Review splicing literature for salt stress effects

Reviews stresses and epigenetics: https://academic.oup.com/jxb/article-abstract/72/20/6836/6327780?redirectedFrom=fulltext

Dissecting molecular mechanisms underlying salt tolerance in rice: a comparative transcriptional profiling of the contrasting genotypes

FL478 is a salt tolerant indica recombinant inbred line, which can be a good source of salt tolerance at the seedling stage in rice. To learn about the genetic basis of its tolerance to salinity, we compared transcriptome profiles of FL478 and its sensitive parent (IR29) using RNA-seq technique.

A total of 1714 and 2670 genes were found differentially expressed (DEGs) under salt stress compared to normal conditions in FL478 and IR29, respectively. Gene ontology analysis revealed the enrichment of transcripts involved in salinity response, regulation of gene expression, and transport in both genotypes.

A total of 1135 and 1894 alternative splicing events were identified in transcripts of FL478 and IR29, respectively. Transcripts encoding two potassium transporters and two major facilitator family transporters were specifically up-regulated in FL478 under salt stress but not in the salt sensitive genotype.

Consistent with the previous reports (Razzaque et al. 2017), the number of significant DEGs was higher in the salt-sensitive genotype.

Ann's note: 150 bp paired end reads. Limitations compared to our study: Only two replicates for treatment and control. Only root samples tested.

Genome-wide discovery of natural variation in pre-mRNA splicing and prioritising causal alternative splicing to salt stress response in rice

We developed VaSP (Fig. 1a) to detect variations in splicing events, especially IntronR, in RNA-seq data from a diverse set of genotypes. For each candidate junction in the sample, VaSP quantifies the splicing event by assigning a 3S score (Fig. 1b), which is the count of junction reads normalised by gene-level read coverage. As the 3S score for each intron splicing is based on the normalisation of gene-level average coverage, 3S scores for an intron in different samples are comparable.

We tested VaSP on an RNA-seq data set of 328 RNA samples for 82 genotypes from the rice diversity panel 1 (RDP1) (Famoso et al., 2011; Zhao et al., 2011; Eizenga et al., 2014; McCouch et al., 2016; Wang et al., 2018). Shoot tissues were collected for RNA-seq with two replicates for plants grown under control and salt stress conditions (Du et al., 2019).

In total, 764 significant GSS events were identified in salt stress conditions. GSS events were used as markers for a GWAS with the shoot Na+ accumulation, which identified six GSS events in five genes significantly associated with the shoot Na+ content. Two of these genes, OsNUC1 and OsRAD23 emerged as top candidate genes with splice variants that exhibited significant divergence between the variants for shoot growth under salt stress conditions.

One of the anchor authors is Harkamal Walia, who provided the agami seeds for our study. He has done a lot of very good work on rice.

rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-Seq data

Here we describe a new statistical model and computer program, replicate MATS (rMATS), designed for detection of differential alternative splicing from replicate RNA-Seq data.

Not related to salt stress, but I have seen this paper cited several times by papers analyzing alternative splicing in rice.

The Full-Length Transcriptome of Spartina alterniflora Reveals the Complexity of High Salt Tolerance in Monocotyledonous Halophyte

We performed comparative transcriptome analysis to uncover the distinct features of Spartina under high salt stress compared with rice. The involvement of AS in high salt tolerance was also documented.

...we performed RNA-seq analysis for rice seedling exposed to 300 mM NaCl for 24 h. DE genes were then identified.

The number of AS events is increased with growing salinity stress (Fig. 7C), suggesting that high salt stress induces AS events in Spartina. In particular, IR remains the major AS type associated with salt stress (Fig. 7C) and 233 IR events were identified in response to high salt stress (Supplementary Table S12).

Next, we also analyzed AS in 2-week rice whole seedlings under high salt stress (300 mM for 24 h) using rMATs (Shen et al. 2014a). Interestingly, GO analysis did not detect any significant functional enrichment for high salt stress-associated AS genes in rice (Supplementary Fig. S8B).

This paper was primarily interested in Spartina because it is a halophyte. They used rice (Oryza sativa L. ssp. japonica cv. Nipponbare) as a comparison.

Comparative Transcriptome Analysis Reveals the Mechanisms Underlying Differences in Salt Tolerance Between indica and japonica Rice at Seedling Stage

The salt stress tolerance of rice is related to the genotype, and different genotypes show discrepant salt stress tolerance (Ganie et al., 2016; Mansuri et al., 2019). Hundreds of salt stress-related QTLs have been identified among different rice populations to date (Kong et al., 2019b; Mansuri et al., 2020). However, only four salt stress-related genes have been cloned and experimentally verified in rice, namely, SKC1 (Lin et al., 2004; Hauser and Horie, 2010), HST1 (Takagi et al., 2015), OsHAK21 (He et al., 2019), and OsSTLK (Lin et al., 2020).

We herein performed a comparative transcriptomic analysis under salt stress in contrasting two rice genotypes, namely RPY geng (japonica, tolerant variety) and Luohui 9 (named as Chao 2R in this study, indica, susceptible variety).

In total there were 2714 DEGs (G2) shared by Chao 2R and RPY geng, 4494 DEGs (G1) unique to Chao 2R and 1160 DEGs (G3) unique to RPY geng (Figure 3A). The top 20 GO terms of G1, G2, G3 were compared in Figures 3B–D. G2 were significantly enriched in well-known GO terms related to abiotic stresses, such as plant-type cell wall organization (GO:0042744), hydrogen peroxide metabolic process (GO:0042743), response to hydrogen peroxide (GO:0042542), and so on (Figure 3C).

The susceptible variety has more unique DEGs than the tolerant variety.

Stress-Induced Changes in Alternative Splicing Landscape in Rice: Functional Significance of Splice Isoforms in Stress Tolerance

Another transcriptomic study associated with salt stress in contrasting rice genotypes has revealed down-regulation of AS, specifically in sensitive genotype, which might contribute to its susceptibility to salt stress [54]. A systematic comparison of transcriptomic profiles of contrasting rice genotypes under salt stress has uncovered AS to be a crucial mechanism underlying salt tolerance in salt-tolerant genotype FL478 [57]. Although more AS events were identified in susceptible genotype than the tolerant, AS events in genes involved in crucial salt tolerance-associated processes of ion-transport and signaling pathways were more frequent in tolerant genotype. These alternatively spliced variants were either highly expressed (bZIP, PP2C variants) or specifically expressed (WRKY30, MAPK kinase, and HAK25, ABC and ZIFL transporter variants) in the tolerant genotype [57]. Similar results were reported in the case of salt-tolerant barley and susceptible rice genotypes under salt stress. Although more AS events were found in rice than barley, a higher level of AS in the barley genes related to ion-transporters and transcription factors contribute to the higher salt tolerance in barley, due to the superior K+/Na+ homeostasis [58]. Moreover, one of the SR splicing factors was specifically expressed in barley. Likewise, a separate transcriptomic analysis revealed higher AS events in salt-sensitive than salt-tolerant genotype, but a higher number of alternatively spliced isoforms were specifically expressed in tolerant genotype as compared to sensitive genotype under salt stress [59].

Collectively, the above five studies indicate that higher stress-induced AS in a genotype does not necessarily correlate with stress tolerance, but the frequency of genotype-specific events, the abundance of AS transcripts related to key stress-responsive processes, and the nature of splicing events are likely to contribute to stress tolerance. It is possible that the bulk of alternatively spliced transcripts may be subjected to degradation by NMD and may not be translated. In fact, only about 14% of the total alternatively spliced transcripts are translated into peptides under hypoxic stress in rice [61]. In a related study, treatment with a salt-tolerant endophyte was shown to confer salt stress adaptation and decrease the AS in salt-sensitive rice plants as compared to their untreated counterparts [62]. It was proposed that treatment with the endophyte causes a lowered perception of salt stress signaling, which might not activate the stress-responsive mechanism of AS in the treated plants, underscoring the importance of AS in response to stress.

Review by Showkat Ahmad Ganie and Anireddy S. N. Reddy

Gene Expression analysis associated with salt stress in a reciprocally crossed rice population

In this study, we reciprocally crossed Horkuch (salt tolerant) with high yielding but salt sensitive IR29 to detect the complement of genes that were responsible for conferring salt tolerance versus sensitivity at the seedling developmental stage.

In general, we observed higher numbers of genes differentially expressed in leaves compared to root tissues. This included both upregulation and downregulation of gene expression across our experimental factors. Gene expression decreased in sensitive leaf after stress exposure where tolerant plants showed the opposite trend. In root, tolerant plants expression decreased at higher time points of stress exposure.

Interestingly, more genes were downregulated in the stress condition compared to their control counterpart in leaf and root tissue.

The review by ASN Reddy cites this paper and states that, "Another transcriptomic study associated with salt stress in contrasting rice genotypes has revealed down-regulation of AS, specifically in sensitive genotype, which might contribute to its susceptibility to salt stress". However, in the paper itself the only reference to alternative splicing states that, "We found 183 GO associations from downregulated genes that were unique to sensitive plants. These were enriched (p-value < 0.05) in... RNA splicing"

Transcriptomic and alternative splicing analyses reveal mechanisms of the difference in salt tolerance between barley and rice

Physiological study showed that XZ26 (salt tolerant barley) had higher salt tolerance than Nipponbare, as reflected by less growth inhibition, lower shoot Na+ concentration and higher K+/Na+ ratio when exposed to salt stress.

Root Na+ concentration in XZ26 was 1.33 fold higher than that in Nipponbare under salt condition (Fig. 1C). However, shoot Na+ concentration was just opposite, being 1.79 fold higher in Nipponbare than in XZ26 (Fig. 1D). Correspondingly, Nipponbare had more Na+ translocation from roots to shoots than XZ26 (Fig. S2). On the other hand, K+ concentration in the roots and shoots of XZ26 and Nipponbare was markedly reduced under salt condition (Fig. 1E and F). But XZ26 had significantly higher K+ concentration than Nipponbare, being 2.64 and 1.60 fold in the roots and shoots, respectively (Fig. 1E and F).

Transcriptomic analysis identified 606 and 186 DEGs in the roots and shoots for XZ26, and 4667 and 2817 DEGs for Nipponbare, respectively. While alternative splicing analysis identified 40 and 33 AS genes (salt-responsive DEGs) in the roots and shoots of XZ26. Moreover, AS genes related to ion transporters and transcription factors could enhance and amplify K+/Na+ homeostasis in barley.

Transcriptome analysis in different rice cultivars provides novel insights into desiccation and salinity stress responses

In this study, the comparison of transcriptomes of a drought-tolerant [Nagina 22 (N22)] and a salinity-tolerant (Pokkali) rice cultivar with IR64 (susceptible cultivar) revealed variable transcriptional responses under control and stress conditions.

The frequency of different AS events under control and stress conditions within each rice cultivar was found to be similar to the AS events predicted at the whole transcriptome level (Supplementary Table S2). Interestingly, the frequency of total number of AS events was lesser under stress as compared to control condition in all the rice cultivars. IR represented the major AS events (~40%) under all the conditions in each rice cultivar. However, a reduced frequency of IR AS events was observed in IR64 and N22 under stress conditions as compared to control, with the exception of Pokkali cultivar (Supplementary Table S2). However, the frequency of ES and ES1 + ES2 events increased under stress conditions in all rice cultivars.

The analysis revealed a total of 3119 and 756 significantly differentially expressed transcripts in IR64 cultivar under desiccation and salinity stress, respectively. Similarly, a total of 2510 and 1809 transcripts in N22, and 3132 and 1036 transcripts in Pokkali were differentially expressed under desiccation and salinity stresses, respectively. In addition, we identified a total of 859 and 587 differentially expressed transcripts in N22 and Pokkali, respectively, as compared to IR64 under control condition (Supplementary Table S3).

A large number of the transcripts generated as a result of AS were found to be differentially expressed in the rice cultivars under desiccation and/or salinity stresses (Supplementary Table S4).

Comparative transcriptome and translatome analysis in contrasting rice genotypes reveals differential mRNA translation in salt-tolerant Pokkali under salt stress

Pokkali (Pok) is a highly salt-tolerant landrace, whereas IR29 is a salt-sensitive but widely cultivated genotype.

In response to salt stress, 57 differentially expressed genes (DEGs) were commonly upregulated at both transcriptional and translational levels in both genotypes; the overall number of up/downregulated DEGs in IR29 was comparable at both transcriptional and translational levels, whereas in Pok, the number of upregulated DEGs was considerably higher at the translational level (544 DEGs) than at the transcriptional level (219 DEGs); in contrast, the number of downregulated DEGs (58) was significantly less at the translational level than at the transcriptional level (397 DEGs).

Importantly, under salt stress, Pok seemed to stabilize mRNAs of the stress-adaptive genes and also efficiently load mRNAs onto the polysomes for translation. The overrepresented Gene Ontology (GO) terms for DEGs of Pok compared to IR29 include maintaining cell wall integrity and photosynthesis, translocating molecules, and detoxifying ROS under salt stress.

These observations unambiguously suggest a greater number of upregulated genes were regulated at the translational level in Pok under salt stress. Notably, most of these translationally upregulated genes were unresponsive at the transcriptional level, implying that these genes were more efficiently loaded onto the polysomes for translation.

By contrast, none of the downregulated genes (transcriptionally and translationally) were shared between the genotypes under salt stress.

UPDATE

Here's another interesting paper that talks about the role of UTR in attracting ribosomes, which I think could be very interesting when viewed from the lens of alternative splicing: https://pubmed.ncbi.nlm.nih.gov/35041803/

A two-staged model of Na+ exclusion in rice explained by 3D modeling of HKT transporters and alternative splicing

Whereas the lowest observed band of 522 bp was characteristic of the OsHKT1;4 splicing prediction, two other spliced variants of 104 and 186 bp larger than the predicted spliced form were also found

Interestingly, Pokkali is able to maintain a much higher functional ratio of OsHKT1;4 transcripts in younger sheath as opposed to Nipponbare.

A new AOX homologous gene OsIM1 from rice (Oryza sativa L.) with an alternative splicing mechanism under salt stress

Comparison of the OsIM cDNA and genomic DNA sequence revealed that normal splicing resulted in transcript OsIM1, and the abnormal splicing led to the pseudo-transcript OsIM2. Under normal conditions both transcripts were present, indicating that two splicing mechanisms existed even without stress. The reason for this phenomenon was not clear. Upon salt-stress, OsIM1 was induced in both salt-tolerant varieties and maintained at a high level, while the increase of the ratio of OsIM1/OsIM2 mainly resulted from the decrease of OsIM2; whereas in the salt-sensitive variety, the quantity of OsIM1 transcript went up in 24 h and decreased more quickly than OsIM2, which resulted in a decrease of the ratio of OsIM1/OsIM2. The difference in the induction pattern of OsIM1/OsIM2 may reflect the competition between the two splicing mechanisms during salt stress, and the salt-tolerance probably not only resulted from an increased expression level of OsIM1, but also due to the ability to regulate the ratio of OsIM1/OsIM2 in plants.

Distinct Evolutionary Origins of Intron Retention Splicing Events in NHX1 Antiporter Transcripts Relate to Sequence Specific Distinctions in Oryza Species

We demonstrate that the IR event involving the 5' UTR is present only in more recently evolved Oryza AA genomes while the IR event governing retention of the 13th intron of Oryza NHX1 (terminal intron) is more ancient in origin, also occurring in halophytic wild rice, Oryza coarctata (KKLL). We also report presence of a retro-copy of the OcNHX1 cDNA in the genome of O. coarctata (rOcNHX1). Preferential species and tissue specific up- or down-regulation of the correctly spliced NHX1 transcript/5' UTR/13th intron-retaining splice variants under salinity was observed.

MSU annotation of the Os07g47100.3 (OsNHX1) transcript suggests partial retention of the 13th intron. However, the O. coarctata OcNHX1i13 transcript not only shows complete retention of the 13th intron but also the entire last is exon (confirmed by 3' RACE-PCR; Supplementary Figure S7).

In O. coarctata leaf tissues, expression of the correctly spliced OcNHX1 cDNA and its retrocopy, rOcNHX1 is significantly reduced (P < 0.001 and P < 0.0001) under salinity stress while the 13th intron retaining transcript, OcNHX1i13, shows increased expression (Figure 6H). In contrast, in root tissues under salinity stress, expression of the correctly spliced OcNHX1 cDNA and rOcNHX1 (P < 0.0001) is upregulated and the 13th intron retaining transcript, OcNHX1i13 is downregulated (P < 0.001; Figure 6H).

Our data shows preferential tissue specific up- or downregulation of the correctly spliced NHX1 transcript as well as splice variants under salinity in Oryza AA species as well as in O. coarctata. This suggests the spliceosome machinery governing alternative splicing events involving NHX1 has tissue specific effects under salinity in Oryza species.

Overexpression of a partial fragment of the salt-responsive gene OsNUC1 enhances salt adaptation in transgenic Arabidopsis thaliana and rice (Oryza sativa L.) during salt stress

The rice (Oryza sativa L.) nucleolin gene, OsNUC1, transcripts were expressed in rice leaves, flowers, seeds and roots but differentially expressed within and between two pairs of salt-sensitive and salt-resistant rice lines when subjected to salt stress. Salt-resistant lines exhibited higher OsNUC1 transcript expression levels than salt-sensitive lines

Salt-stress was found to induce OsNUC1 transcript expression (both OsNUC1-S and OsNUC1-L combined) in the leaf blades in all four rice lines investigated, that is the two salt-sensitive WT lines (LPT123 and KDML105) and the two derived isogenic salt-resistant lines (LPT123-TC171 and FL530) (Fig. 2). However, the OsNUC1 transcript expression levels were different between the four lines. Twenty-one-day-old rice seedlings transferred to WP nutrient solution alone or with 0.5% (w/v) NaCl revealed a significantly increased level of OsNUC1 expression after 1–2 d of salt stress compared to those without salt. However, the salt-resistant lines, LPT123-TC171 and FL530, showed a much higher level of gene expression when compared to their salt-sensitive counterparts with the same genetic background (Fig. 2).

Over-expression of OsNUC1-S induces salt tolerance in rice. No significant change in the shoot FW (fresh weight) was found between the wild type and the transgenic lines, but the shoot FWs of all transgenic lines were significantly higher than those of the WT after salt stress for 3 d (Fig. 10B), although not as high as in the transgenic lines without salt stress.

Enhanced Salt Tolerance Conferred by the Complete 2.3 kb cDNA of the Rice Vacuolar Na+/H+ Antiporter Gene Compared to 1.9 kb Coding Region with 5' UTR in Transgenic Lines of Rice

This study shows constitutive over-expression of the vacuolar Na+/H+ antiporter gene (OsNHX1) from the rice landrace (Pokkali) and attainment of enhanced level of salinity tolerance in transgenic rice plants. It also shows that inclusion of the complete un-translated regions (UTRs) of the alternatively spliced OsNHX1 gene provides a higher level of tolerance to the transgenic rice. Two separate transformation events of the OsNHX1 gene, one with 1.9 kb region containing the 5' UTR with CDS and the other of 2.3 kb, including 5' UTR, CDS, and the 3' UTR regions were performed. Both transgenic lines were observed to be tolerant compared to WT plants at both physiological stages. However, the transgenic lines containing the CDS with both the 5' and 3' UTR were significantly more tolerant compared to the transgenic lines containing OsNHX1 gene without the 3' UTR.

Alternative splicing of the OsBWMK1 gene generates three transcript variants showing differential subcellular localizations

Here, we show that alternative splicing of the OsBWMK1, a member of the rice MAPK family, generates three transcript variants, OsBWMK1L, OsBWMK1M, and OsBWMK1S. The OsBWMK1L transcript variant was highly and constitutively expressed in all rice tissues tested and its expression was not altered by various stress conditions, whereas OsBWMK1M and OsBWMK1S were normally expressed at low levels but were induced by various stresses.

Abiotic Stresses Cause Differential Regulation of Alternative Splice Forms of GATA Transcription Factor in Rice

In this study, we present an overview of the GATA gene family in rice (OsGATA)

Transcript abundance analysis in contrasting genotypes of rice-IR64 (salt sensitive) and Pokkali (salt tolerant), for individual GATA members indicated their differential expression in response to various abiotic stresses such as salinity, drought, and exogenous ABA. One of the members of subfamily VII-OsGATA23a, emerged as a multi-stress responsive transcription factor giving elevated expression levels in response to salinity and drought.

Salt tolerance involved candidate genes in rice: an integrative meta-analysis approach

Herein, a genome-wide meta-analysis, using microarray and RNA-seq data was conducted which resulted in the identification of differentially expressed genes (DEGs) under salinity stress at tolerant rice genotypes.

Briefly, the young seedlings of FL478 (Salt tolerant) and IR29 (Salt sensitive) were treated with 150 mM NaCl and the root samples were collected 24 h after inception of the salt stress.

A total of 3449 DEGs were detected in 46 meta-QTL positions, among which 1286, 86, 1729 and 348 DEGs were observed in root, shoot, seedling, and leaves tissues, respectively.

23 potential candidate genes were detected in Saltol and hotspot-regions overlying original QTLs for both yield components and ion homeostasis traits; among which, there were many unreported salinity-responsive genes.

I have created an endnote library (IGBF3041.enl) containing the above references and attached it to this ticket.

Nowlan Freese notes: A google search for "rice" and "splicing" and "salt" yields only few results.