheng et al. 2017). Interestingly, it was reported that Arabidopsis CPR5 also interacts with the ETR1 receptor and participates in ethylene signaling (Wang et al. 2017). CPR5 was initially isolated from the research on plant systemic acquired resistance (Bowling et al. 1997;Boch et al. 1998). Further study indicated that Arabidopsis CPR5 can act as a nucleoporin to regulate immunity triggering and programmed cell death (Gu et al. 2016). Recently, we reported that CPR5 can regulate nucleocytoplasmic transport of mRNAs in ethylene signaling pathway (Chen et al. 2022).
It is known that the two-way transport of mRNAs between the nucleus and cytoplasm is controlled by the nuclear pore complex (NPC). In yeast cells, the NPC is composed of 35-50 proteins (Yang et al. 1998;Rout et al. 2000). The mammalian NPC is a larger complex consisting of 80-100 proteins (Görlich and Kutay 1999). In higher plants, the NPC contains at least 30 nucleoporins (Meier and Brkljacic 2009;Tamura et al. 2010). The Arabidopsis nucleoporins are involved in various biological processes such as pathogen interaction, nodulation, cold response, flowering, and hormone signaling (Zhang and Li 2005;Dong et al. 2006;Parry et al. 2006;Meier and Brkljacic 2009;Tamura et al. 2010). Compared to the Arabidopsis CPR5 which was thought to be a plant-specific transmembrane nucleoporin and may contribute to the stability of the NPC core scaffold (Gu et al. 2016), the Arabidopsis nucleoporin NUP160 and NUP96 are the plant homologs of the vertebrate nucleoporins (Zhang and Li 2005;Dong et al. 2006). Unfortunately, less has been known about the regulatory functions of the nucleoporins NUP160 and NUP96 in nucleocytoplasmic transport of mRNAs and ethylene signaling.
In the present study, we provided evidence demonstrating that the Arabidopsis nucleoporin NUP160 and NUP96 play a role in the nucleocytoplasmic transport of mRNAs. The Arabidopsis nucleoporin mutants (nup160, nup96-1, nup96-2) exhibited enhanced ethylene sensitivity. Using poly(A)-mRNA in situ hybridization and nuclear qRT-PCR analysis, we detected the impeded export of ethylene-related mRNAs out of the nucleus in the nucleoporin mutants. All the examined mRNAs of the ethylene signaling-related components were highly accumulated in the nucleus. These results advanced our understanding that the nucleoporin NUP160 and NUP96 may mediate the mRNAs nucleocytoplasmic transport to participate in ethylene signaling.

Results
Ethylene sensitivity assays of the nucleoporin mutants (nup160, nup96-1, nup96-2) To know whether the ethylene sensitivity of the nuleoporin mutants (nup160, nup96-1 and nup96-2) was altered, the etiolated seedling phenotypes of the mutants were examined. When treated with different concentrations of ACC, the seedling hypocotyl lengths of the nucleoporin mutant nup160, nup96-1 and nup96-2 were significantly shorter than that of WT, suggesting that the Arabidopsis nuleoporin mutants could be hypersensitive to ethylene.
Interestingly, it was observed that the seedling hypocotyl lengths of nup160, nup96-1 and nup96-2 were shorter than that of WT even without application of exogenous ACC, suggesting that ethylene levels may be endogenously elevated in the nucleoporin mutants. To block the effect of endogenous ethylene on the etiolated seedling phenotype of the nucleoporin mutants, 1-methylcyclopropene (1-MCP), an ethylene action inhibitor, was added to the culture medium. As shown in Fig. 1A, relative decreases of the seedling hypocotyl lengths of the nucleoporin mutants were obtained, supporting that the nucleoporin mutants (nup160, nup96-1, nup96-2) were more sensitive to ethylene.
In Arabidopsis, ethylene signaling has been shown to promote seed germination in the presence of salt stress (Wilson et al. 2014). We next examined the ethylene sensitivity of the nucleoporin mutants (nup160, nup96-1, nup96-2) by measuring seed germination rates of the mutants under salt stress. To determine the individual contribution of each nucleoporin to seed germination in the presence of salt stress, the time course of germination was examined ( Fig. 1B and C). In the absence of NaCl, the germination rate of all seed lines reached about 90%, with the exception of nup160. It appeared that nup160 had a stronger effect on seed germination than that of nup96.
In the presence of 150 mM NaCl, either the nucleoporin mutant nup160 or the nucleoporin mutant nup96 (nup96-1, nup96-2) had significant effect on the time course of seed germination compared to the WT. All the nucleoporin mutants (nup160, nup96-1, nup96-2) germinated later and slower than wild-type seeds. The nup160 was more sensitive than either of the nup96 mutants. These data suggest that both NUP160 and NUP96 play a positive role in seed germination under salt stress.

Nucleocytoplasmic export of mRNAs was impeded in the nucleoporin mutants (nup160, nup96-1, nup96-2)
We further analyzed the nucleocytoplasmic transport of mRNAs in the nucleoporin mutant nup160, nup96-1 and nup96-2. As described previously (Dong et al. 2006), in situ poly(A)-mRNA hybridization analysis was employed, and obvious nuclear poly(A) signal aggregations were observed in the nucleus structures of nup160, nup96-1 and nup96-2, respectively ( Fig. 2). In contrast, no such aggregated signal was detected in the wild type plant (Col-0). To confirm that the poly(A)-tagged aggregations localized to the nucleus, co-staining of the samples with fluorescein-labelled oligo(dT) and DAPI was performed. As shown in Fig. 3, nuclear localization of the aggregated mRNAs tagged by the fluorescein-labelled oligo(dT) was detected in the cells of the nucleoporin mutants (nup160-1, nup96-1) but not in the wild type (Col-0).

Nucleocytoplasmic transport of ethylene signaling-related mRNAs was affected in the nucleoporin mutants
To examine whether the transcript levels of the ethylene signaling-related mRNAs were affected when nucleocytoplasmic export of mRNAs was impeded in the nucleoporin mutants, we firstly measured the nuclear accumulation of the ETR1 receptor mRNA in the mutant plants. Total RNA and nuclear RNA were extracted from the 10-day-old seedlings of WT, nup160, nup96-1 and nup96-2, respectively. qRT-PCR analysis revealed that nuclear accumulation of the ETR1 mRNA in the nucleoporin mutants was significantly higher than that of WT (Fig. 4).
We next analyzed the accumulation of the other ethylene signaling-related mRNAs in the nucleoporin mutant nup160, nup96-1 and nup96-2. As shown in Fig. 4, the ethylene receptor genes (ETR2, ERS1, ERS2 and EIN4), the downstream components (CTR1, EIN2, EIN3), and some of the ETHYLENE RESPONSIVE FACTORS (ERFs) in ethylene signaling pathway were examined. The results indicated that all the mRNAs of the ethylene receptor genes (ETR2, ERS1, ERS2, EIN4) were highly aggregated in the nucleus of the nucleoporin mutants (nup160, nup96-1 and nup96-2), being similar to that of the ETR1 receptor. Meanwhile, we examined the mRNA aggregation of the Arabidopsis RTE1 which encodes an activator of the ETR1 receptor, and the nuclear accumulation of the RTE1 mRNA was observed in the nucleoporin mutants. In addition, the mRNA nuclear aggregations of the ETR1 receptor associated protein genes including CPR5, CTR1, and EIN2 were detected in the nucleoporin mutants (nup160, nup96-1 and nup96-2). Similarly, the downstream components of ethylene signaling pathway such as EIN3 and the ERFs (ERF1, ERF2, ERF4, ERF11, ERF105) exhibited highly accumulation of the mRNAs in the nucleus of the nucleoporin mutants compared to that of WT (Fig. 4). Moreover, we examined the mRNAs accumulation of the genes which were involved in the other pathways such as flowering pathway, including REM16, LEY, GI and AP1 (Yu et al. 2020), and experiments showed that increased nuclear aggregations of the mRNAs were detected in the nucleoporin mutants (nup160, nup96-1 and nup96-2) (Fig. 4). These results suggest that nucleoporins are also involved in other pathways in Arabidopsis.

Elevated mRNA levels of the ethylene-induced ERFs in the nucleoporin mutants
As expression levels of the downstream ethylene-induced ERFs can be used as a reference for ethylene sensitivity as described previously (Wang et al. 2017;Zheng et al. 2017), we analyzed the relative expression levels of the ethyleneinduced ERFs (ERF1, ERF2, ERF4, ERF11 and ERF105) in WT, nup160, nup96-1 and nup96-2. Without ACC treatment, the expression levels of all the examined ERFs in WT and the nucleoporin mutants were low. When treated with ACC at a low concentration (5 μM), the expression levels of all the examined ERFs were elevated more in the mutants than in the WT (Fig. 5). As a control, the gene which was not the component of ethylene signaling pathway such as ADF1, encoded an actin-depolymerization factor in Arabidopsis (Dong et al. 2001), was examined in the nucleoporin mutants, and data showed no ethylene-induced expression in the plants (Fig. 5).

Transcriptomic examination of ethylene-induced gene expressions in the nucleoporin mutants
To know global changes of the ethylene-induced transcripts in the nucleoporin mutants, transcriptome analysis was employed. The 2-week-old seedlings treated with water (control) or 100 μM ACC were collected and used for RNA-seq analysis. Based on the comparisons between the control and the ACC treated samples, the transcripts with fold change ≥ 2 and FDR < 0.05 were defined as significant WT-vs-WT-ACC (WT treated with ACC), nup96-vs-nup96-ACC (nup96 treated with ACC), and nup160-vs-nup160-ACC (nup160 treated with ACC) showed overlapped DEGs among the comparisons ( Fig. 6a; Supplementary s1-s3). The heat map represented relative expression of mostly overlapped DEGs among the ethylene-induced genes in both nup160 and nup96 ( Fig. 6b; Supplementary s4). The GO and KEGG enrichment analysis indicated that the DEGs among the ethylene-induced genes in the nucleoporin mutants were mainly involved in biosynthesis of secondary metabolites, metabolisms of amino acids, cofactors, vitamins and carbohydrate, environmental adaptation, hormone responses and signal transduction (Fig. 6c and d; Supplementary s5).

Discussion
In a previous study (Chen et al. 2022), we reported that the nucleoporin CPR5, a plant-specific transmembrane nucleoporin, plays an important role in regulation of the nucleocytoplasmic transport of the mRNAs in ethylene signaling pathway. CPR5 was thought to contribute to the stability of the NPC core scaffold (Gu et al. 2016). Knockout of CPR5 leads to mRNAs aggregation in the nucleus of cpr5-T3. qRT-PCR examination revealed that the ethylene signalingrelated mRNAs including those of ETR1, ETR2, ERS1, ERS2, EIN4 and CTR1 were dramatically accumulated in the nucleus of cpr5-T3. However, no significant accumulation of the mRNAs of EIN2, EIN3 and some ERFs was observed in the nucleus of cpr5-T3. It was suggested that CPR5 may selectively regulate the mRNAs nucleocytoplasmic transport in ethylene signaling pathway. Compared to the cpr5 mutant, the nucleoporin mutant nup160, nup96-1 1 3 and nup96-2 restricted nucleocytoplasmic transport of all the examined ethylene signaling-related mRNAs, including the receptor ERT1, ETR2, ERS1, ERS2 and EIN4, and the downstream CTR1, EIN2, EIN3, and ERFs (Fig. 4), none of them seem to be compromised in their export out of the nucleus. The observations suggest that the nucleoporin NUP160 and NUP96 may function differently from CPR5 in regulating the mRNAs nucleocytoplasmic transport in ethylene signaling pathway.
In ethylene signaling pathway, there are both negative and positive regulators. For example, the ethylene receptor proteins ETR1, ETR2, ERS1, ERS2, EIN4 and the receptor associated CTR1 negatively regulate ethylene signaling and ethylene responses in Arabidopsis, whereas the EIN2 and EIN3 play positive roles in ethylene signaling pathway. In addition, it was reported that the downstream ERFs can be functional as positive or negative regulators in ethylene signaling (Lyons et al 2013). The results from our study showing that all the examined nucleoporin mutants (nup160, nup96-1, nup96-2, and cpr5-T3) displayed enhanced ethylene sensitivity ( Fig. 1; Chen et al. 2022), suggesting the negative regulators of ethylene signaling may be mainly affected when the mRNAs nucleocytoplasmic transport were impeded in the mutant plants. When a bunk of mRNAs export out of nucleus were affected in ethylene signaling pathway in the nucleoporin mutants, restriction of the negative regulators such as ETR1, ETR2, ERS1, ERS2, EIN4 and CTR1 may largely contribute to the enhanced ethylene sensitivity of the plants. Undoubtedly, the nucleocytoplasmic export of the ethylene signaling-related mRNAs in the nucleoporin mutants (nup160, nup96-1 and nup96-2) advanced our understanding of the ethylene signaling and its molecular regulations in plants.
In addition to ethylene signaling pathway, the increased aggregations of the nuclear mRNAs were observed for REM16, LEY, GI and AP1 in flowering pathway (Fig. 4), suggesting that the mRNAs nucleocytoplasmic transport may participate in the other pathways. As supported, it was observed that the nucleoporin mutants exhibited an early flowering phenotype, similarly as previously reported in the Arabidopsis nucleoporin mutants, including nup96 (Parry et al. 2006), nup160 (Dong et al. 2006;Parry et al. 2006), nup136 (Tamura et al. 2010), andtpr/nua (Jacob et al. 2007;Xu et al. 2007). It is suggested that early flowering phenotype may be largely attributed to the nuclear aggregations of the flowering-related mRNAs such as REM16, LEY, GI and AP1 in flowering pathway (Fig. 4).
To understand more about the regulation functions of the nucleoporin NUP160 and NUP96 in ethylene signaling, a transcriptome analysis was performed (Fig. 6). The transcriptomes of 2-week-old seedlings of the nup160 and nup96 treated with 100 μM ACC were compared with those of the wild type samples. The multiple comparisons of WT-vs-WT-ACC, nup160-vs-nup160-ACC, and nup96-vs-nup96-ACC showed that the expressions of the genes such as ARGOS (AT3G59900) which is a negative regulator of ethylene signaling (Shi et al. 2016), ERF014 (AT1G44830) which is a member of group II in the ERF family (Zhang et al. 2016), ACS4 (AT2G22810, ethylene biosynthetic gene ACC SYNTHASE4), DCR (AT2G39980) encoding a member of the BAHD family of acyltransferases, LTL1 (AT3G04290) which is a salt-induced gene encoding a GDSL-motif lipase (Naranjo et al. 2006), ARF14 (AT3G25730) encoding a member of the APETALA 2/Ethylene Response Factor (AP2/ERF) family (Vogel et al. 2012), and EARLI1 and EARLI1-like (AT4G12510, AT4G12520, AT5G46890, AT5G46900) genes that contains signal peptide sequences and are also classified into the lipid transfer protein (LTP) family (Shi et al. 2011), were significantly changed ( Fig. 6; Supplementary s4-5). These observations provide clues suggesting that the nucleoporin NUP160 and NUP96 may participate in ethylene signaling and ethylene responses via these genes.
The eukaryotic nucleus is enclosed by the nuclear envelope. The spatial separation of mRNA synthesis in the nucleus and translation in the cytoplasm has forced the development of nucleocytoplasmic RNA transport pathways through the nuclear pores. In early studies, it was reported that the Arabidopsis MOS3, belonging to the nucleoporin Nup107-160 complex, functions in mRNAs export pathway (Germain et al. 2010;Ehrnsberger et al. 2019a). In the present study, we provided evidence indicating that both the nucleoporin NUP160 and NUP96 participate in the ethylene signaling-related mRNAs nucleocytoplasmic transport and affect the ethylene sensitivity of the mutants (Figs. 1  and 4). In addition to nucleoporins, it was reported that the RNA helicase UAP56 can interacts with ALY and UIEF1/2 (UAP56-INTERACTING EXPORT FACTOR1 and 2), and participate in the regulation of mRNAs nucleocytosolic transport (Pfaff et al. 2018;Ehrnsberger et al. 2019b). Interestingly, a recent study reported that post-transcriptional splicing of mRNAs was involved in their nucleocytosolic exporting (Rudzka et al. 2022). Without a doubt, more efforts are needed to study the effects of pre-mRNA splicing on its nuclear exportation in the future research.

Plant materials and ethylene response assays
The Arabidopsis nucleoporin mutant nup160-1 had a C-to-T substitution which created a premature stop codon (CAA to TAA) at exon 16 as shown in our previous study (Dong et al. 2006). The mutants nup96-1 and nup96-2 were kindly provided by Dr. Xin Li (Zhang and Li 2005). A single A-to-C point mutation was found in nup96-1 (mos3-1). The nup96-2 (mos3-2) contained a T-DNA insertion in the fifth exon (Salk_109959). Seeds of the wild type (Col-0) and the nucleoporin mutants were surface sterilized and then sowed on 1/2 MS (Murashige and Skoog) medium or in soil in a controlled environment growth chamber at 21 ℃ under 16 h light/8 h dark.
The ethylene response assay of Arabidopsis seedlings was as previously described (Wang et al. 2017). Seeds were surface sterilized and then sowed on 1/2 MS medium containing ACC at different concentrations (0, 0.5, 5, 10, 20, 100 μM). After treatment at 4 ℃ for 3 days, plates were moved to a growth chamber for 8 h under white light, and then wrapped with aluminum foil and placed in a growth chamber for indicated periods. Measurement of hypocotyl length and statistical data were evaluated by Student's t test.

Seed germination experiments
Seed germination experiments were according to Wilson et al (2014). In brief, seeds were surface sterilized in 70% (v/v) ethanol for 30 s, dried on filter paper, and then placed on agar plates with 1/2 MS medium containing NaCl at concentration of 150 mM (pH 5.7, 0.8% (w/v) agar). At least twenty seeds of one genotype were plated per plate. Three plates per genotype were used in each experiment. The plates were placed vertically in a controlled environment chamber at 21 ℃ under 16 h light/8 h dark. Germination was evaluated at the indicated times. Seeds germination rates were measured and the values were analyzed by Student's t test.

qRT-PCR analysis
Two week-old Arabidopsis seedlings were used for total RNA extraction according to TRNzol (TIANGEN, China). Half of the same seedling sample (3 g) was used for the nuclear RNA extraction as previously described by Chen et al (2022). To remove any contaminating genomic DNA, the RNA was first treated using the gDNA Eraser in the kit, and then used for cDNA synthesis with PrimeScript RT Enzyme in the kit (Takara). Real-time qPCR was performed using Applied Biosystems QuantStudio 5 (ABI, https:// www. therm ofish er. com/). The qRT-PCR was conducted at 95 ℃ for 10 min, followed by 40 cycles at 95 ℃ for 30 s, 55 ℃ for 30 s, and 72 ℃ for 20 s. Biological replicates for each set of experiments were carried out three times, and the mean value of three replicates was normalized using Tubulin8 as an internal control. Gene-specific primers used for qRT-PCR are provided in supplementary material (Supplementary table s6).

Poly(A)-mRNA in situ hybridization
Poly(A)-mRNA in situ hybridization was performed as previously described (Gong et al. 2005;Chen et al. 2022). The samples were observed using a laser scanning confocal microscopy (Leica TCS SP5, Germany) with a 488 nm excitation laser, and photopictures were taken from the leaf mesophyll cells.
For DAPI co-staining, the young leaves used for in situ hybridization with fluorescein-labeled oligo (dT) probe were stained with DAPI (4′,6-diamidino-2-phenylindole, 0.1 mg/ mL) according to instructions (Sigma-Aldrich). Florescent signal was examined by a laser scanning confocal microscopy (Leica TCS SP5) at emission 505-530 nm for green signal or 435-500 nm for the blue.

Transcriptome sequencing
The 2-week-old seedlings treated with 100 μM ACC for 6 h or untreated were collected and used for sequencing by Genedenovo Biotechnology Co., Ltd (Guangzhou, China). The genes with fold change ≥ 2 and False Discovery Rate (FDR) < 0.05 were defined as significant Differentially Expressed Genes (DEGs). Their Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were further detected according to the GO database (http:// www. geneo ntolo gy.org/) and KEGG database (http:// www. genom e.jp/kegg/), respectively.