SKIP Confers Osmotic Tolerance during Salt Stress by Controlling Alternative Gene Splicing in Arabidopsis

Please cite this article in press as: Feng et al., SKIP Confers Osmotic Tolerance during Salt Stress by Controlling Alternative Gene Splicing in Arabidopsis, Molecular Plant (2015), http://dx.doi.org/10.1016/j.molp.2015.01.011

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SKIP Confers Osmotic Tolerance during Salt Stress by Controlling Alternative Gene Splicing in Arabidopsis Jinlin Feng1,2,4, Jingjing Li1,4, Zhaoxu Gao1,3,4, Yaru Lu1, Junya Yu2, Qian Zheng1, Shuning Yan1, Wenjiao Zhang1, Hang He3, Ligeng Ma2,* and Zhengge Zhu1,* 1

College of Life Sciences, Hebei Normal University, Shijiazhuang, Hebei 050021, China

2

College of Life Sciences, Capital Normal University, Beijing 100048, China

3

College of Life Sciences, Peking University, Beijing 100871, China

4These

authors contributed equally to this article.

*Correspondence: Ligeng Ma ([email protected]), Zhengge Zhu ([email protected]) http://dx.doi.org/10.1016/j.molp.2015.01.011

ABSTRACT Deciphering the mechanisms underlying plant responses to abiotic stress is key for improving plant stress resistance. Much is known about the regulation of gene expression in response to salt stress at the transcriptional level; however, little is known about this process at the posttranscriptional level. Recently, we demonstrated that SKIP is a component of spliceosome that interacts with clock gene pre-mRNAs and is essential for regulating their alternative splicing and mRNA maturation. In this study, we found that skip-1 plants are hypersensitive to both salt and osmotic stresses, and that SKIP is required for the alternative splicing and mRNA maturation of several salt-tolerance genes, including NHX1, CBL1, P5CS1, RCI2A, and PAT10. A genome-wide analysis revealed that SKIP mediates the alternative splicing of many genes under salt-stress conditions, and that most of the alternative splicing events in skip-1 involve intron retention and can generate a premature termination codon in the transcribed mRNA. SKIP also controls alternative splicing by modulating the recognition or cleavage of 50 and 30 splice donor and acceptor sites under salt-stress conditions. Therefore, this study addresses the fundamental question of how the mRNA splicing machinery in plants contributes to salt-stress responses at the posttranscriptional level, and provides a link between alternative splicing and salt tolerance. Key words: salt response, osmotic tolerance, SKIP, alternative splicing, posttranscriptional regulation Feng J., Li J., Gao Z., Lu Y., Yu J., Zheng Q., Yan S., Zhang W., He H., Ma L., and Zhu Z. (2015). SKIP Confers Osmotic Tolerance during Salt Stress by Controlling Alternative Gene Splicing in Arabidopsis. Mol. Plant. --, 1–15.

INTRODUCTION Given their sessile nature, plants are dependent on their immediate environment for survival, and the growth and development of plants are heavily affected by environmental cues (Xiong et al., 2002; Zhu, 2002; Munns and Tester, 2008; Ji et al., 2013). Soil salinity is a major environmental stress that greatly affects the growth and development of plants, and which decreases crop productivity and quality (Zhu, 2002; Hussain et al., 2008). High levels of salt induce both hyperionic and hyperosmotic stresses, and can result in plant damage due to membrane disorganization, the generation of reactive oxygen species, metabolic toxicity, inhibition of photosynthesis, and the attenuation of nutrient acquisition (Hasegawa et al., 2000; Parida and Das, 2005; Cheong and Yun, 2007; Ji et al., 2013).

Plants adapt their growth and development in response to salt stress via the regulation of gene expression. Salt stress induces the expression of many stress-responsive genes, including those encoding transcription factors (Yamaguchi-Shinozaki and Shinozaki, 2006). Some of these genes are rapidly induced (early-response genes, including CBF/DREB family genes, RD22BP, MYBs, and ABF/AREB), and their expression regulates and activates downstream delayed-response genes (Kizis et al., 2001; Zhu, 2002; Yamaguchi-Shinozaki and Shinozaki, 2006). Many salt-induced genes encode proteins that function in ion homeostasis, including Na+/H+ antiporters

Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.

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SKIP Confers Salt-stress by Mediating Alternative Splicing

(Apse et al., 1999; Shi et al., 2000). Other salt-induced genes encode proteins that appear to function in damage limitation or repair, including osmolyte biosynthesis genes, LEA/dehydrintype genes, and genes encoding detoxification enzymes, chaperones, proteases, and ubiquitination-related enzymes (Xiong et al., 2001; Zhu, 2001; Munns and Tester, 2008; Ji et al., 2013). However, most previous studies have focused on the regulation of gene expression at the transcriptional level. Thus, little is known about the regulation of salt-induced gene expression at the posttranscriptional level.

In the present study, we analyzed the function and mechanism of action of SKIP in the response of Arabidopsis to salt stress. We found that a mutation in SKIP caused hypersensitivity to both salt and osmotic stresses, and that SKIP was required for the accurate splicing of pre-mRNAs and the maturation of mRNAs encoded by several salt-tolerance genes. Our results indicate that SKIP confers osmotic tolerance under salt stress by controlling the alternative splicing of salt-tolerance genes, providing a link between alternative gene splicing and plant salt responses.

Almost immediately after the initiation of transcription, premRNAs are subjected to a series of modifications that are essential for their nuclear export, maturation, and subsequent translation in eukaryotes (Maniatis and Reed, 2002; Reddy, 2007; Moore and Proudfoot, 2009). One of these modifications, pre-mRNA splicing, plays a key role in gene expression. It not only affects the abundance of mature mRNAs, it is also intimately interconnected with transcription, translation, and downstream mRNA metabolic events, including mRNA export and turnover, in eukaryotes (Black, 2003; Moore and Proudfoot, 2009).

RESULTS

Alternative splicing creates multiple mRNA transcripts from a single gene through the selection and utilization of alternative splice sites in the pre-mRNA; thus, it contributes to increased protein diversity from a limited number of genes in a genome (Reddy, 2007; Zhang et al., 2010; Syed et al., 2012; Braunschweig et al., 2013). A lack of precision during splicing can produce aberrant or nonfunctional mRNAs that are not only wasteful due to the decreased amount of functional mRNA but can also lead to the production of harmful proteins that may perturb normal cellular processes (Reddy, 2007; Kalyna et al., 2012; Braunschweig et al., 2013). Thus, the efficiency and precision of pre-mRNA splicing are critical for gene function (Reddy, 2004; Moore and Proudfoot, 2009; Syed et al., 2012). Pre-mRNA splicing is carried out by the spliceosome, a large dynamic macromolecular machine composed of five small nuclear riboproteins (snRNPs) and many non-snRNP proteins, including serine/arginine-rich (SR) proteins (Staley and Guthrie, 1998). The accuracy and efficiency of pre-mRNA splicing are dependent on the spliceosome’s components, which associate with and dissociate from the spliceosome sub-complex in a dynamic manner (Braunschweig et al., 2013). Thus, regulation of the precision and efficiency of splicing by the spliceosome is necessary for gene function. The machinery and mechanism by which splicing occurs are conserved from yeasts to humans and plants (Black, 2003). Plants employ splicing to modulate gene expression and development (Reddy, 2007). For example, splicing factors are involved in the floral transition, disease resistance, and the circadian clock regulation (Dinesh-Kumar and Baker, 2000; Quesada et al., 2003; Palusa et al., 2007; Zhang and Mount, 2009; Hong et al., 2010; Sanchez et al., 2010; Wang et al., 2012). In a previous report, we verified that Ski-interacting protein (SKIP) is a splicing factor and a component of the spliceosome that is required for the accurate splicing of clock genes in Arabidopsis (Wang et al., 2012). 2

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The Mutation of SKIP Causes Salt-Tolerance Defects in Arabidopsis We previously showed that the Arabidopsis mutant skip-1 has a lengthened circadian period due to a defect in the alternative splicing of clock genes (Wang et al., 2012). As the transcriptional and posttranscriptional regulation of gene expression is key for long-term salt tolerance (Kizis et al., 2001; Zhu, 2002; Yamaguchi-Shinozaki and Shinozaki, 2006), we examined whether SKIP is involved in the response of plants to salt stress. For this purpose, we first compared the germination rate between wild-type and skip-1 plants under conditions of salt stress. There was no difference in germination rate between wild-type and skip-1 seeds plated on Murashige and Skoog (MS) medium without sodium chloride (NaCl), whereas the germination rate was significantly decreased in skip-1 compared with that in wild-type when the seeds were germinated on MS medium containing NaCl (Figure 1A). The aforementioned result suggested that skip-1 was more sensitive than wild-type to NaCl. To confirm this, we examined the root growth and survival rate of wild-type and skip-1 plants under salt-stress conditions. We found that root growth was inhibited significantly by NaCl in skip-1 compared with that in wild-type plants (Figure 1B and 1E). In addition, the survival rate was significantly higher in wild-type than in skip-1 plants on NaClcontaining medium (Figure 1C and 1F). To determine whether skip-1 is sensitive to Na+, Cl, or other ions, wild-type and skip-1 plants were grown on NaNO3-, KCl-, or KNO3-containing medium. The skip-1 plants were hypersensitive to NaNO3, KCl, and KNO3 (Figure 1D and 1G), indicating that skip-1 is hypersensitive to both Na+ and K+. The skip-1 plants were sensitive not only to NaCl and NaNO3 but also to high concentrations of KCl or KNO3 (Figure 1D), indicating that the salt-sensitive phenotype of skip-1 might result from osmotic stress. To address this possibility, wild-type and skip-1 plants were grown under conditions that mimicked osmotic stress (i.e., in the presence of a high concentration of PEG 8000). The skip-1 plants were sensitive to osmotic stress in terms of root growth compared with the wild-type plants (Figure 2A– 2C). However, when the osmotically stressed skip-1 plants were transferred to normal growth conditions, there was no difference in survival rate between the wild-type and skip-1 plants; almost all of the osmotically stressed skip-1 plants survived after being transferred to normal growth conditions (Figure 2D–2F). Considering that about 90% of the skip-1 plants died following exposure to 150 mM NaCl (Figure 1C and 1F), which is roughly equal to an osmotic potential of 0.7 MPa, we

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SKIP Confers Salt-stress by Mediating Alternative Splicing

Figure 1. The Mutation of SKIP Leads to a Salt-Hypersensitive Phenotype. (A) The skip-1 mutant was hypersensitive to NaCl compared with wild-type plants in terms of seed germination. The values are the mean ± SD of three biological replicates. (B and E) The skip-1 mutant was hypersensitive to NaCl compared with wild-type plants in terms of root growth. Images of the roots are shown in (B); the relative root lengths are shown in (E). The relative root growth was calculated based on a root length of 100% for plants of each genotype grown on MS medium without NaCl. The values in (E) are the mean ± SD from three biological replicates. (C and F) The skip-1 mutant was hypersensitive to NaCl compared with wild-type plants in terms of survival. Images of the plants are shown in (C); the survival rates are given in (F). The values in (F) are the mean ± SD from three biological replicates. (D and G) The skip-1 mutant was hypersensitive to NaNO3, KCl, and KNO3 compared with wild-type plants in terms of root growth. Images of the roots are shown in (D); the relative root lengths are shown in (G). The relative root growth was calculated based on a root length of 100% for plants of each genotype grown on MS medium without NaCl. The values in (G) are the mean ± SD from three biological replicates. Significant differences detected using Student’s t-test: **p < 0.01; ***p < 0.001.

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SKIP Confers Salt-stress by Mediating Alternative Splicing Figure 2. The Mutation of SKIP Leads to an Osmotic Hypersensitive Phenotype. (A–C) The skip-1 mutant was hypersensitive to osmotic stress induced using PEG 8000 compared with wild-type plants in terms of root growth. Images of the roots are shown in (A) and (B); the relative root lengths are shown in (C). The relative root growth was calculated based on a root length of 100% for plants of each genotype grown on 1/2 MS medium without PEG 8000. The values are the mean ± SD of three biological replicates. (D–F) The skip-1 mutant was not hypersensitive to osmotic stress compared with wild-type plants in terms of survival. Images of the plants are shown in (D) and (E); the survival rate was calculated in (F). The values in (F) are the mean ± SD from three biological replicates. Significant difference detected using Student’s t-test: **p < 0.01.

concluded that loss of SKIP function leads either to increased osmotic stress that indirectly increases ion toxicity, or directly to both enhanced osmotic stress and ion toxicity. We further observed that the skip-1 plants contained slightly more Na+ and significantly less K+ in their roots compared with wild-type plants under conditions of NaCl stress (Supplemental Figure 1); thus, the skip-1 plants exhibited an increased Na+/K+ ratio in their roots under NaCl-stress conditions (Supplemental Figure 1). To confirm that the salt-hypersensitive phenotype of skip-1 was caused by the mutation in SKIP, we transformed wild-type genomic SKIP DNA into skip-1 driven by its native promoter, and two of the complementation lines, Com-12 and Com-29, were selected for further analysis. All of the salt-hypersensitive phenotypes observed in skip-1, including altered seed germination (Figure 3A), root growth (Figure 3B and 3E), seedling survival (Figure 3C and 3F), and responses to Na+ and K+ (Figure 3D and 3G), were rescued by the transformation of SKIP into skip-1. These results suggest that SKIP is essential for salt tolerance in Arabidopsis.

Salt Stress Induces Genome-Wide Alternative Splicing in Arabidopsis Plants respond to salt stress and adapt to environmental stresses through the differential expression of salt-responsive genes (Kizis et al., 2001; Yamaguchi-Shinozaki and Shinozaki, 2006). In addition, alternative gene splicing is a key step in the regulation of gene expression at the posttranscriptional level (Reddy, 2007; Syed et al., 2012). To assess whether alternative gene splicing is involved in the response of Arabidopsis to salt stress, we performed ultra-high-throughput RNA sequencing (RNA-seq) using wild-type plants grown under normal and salt-stress conditions. About 23‒24 million reads with an average length of 101 bp were aligned to the Arabidopsis reference genome (TAIR10) (Table 1). About 90% of the reads mapped to unique loci (Table 1). Two biological replicates for each growth condition were sequenced; the correlation coefficients (R2) were 0.934 and 0.924 for the replicates produced under normal and salt-stress conditions, respectively. 4

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Compared with the wild-type plants under normal growth conditions, under salt-stress conditions we identified 12 218 novel splicing events (corresponding to 6188 genes, Supplemental Table 1), which could be sorted into five categories: intron retention (IR), exon skipping (ES), an alternative 50 or 30 splice site (AltD or AltA, respectively), and alternative 50 and 30 splice sites (AltDA) (Figure 4A). Although examples of other types of alternative splicing were detected, most of the altered splicing events involved IR (Figure 4A), indicating that salt stress induces genome-wide alternative splicing and that IR is the major alternative splicing event in plants exposed to salt stress. These new splicing events were validated by detecting representative novel splicing events (using IR as an example, since it was the dominant type of splicing event) in plants grown under saltstress conditions through RT–PCR using primers flanking the splice sites. New splicing events were detected in each chromosome for those samples grown under salt-stress conditions, but not for those samples grown under normal conditions (Figure 5A and 5B). The aforementioned results indicated the presence of alternative gene splicing in salt-stressed plants. One of the possibilities for causing this result is that the salt treatment might delay the pre-mRNA splicing, and the pre-mRNA splicing may be not completed in such short-term salt treatment (4 h). To clarify this issue, we conducted a time-course experiment to detect the salt-induced alternative gene splicing using five genes randomly selected from our genome-wide analysis (Supplemental Tables 1 and 2) as examples. It was observed that the aberrantly spliced variant was produced within 1 h of salt treatment, and the abundance of aberrantly spliced variant was not obviously increased after 4 h of salt treatment (Supplemental Figure 2). We also observed that the abundance of aberrantly spliced variant was increased as the NaCl concentration increased (Supplemental Figure 3). These results indicate that salt stress could induce alternative pre-mRNA splicing. The induction of alternative gene splicing by salt stress was present in both shoot and root tissue for those genes normally expressed in shoot and root tissues (Supplemental Figure 4), suggesting that there was

Please cite this article in press as: Feng et al., SKIP Confers Osmotic Tolerance during Salt Stress by Controlling Alternative Gene Splicing in Arabidopsis, Molecular Plant (2015), http://dx.doi.org/10.1016/j.molp.2015.01.011

SKIP Confers Salt-stress by Mediating Alternative Splicing

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Figure 3. The Salt-Hypersensitive Phenotypes of skip-1 Were Rescued by Transformation with SKIP Genomic DNA. (A) The transformation of SKIP genomic DNA into skip-1 rescued the salt-hypersensitive response of the mutant in terms of seed germination. (B and E) The transformation of SKIP genomic DNA into skip-1 rescued the salt-hypersensitive response of the mutant in terms of root growth. Images of the plants are shown in (B); the relative root lengths are shown in (E). The relative root growth was calculated based on a root length of 100% for plants of each genotype grown on MS medium without NaCl. (C and F) The transformation of SKIP genomic DNA into skip-1 rescued the salt-hypersensitive response of the mutant in terms of plant survival. Images of the plants are shown in (C); the survival rates are given in (F). (D and G) The transformation of SKIP genomic DNA into skip-1 rescued the hypersensitivity response of the mutant to NaNO3, KCl, and KNO3. Images of the plants are shown in (D); the relative root lengths are shown in (G). The relative root growth was calculated based on a root length of 100% for plants of each genotype grown on MS medium without NaCl. Com-12 and Com-29 are two representative complementation lines. The values in (A), (E), (F), and (G) are the mean ± SD from three biological replicates. Significant differences detected using Student’s t-test: **p < 0.01; ***p < 0.001.

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Raw reads

SKIP Confers Salt-stress by Mediating Alternative Splicing WT-0 Rep. 1

WT-0 Rep. 2

WT-S Rep. 1

WT-S Rep. 2

skip-S Rep. 1

skip-S Rep. 2

23 349 414

22 743 250

23 161 945

24 485 256

23 880 968

22 928 099

Mapped reads

22 443 888

21 683 016

22 402 501

23 758 308

23 008 231

22 232 836

No. of mapped paired-end reads

20 983 044

20 045 987

20 539 025

22 019 768

21 562 504

20 980 180

Proportion of mapped paired-end reads

89.87%

88.14%

88.68%

89.93%

90.29%

91.50%

Table 1. Summary of the RNA-Seq Data. WT-0, wild-type plants grown under normal conditions were transferred to MS medium without NaCl and grown for an additional 4 h before being harvested. skip-S and WT-S, skip-1 and wild-type plants grown under normal conditions were transferred to MS medium containing 200 mM NaCl and grown for an additional 4 h before being harvested. Rep., replicate.

no shoot or root tissue-specific alternative gene splicing for those selected genes.

the first intron, 7301 (93.2%) contained a premature termination codon (Table 2).

We further examined the effect of alternative splicing on gene expression under conditions of salt stress. As most of the new splicing events under salt stress involved IR, we focused on the effect of IR on mRNA maturation and translation. One of the consequences of IR is the generation of a premature termination codon in the encoded mRNA. As most genes in Arabidopsis contain more than one intron, and since more than one intron may be retained in the new version of an mRNA following alternative splicing, we reasoned that it would be too complicated to calculate the number of premature termination codons that would be produced if we were to consider all of the possible outcomes of IR for those genes containing more than one intron. To make our analysis more straightforward, we considered only those new splicing events in which the first intron was retained. Among 5527 genes with IR under salt-stress conditions, 2239 (40.5%) retained the first intron (Table 2). Among 2239 aberrantly spliced mRNAs in which the first intron was retained, 2101 (93.8%) carried a new premature termination codon (Table 2). These results indicate that alternative splicing induced by salt stress may lead to the generation of nonfunctional mRNAs.

We also examined salt stress-induced and SKIP-mediated alternative splicing events involving AltD and AltA (Supplemental Tables 1 and 2). Consensus sequences at the 50 and 30 splice sites are important for the accurate splicing of pre-mRNAs in plants (Reddy, 2007). We found that the nucleotide sequences of the 50 and 30 splice sites were well conserved when compared with control 50 and 30 splice sites (50 and 30 splicing events out of all of the GT_AG_U2 splicing events in wild-type plants grown under normal conditions, wild-type plants grown under salt-stress conditions, and skip-1 plants grown under salt-stress conditions) (Figure 4C). However, although most of the splice sites used in salt stress-grown wild-type and skip-1 plants conformed to the consensus sequence, the 50 and 30 splice sites used in the novel splicing events from both WT-0 vs. WT-S and WT-S vs. skip-S detected differed from the consensus sequence, with obvious decreases in the frequency of the dominant G and T at donor positions 0 and +1 and of the A and G at acceptor positions 1 and 0 (Figure 4C). These results indicate that salt stress decreases the ability of the spliceosome to accurately recognize splice sites, and that SKIP plays an important role in the recognition or cleavage of 50 alternative donor sites and 30 alternative acceptor sites by the spliceosome.

SKIP Is Necessary for Alternative Gene Splicing under Salt-Stress Conditions To determine the function of SKIP in the regulation of gene expression under salt-stress conditions, we performed RNAseq using skip-1 and wild-type plants grown in the presence of NaCl. About 22.9 and 23.9 million reads, respectively, with an average length of 101 bp were aligned to the Arabidopsis reference genome (TAIR10) for the two replicate samples (Table 1). Among them, about 90% of the reads mapped to unique loci in the Arabidopsis genome (Table 1). The R2 value for the two replicates was 0.902. Compared with the wild-type plants, 46 389 novel splicing events (corresponding to 12 865 genes, Supplemental Table 2) were identified in skip-1 under salt-stress conditions (Figure 4B). Again, five categories of alternative splicing events, including IR, ES, AltD or AltA, and AltDA, were observed (Figure 4B). Consistent with our observation of salt stress-induced alternative gene splicing, the majority of the new splicing events involved IR (Figure 4B). These new splicing events were validated by RT– PCR using our RNA-seq data (Figure 5C and 5D). Among 12 839 genes with IR, 7831 (61.0%) retained the first intron; moreover, among those aberrantly spliced mRNAs that retained 6

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SKIP Is Involved in Mediating Salt-Induced Alternative Splicing We further compared the salt-induced novel splicing events and SKIP-mediated splicing events (Supplemental Tables 1 and 2), and found that the number of IR events mediated by SKIP was greater than that induced by salt on a genome-wide scale (Figure 6A). In addition, 86.8% of the salt-induced IR events were included in the novel IR events identified in skip-1 plants (Figure 6A). To further evaluate the effect of salt stress on alternative gene splicing and the roles of SKIP in maintaining the accuracy of gene splicing and in salt-stress responses, we examined the retained intron number for those genes that exhibited both aberrant salt-induced and SKIP-mediated gene splicing (Figure 6A). We found that the aberrantly spliced variants induced by salt in skip-1 tended to retain more introns than did the aberrantly spliced variants in wild-type plants (Figure 6B). Next, we assessed the abundance of aberrantly spliced variants (mRNAs with IR) for those genes showing aberrant splicing in

Please cite this article in press as: Feng et al., SKIP Confers Osmotic Tolerance during Salt Stress by Controlling Alternative Gene Splicing in Arabidopsis, Molecular Plant (2015), http://dx.doi.org/10.1016/j.molp.2015.01.011

SKIP Confers Salt-stress by Mediating Alternative Splicing both salt-stressed wild-type and skip-1 plants. As mentioned above, it was deemed too complicated to calculate the abundance of aberrantly spliced variants from all possible variants with retained introns for those genes containing more than one intron. Instead, we examined the abundance of aberrantly spliced variants containing a specific intron. For this purpose, we first calculated the average intron number for all of the annotated genes in the Arabidopsis genome, and found that the average intron number was about six (Figure 6C). Further, we calculated the number of genes showing aberrant splicing in salt-induced wild-type and skip-1 plants with retention of the same intron (from the first to the sixth intron; Figure 6D). The abundance of aberrantly spliced variants (mRNAs with IR) in salt-induced skip-1 was higher than that in salt-induced wild-type plants (Figure 6E). These results suggest that SKIP plays a key role in ensuring the accurate splicing of pre-mRNAs under salt-stress conditions.

SKIP Is Required for the Splicing of Pre-mRNAs Encoded by Salt-Tolerance Genes in Arabidopsis To determine whether SKIP is required for the splicing of genes involved in salt tolerance, we used RT–PCR to examine the splicing patterns of those genes known to be required for salt responses and which exhibited a splicing defect in skip-1 under salt-stress conditions in our genome-wide analysis (Supplemental Tables 1 and 2). Aberrantly spliced variants for all of the selected genes, including NHX1 (Apse et al., 2003), CBL1 (Cheong et al., 2003), P5CS1 (Sze´kely et al., 2008), RCI2A (Mitsuya et al., 2005), PAT10 (Zhou et al., 2013), and RD29A, were detected in salt-grown skip-1, and these variants were further validated by sequencing (Figure 7A). The increase in these aberrantly spliced variants was further confirmed by quantitative (q) RT–PCR (Figure 7B). All of the aberrantly spliced variants retained introns that would cause premature termination during translation (Figure 7A); thus, no functional proteins could be produced from these aberrantly spliced variants. The increase in aberrantly spliced variants could lower the abundance of the fully spliced isoform encoded by these salt-tolerance genes. To confirm this notion, we performed qRT–PCR to detect the expression of the fully spliced isoforms (i.e., those without retention of the detected introns) of these salt-tolerance genes. Increased accumulation of aberrantly spliced variants and decreased accumulation of the fully spliced isoforms were detected in skip-1 for each of the tested salt-tolerance genes (Figure 7B and 7C). These observations were true when NaCl stress was applied for different time periods (from 1 to 8 h; Supplemental Figure 5). These results indicate that under saltstress conditions, SKIP is required for the accurate splicing of pre-mRNAs encoded by salt-tolerance genes.

DISCUSSION To date, most studies of the salt response network in plants have emphasized ion transport and transcriptional regulatory mechanisms (Yamaguchi-Shinozaki and Shinozaki, 2006; Ji et al., 2013). Recent genome-wide gene expression analyses have suggested that mRNA splicing is under the control of developmental and stress signals (Filichkin et al., 2010; Marquez et al., 2012),

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and that PRMT5- and LSM5-mediated pre-mRNA splicing is important for plant salt responses (Zhang et al., 2011; Cui et al., 2014). Our study identified SKIP as a splicing factor required for the complete splicing of pre-mRNAs encoded by salt-tolerance genes and for osmotic tolerance under salt stress in plants.

SKIP Is Required for Normal Functioning of the Salt-Tolerance Response SKIP is an evolutionarily conserved protein, with close homologs in yeast and mammals (Wang et al., 2012). Prp45 is the SKIP homolog in yeast (Gahura et al., 2009). Weak prp45 alleles exhibit a temperature-sensitive growth phenotype, while strong prp45 alleles are lethal (Figueroa and Hayman, 2004; Gahura et al., 2009). Human SKIP is necessary for the expression of p21, and defects in SKIP result in subsequent p53-mediated apoptosis (Chen et al., 2011). Among plants, Hou et al. (2009) observed that transgenic rice plants overexpressing OsSKIP exhibited salt and drought tolerance. In comparison, Lim et al. (2010) observed that Arabidopsis plants overexpressing AtSKIP exhibited salt and drought resistance, while AtSKIP antisense plants showed enhanced sensitivity to salt and drought exposure. However, Zhang et al. (2013) discovered that the overexpression of GmGBP1, the SKIP homolog in soybean, led to salt hypersensitivity in Arabidopsis. Thus, the role of SKIP in salt tolerance in plants is not completely clear. No rice or soybean knockout mutant for SKIP is currently available, and the Arabidopsis mutant skip-2, a T-DNA insertion line, is very weak and cannot complete its life cycle (Wang et al., 2012). It is thus difficult to use overexpression lines to clarify the function of SKIP and its molecular mechanisms in plants. By screening a T-DNA insertion library, we previously isolated a weak skip allele (skip-1) that has a 22 bp deletion at its C terminus (the mutation is not linked to the T-DNA) and which is viable, although it exhibits defects in flowering time and the circadian clock (Wang et al., 2012). This weak skip allele (skip-1) has given us the unique opportunity to clarify the function and molecular mechanisms of SKIP in plants exposed to salt stress. In this study, we verified that skip-1 is hypersensitive to NaCl, NaNO3, KCl, and KNO3 (Figure 1), and that the hypersensitive phenotype observed in skip-1 could be completely rescued by transforming skip-1 plants with genomic SKIP DNA (Figure 3). The skip-1 mutant was found to be hypersensitive to both Na+ and K+ (Figures 1 and 3). In addition, it has been reported that SKIP overexpression increases drought resistance in rice and Arabidopsis (Hou et al., 2009; Lim et al., 2010). Together, these findings suggest that the hypersensitivity of skip-1 to salt is likely the result of osmotic stress. To clarify this issue, we conducted an experiment under conditions that mimicked osmotic stress. Salt stress induced both growth inhibition and seedling death in skip-1 (Figures 1 and 3), while an equal strength of osmotic stress induced only growth inhibition (Figure 2). As high salinity leads to both osmotic stress and ion toxicity (Munns and Tester, 2008), the aforementioned results suggest that the loss of SKIP function leads either to increased osmotic stress that indirectly increases ion toxicity, or directly Molecular Plant --, 1–15, -- 2015 ª The Author 2015.

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SKIP Confers Salt-stress by Mediating Alternative Splicing

Figure 4. Genome-Wide Effects of Salt Stress and the Mutation of SKIP on Alternative Gene Splicing. (A) Salt stress induced alternative splicing events in wild-type plants. WT-S: wild-type plants grown under normal conditions were transferred to MS medium containing 200 mM NaCl and grown for an additional 4 h before being harvested. WT-0: wild-type plants grown under normal conditions were transferred to MS medium without NaCl and grown for an additional 4 h before being harvested. (B) SKIP-mediated alternative splicing events. skip-S: skip-1 plants grown under normal conditions were transferred to MS medium containing 200 mM NaCl and grown for an additional 4 h before being harvested. WT-S: wild-type plants grown under normal conditions were transferred to MS medium containing 200 mM NaCl and grown for an additional 4 h before being harvested. (legend continued on next page)

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SKIP Confers Salt-stress by Mediating Alternative Splicing

Figure 5. Validation of IR Splicing Defects as Detected by RNA-Seq through RT–PCR. (A) Pre-mRNAs with IR induced by salt stress as detected by RNA-seq. Genes from each chromosome were randomly selected from the list of saltinduced IR events as representatives. The retained intron is indicated by a box over the cartoon. (B) Validation of the selected genes with IR splicing defects by RT–PCR in salt-stressed wild-type plants. The left panel is non-RT controls. (C) Pre-mRNAs with IR splicing defects in salt-stressed skip-1 as detected by RNA-seq. Genes from each chromosome were randomly selected from the IR list of SKIP-mediated IR events as representatives. The retained intron is indicated by a box over the cartoon. (D) Validation of the selected genes with IR splicing defects by RT–PCR in salt-stressed skip-1 plants. The left panel is non-RT controls. WT-0: wild-type plants grown under normal conditions were transferred to MS medium without NaCl and grown for an additional 4 h before being harvested. WT-S: wild-type plants grown under normal conditions were transferred to MS medium containing 200 mM NaCl and grown for an additional 4 h before being harvested. skip-S: skip-1 plants grown under normal conditions were transferred to MS medium containing 200 mM NaCl and grown for an additional 4 h before being harvested.

to both enhanced osmotic stress and ion toxicity. These results indicate that the mutation in SKIP conferred a hypersensitive response to salt stress. Considering the results of the present study and the aforementioned previous studies (Hou et al., 2009; Lim et al., 2010; Zhang et al., 2013), we conclude that SKIP is essential for the response of plants to salt stress and that it confers osmotic tolerance under salt stress in plants.

SKIP Confers Salt Tolerance by Modulating the Alternative Splicing of Salt-Tolerance Genes SKIP functions as a transcriptional coactivator/corepressor in mammals (Bres et al., 2005; Scott and Plon, 2005). In addition, a proteomic analysis of the spliceosome in yeast identified SKIP as part of the Prp19-related complex, which is part of the spliceosome (Bessonov et al., 2008). Furthermore, yeast SKIP

(C) The frequency distribution of nucleotides at consensus 50 and 30 splice sites. Sequence logos illustrate consensus sequences for the total splice sites in wild-type plants grown without salt stress (top panel), wild-type plants grown in the presence of 200 mM NaCl (second panel from the top), salt-induced new splicing events (third panel from the top), the total splice sites in skip-1 (fourth panel from the top), and novel splice sites detected in skip-1 (bottom panel) plants grown in the presence of 200 mM NaCl.

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Molecular Plant

SKIP Confers Salt-stress by Mediating Alternative Splicing WT-S versus WT-0

skip-S versus WT-S

No. of IR events

9301

45 449

Genes showing IR

5527

12 839

Retention of the first intron

2239 (40.51%)

7831 (60.99%)

Identification of a premature termination codon in the retained intron

2101 (93.84%)

7301 (93.23%)

Table 2. Summary of New Splicing Events Mediated by SKIP and Induced by Salt Stress. WT-0, wild-type plants grown under normal conditions were transferred to MS medium without NaCl and grown for an additional 4 h before being harvested. skip-S and WT-S, skip-1 and wild-type plants grown under normal conditions were transferred to MS medium containing 200 mM NaCl and grown for an additional 4 h before being harvested.

(Prp45) has been shown to be essential for the splicing of actin and other genes (Gahura et al., 2009). It was also observed that human SKIP acts as a splicing factor that specifically regulates the alternative splicing and expression of p21 (Chen et al., 2011). These results suggest that SKIP has dual functions as a transcriptional coactivator/corepressor and as a splicing factor. We previously verified that SKIP is a component of the spliceosome in plants, and that SKIP interacts directly with another component of the spliceosome, SR45 (Wang et al., 2012). SKIP functions as a splicing factor for clock genes, and is essential for the accurate splicing of their pre-mRNAs and for the maturation of their mRNAs; thus, SKIP is required for the normal functioning of the circadian clock in Arabidopsis (Wang et al., 2012). However, the mechanism underlying the effect of SKIP on the salt response of plants is unknown. Based on the observation that plant SKIP is cable of inducing reporter genes in yeast, both Lim et al. (2010) and Zhang et al. (2013) concluded that plant SKIP works as a transcriptional cofactor in salt responses. Hou et al. (2009) also suggested that OsSKIP mediates stress responses through the regulation of numerous stress-related genes at the transcriptional level. In the present work, we verified that the mutation of SKIP led to a defect in the alternative splicing of a portion of the Arabidopsis genome (Figures 4–6 and Table 1). SKIP is necessary for the spliceosome to recognize or cleave the 50 alternative donor site and 30 alternative acceptor site in pre-mRNAs (Figure 4C). In addition, SKIP is required for the accurate splicing of salttolerance genes, including NHX1, CBL1, P5CS1, RCI2A, and PAT10 (Figure 7). The abundance of the fully spliced isoforms of these genes was decreased while the abundance of aberrantly spliced variants was increased in skip-1 plants (Figure 7). The aberrantly spliced variants contained a premature termination codon (Figure 7A and Table 2), which would target them for degradation via the nonsense-mediated decay pathway (Reddy, 2007; Kalyna et al., 2012); thus, they were nonfunctional mRNAs. It is worth mentioning that genes for which SKIP was required for accurate splicing included genes encoding ion transporters, calcium sensor, protein involved in proline biosynthesis, and protein S-ACYL transferase required for the membrane association of calcineurin B-like proteins. Therefore, SKIP confers salt tolerance not by controlling the precision of 10

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alternative splicing for genes involved in a specific pathway, but for a subset of salt-tolerance genes involved in different pathways. It is worth noting that alternative splicing caused an increase in the production of ineffective transcripts (Figures 6 and 7; Table 2), but its effect on the production of functional transcripts appeared to be limited, given that most of the selected salt-tolerance genes were only slightly affected (although statistically significant) by salt stress (Figure 7). Thus, we cannot rule out the possibility that the salt-sensitive phenotype of skip-1 is a consequence of wasting too much energy on the activation of transcription due to the defect in alternative gene splicing.

Alternative Splicing Is a Key Step in the Regulation of Gene Expression by Salt at the Posttranscriptional Level The regulation of gene expression is important for long-term salt responses and for the adaptation and survival of plants exposed to environmental stresses (Xiong et al., 2001; Zhu, 2002; Yamaguchi-Shinozaki and Shinozaki, 2006). Thus, most studies have focused on the transcriptional regulation of gene expression (Yamaguchi-Shinozaki and Shinozaki, 2006; Ma et al., 2014). The signal transduction pathway for gene expression (Zhu, 2001; Ji et al., 2013; Zhou et al., 2014), the transcription factors that regulate salt-responsive genes (Yang et al., 2012; Schmidt et al., 2013), and the genes involved in salt responses (Chen et al., 2012; Liu et al., 2012; Zahaf et al., 2012) have been well documented; however, questions remain regarding the transcriptional regulation of gene expression in response to salt stress. Indeed, little is known about the regulation of salt stress-specific gene expression at the posttranscriptional level. Marquez et al. (2012) identified developmental cue- and stress (including salt stress)-induced alternative gene splicing. Zhang et al. (2011) verified that SKB1/PRMT5, a protein methyltransferase that modulates spliceosome activity via methylation of the spliceosome component LSM4, is required for salt responses, and that the mutation of PRMT5 resulted in a salt-hypersensitive phenotype. Chen et al. (2013) found that HOS5 and its interacting factors are involved in the alternative splicing of a small portion of the Arabidopsis genome, and that they are required for the response of Arabidopsis plants to salt stress. More recently, Cui et al. (2014) revealed that LSM5, which is required for the response of plants to abscisic acid and drought (Xiong et al., 2001), mediates salt responses through the dynamic regulation of genome-wide pre-mRNA splicing. In the present study, we verified that salt stress induces genomewide alternative splicing by modulating the recognition or cleavage of 50 alternative donor and 30 alternative acceptor sites in genes targeted by the spliceosome (Figure 4C). SKIP is required for the accurate alternative splicing of genes, including salt-tolerance genes, under salt-stress conditions (Figures 4–7). Thus, SKIP is a splicing component necessary for the correct alternative splicing of salt-tolerance genes and, therefore, for normal functioning of the salt response in plants. We further observed that the majority (more than 86%) of salt stress-induced alternative splicing events were

Please cite this article in press as: Feng et al., SKIP Confers Osmotic Tolerance during Salt Stress by Controlling Alternative Gene Splicing in Arabidopsis, Molecular Plant (2015), http://dx.doi.org/10.1016/j.molp.2015.01.011

Molecular Plant

SKIP Confers Salt-stress by Mediating Alternative Splicing

Figure 6. The Correlation between SKIP-Mediated and Salt-Induced IR Events. (A) Common genes from among those SKIP-mediated and salt-induced genes exhibiting IR. (B) The gene number distribution for those genes containing SKIP-mediated and salt-induced IR events. The x axis represents the ratio of the number of retained introns for mRNAs encoded by the same gene from salt-induced skip-1 and salt-induced wild-type plants. (C) The distribution of introns in the Arabidopsis genome. The average intron number per gene was about 5.8. (D) Genes in which the same intron was retained following SKIP-mediated and salt-induced IR events. (E) The relative expression level of aberrantly spliced variants with the indicated intron retained between salt-induced skip-1 and wild-type plants. WT-0: wild-type plants grown under normal conditions were transferred to MS medium without NaCl and grown for an additional 4 h before being harvested. WT-S: wild-type plants grown under normal conditions were transferred to MS medium containing 200 mM NaCl and grown for an additional 4 h before being harvested. skip-S: skip-1 plants grown under normal conditions were transferred to MS medium containing 200 mM NaCl and grown for an additional 4 h before being harvested.

SKIP-mediated alternative splicing events (Figure 6A), and that the defect in pre-mRNA splicing in skip-1 was more severe than that in salt stress-treated wild-type plants (Figure 6). This result

suggests that salt stress induces alternative gene splicing via the regulation of SKIP expression or the SKIP protein level. It was also observed that most of the alternative Molecular Plant --, 1–15, -- 2015 ª The Author 2015.

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Molecular Plant

SKIP Confers Salt-stress by Mediating Alternative Splicing

Figure 7. SKIP Is Required to Control the Alternative Splicing of Salt-Tolerance Genes. (A) Detection of IR splicing defects by RT–PCR in wild-type and skip-1 plants. The left panel shows non-RT controls. (B) Measurement of the abundance of mRNAs with IR by qRT–PCR in wild-type and skip-1. The values are the mean ± SD from three biological replicates. (C) The abundance of splice variants of select salt-tolerance genes with a fully spliced intron as detected by qRT–PCR. The values are the mean ± SD from three biological replicates. The regions from where the primers used for qRT–PCR were designed are indicated by the arrows in the gene models in the right panel of (A). Significant differences detected using Student’s t-test: *p < 0.05; **p < 0.01; ***p < 0.001.

splicing variants induced by salt stress or mediated by SKIP were nonfunctional mRNAs, as they could not be translated into functional proteins due to the presence of a premature stop codon (Table 2). This indicates that salt stress induces the downregulation of some proteins through the production of nonfunctional mRNAs via alternative splicing, while SKIP protects against the salt-induced production of nonfunctional mRNAs, thereby conferring salt tolerance. These results demonstrate that the mRNA splicing machinery in Arabidopsis contributes to salt response at the posttranscriptional level, and that SKIP provides a link between alternative splicing and salt tolerance. 12

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METHODS Plant Materials and Growth Conditions The Arabidopsis thaliana (both wild-type and mutant) plants used in this study were of the Columbia-0 (Col-0) ecotype. The skip-1 mutant was identified from a T-DNA insertion library and characterized as described previously (Wang et al., 2012). The two complementation lines for skip-1 used in this study were described previously (Wang et al., 2012). Seeds were sterilized and then plated on MS medium containing 0.8% agar and 1% sucrose. After stratification in the dark at 4 C for 3 days, the plates were transferred to a Percival CU36L5 growth chamber (Percival Scientific, Perry, IA, USA) at 22 C for 16 h under white light (70 mmol m2 s1) and at 18 C for 8 h under dark conditions.

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Molecular Plant

SKIP Confers Salt-stress by Mediating Alternative Splicing Salt-Stress Treatment and the Plant Response to Salt

Alternative Splicing Event Analysis Using RACKJ

For the germination assay, seeds were germinated on MS agar medium or MS medium containing the indicated concentrations of NaCl. For the survival rate assay, 5-day-old seedlings were transferred from germination medium to MS agar medium containing various concentrations of NaCl in opposite directions between the left part and right part in the plate in Figure 1C, and the top part and bottom part of plate in Figure 3C. For the root growth assay, 5-day-old seedlings were transferred from germination medium to MS agar medium containing NaCl, NaNO3, KCl, or KNO3, after which the seedlings were grown vertically for 10 more days.

Alternative splicing was analyzed using RACKJ software (http://rackj. sourceforge.net; Li et al., 2013; Wu et al., 2014), which constructed results for alternative splicing events by including exon-level reads and intron-level reads. Comparisons were performed by chi-square tests, and significant alternative splicing events were identified by a p value of <0.001. The nucleotide frequencies around novel and known splice sites were determined and displayed visually using sequence logos (Schneider and Stephens, 1990).

PEG 8000 Treatment For the PEG 8000 (0.25, 0.5, and 0.7 MPa) experiment, 5-day-old seedlings were transferred from germination medium to PEG 8000containing medium for 10 days. The PEG 8000 plates were prepared as described by Verslues et al. (2006). In brief, 1.5% agar plates containing 1/2 MS medium and 6 mM MES buffer were solidified and then overlaid with a solution of 0, 250, or 400 g/l PEG 8000, producing an osmotic potential (in MPa) of 0.25, 0.5, and 0.7, respectively. The solution was allowed to sit for 24 h, and excess solution was removed from the plates.

SUPPLEMENTAL INFORMATION Supplemental Information is available at Molecular Plant Online.

FUNDING This work was supported by grants from the National Basic Research Program (973 Program) of the Ministry of Science and Technology of China (2012CB114200 and 2012CB910900), Beijing Municipal Government Science Foundation (CIT&TCD20150102) and from the National Nature Science Foundation of China (31340046).

ACKNOWLEDGMENTS Analysis of the Ion Content Five-day-old seedlings were transferred from germination medium to MS agar medium containing 100 mM NaCl, and grown vertically for 10 days. The shoots and roots were then harvested and dried at 80 C for 2 days. All of the samples were weighed and heated at 300 C for 1 h and at 575 C for 4 h in a muffle furnace. The resulting ground dry matter was dissolved in 0.1 M HCl. The Na+ and K+ contents of the samples were determined with an atomic absorption spectrophotometer (Hitachi Z-5000; Hitachi, Tokyo, Japan).

RNA Extraction and RT–PCR Total RNA was extracted from 11-day-old plants using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and quantified spectrophotometrically at 260 nm. For RT–PCR and qRT–PCR, after RNase-free DNase I (RQ1 RNase-Free DNase; Promega, Madison, WI, USA) treatment, 3 mg of total RNA was used for first strand cDNA synthesis (RevertAid First Strand cDNA Synthesis Kit; Fermentas, Burlington, ON, Canada). Takara SYBR Premix Ex Taq (Takara Bio, Otsu, Japan) and a 7500 Fast Real-Time PCR instrument (Applied Biosystems, Foster City, CA, USA) were used for qRT–PCR. Alternative splicing-specific primers were used to amplify the expected alternatively spliced mRNA isoforms (all primer sequences are given in Supplemental Table 3).

mRNA Sequencing and Bioinformatics Analysis of the RNA-Seq Data Total RNA was isolated using Trizol reagent (Invitrogen) from 11-day-old wild-type and skip-1 plants treated with or without 200 mM NaCl for 4 h. The Arabidopsis RNA samples described above were used for Illumina HiSeq deep sequencing (Illumina HiSeq 2000; Illumina, San Diego, CA, USA). There were six samples in total, with each condition having two biological replicates (Table 1). The RNA-seq analysis workflow depicted in Supplemental Figure 6 was implemented to analyze the data. Those paired-end reads 101 bp in length generated using the Illumina HiSeq 2000 system were initially processed to remove the adapter sequences and low-quality (Q score <20) reads. Next, the high-quality reads were mapped to the Arabidopsis TAIR10 genome using the spliced alignment identification tool TopHat (Trapnell et al., 2009). TopHat first mapped the reads using an unspliced aligner, and the remaining unmapped reads were split into shorter segments and aligned independently. Here, default TopHat parameters were used, and two mismatches per read were allowed. Our raw data and the processed RNA-seq data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GSE55632).

The main part of this work was conducted at Capital Normal University. We thank Dr. Jessica Habashi for critical reading of the manuscript. We thank the staff of the Biodynamic Optical Imaging Center, Peking University (Beijing, China), for performing the RNA-seq analysis. No conflict of interest declared. Received: May 14, 2014 Revised: January 12, 2015 Accepted: January 13, 2015 Published: January 21, 2015

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