# Alternative Splicing Dynamics in Plant Adaptive Responses to Stress

Abdulrahman Alhabsi, Yu Ling, Martin Crespi, Anireddy S N Reddy, Magdy Mahfouz

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Abdulrahman Alhabsi, Yu Ling, Martin Crespi, Anireddy S N Reddy, Magdy Mahfouz. Alternative Splicing Dynamics in Plant Adaptive Responses to Stress. Annual Review of Plant Biology, 2025, 76 (1), pp.687-717. 10.1146/annurev-arplant-083123-090055. hal-05461431

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# Alternative Splicing Dynamics in Plant Adaptive Responses to Stress

Abdulrahman Alhabsi,<fnref n="1" /> Yu Ling,<fnref n="2" /> Martin Crespi,<fnref n="3" />,<fnref n="4" />
Anireddy S.N. Reddy,<fnref n="5" /> and Magdy Mahfouz<fnref n="1" />

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Annu. Rev. Plant Biol. 2025. 76:687–717

First published as a Review in Advance on
February 14, 2025

The *Annual Review of Plant Biology* is online at
plant.annualreviews.org

https://doi.org/10.1146/annurev-arplant-083123-090055

Copyright © 2025 by the author(s). This work is
licensed under a Creative Commons Attribution 4.0
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## Keywords

pre-mRNA, RNA, alternative splicing, transcriptional control, abiotic stress, biotic stress, transcriptome engineering, alternative splicing editing

## Abstract

Plants thrive in dynamic environments by activating sophisticated molecular networks that fine-tune their responses to stress. A key component of these networks is gene regulation at multiple levels, including precursor messenger RNA (pre-mRNA) splicing, which shapes the transcriptome and proteome landscapes. Through the precise action of the spliceosome complex, noncoding introns are removed and coding exons are joined to produce spliced RNA transcripts. While constitutive splicing always generates the same messenger RNA (mRNA), alternative splicing (AS) produces multiple mRNA isoforms from a single pre-mRNA, enriching proteome diversity. Remarkably, 80% of multiexon genes in plants generate multiple isoforms, underscoring the importance of AS in shaping plant development and responses to abiotic and biotic stresses. Recent advances in CRISPR-Cas genome and transcriptome editing technologies offer revolutionary tools to dissect AS regulation at molecular levels, unveiling the functional significance of specific isoforms. In this review, we explore the intricate

<footnote n="1">¹Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia; email: magdy.mahfouz@kaust.edu.sa</footnote>

<footnote n="2">²College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang, People's Republic of China</footnote>

<footnote n="3">³Institute of Plant Sciences Paris-Saclay (IPS2), Université Paris-Saclay, CNRS, INRAE, Gif sur Yvette, France</footnote>

<footnote n="4">⁴Institute of Plant Sciences Paris-Saclay (IPS2), Université Paris Cité, Gif sur Yvette, France</footnote>

<footnote n="5">⁵Department of Biology and Program in Cell and Molecular Biology, Colorado State University, Fort Collins, Colorado, USA</footnote>
mechanisms of pre-mRNA splicing and AS in plants, with a focus on stress responses. Additionally, we examine how leveraging AS insights can unlock new opportunities to engineer stress-resilient crops, paving the way for sustainable agriculture in the face of global environmental challenges.

## Contents

<table><tr><td>1. INTRODUCTION</td><td>688</td></tr><tr><td>1.1. Pre-mRNA Splicing</td><td>689</td></tr><tr><td>1.2. Alternative Splicing and Its Prevalence</td><td>689</td></tr><tr><td>1.3. The Regulation of Alternative Splicing</td><td>690</td></tr><tr><td>2. THE ROLE OF ALTERNATIVE SPLICING IN ABIOTIC STRESS RESPONSES</td><td>692</td></tr><tr><td>2.1. Alternative Splicing Rep programs Gene Expression of Critical Regulators in Response to Cold Stress and Low Temperature</td><td>693</td></tr><tr><td>2.2. Pre-mRNAs of Genes Other Than Transcription Factors Showed Alterations in Splicing Patterns During Cold Stress</td><td>694</td></tr><tr><td>2.3. Salt Stress Generates Novel Splice Variants</td><td>694</td></tr><tr><td>2.4. The Impact of Drought Stress on Alternative Splicing</td><td>695</td></tr><tr><td>3. ALTERNATIVE SPLICING IN RESPONSE TO BIOTIC STRESSES</td><td>697</td></tr><tr><td>3.1. Alternative Splicing in Response to Viruses Confers Resistance</td><td>697</td></tr><tr><td>3.2. Fungal, Oomycete, Bacterial, and Nematode Infection Regulates Alternative Splicing</td><td>697</td></tr><tr><td>4. INVOLVEMENT OF SPLICING FACTORS AND REGULATORS IN STRESS RESPONSES</td><td>698</td></tr><tr><td>5. THE INTERPLAY BETWEEN ALTERNATIVE SPLICING MODULATION AND OTHER TYPES OF REGULATION</td><td>700</td></tr><tr><td>6. SPLICING REPORTERS FOR IN VIVO DETECTION OF SPLICE ISOFORM EXPRESSION AND IDENTIFICATION OF SPLICING REGULATORS</td><td>700</td></tr><tr><td>7. ENGINEERING ALTERNATIVE SPLICING FOR TRAIT IMPROVEMENT</td><td>702</td></tr><tr><td>7.1. Alternative Splicing Modulation at the DNA Level</td><td>702</td></tr><tr><td>7.2. The Modulation of Specific Splice Isoforms at the RNA Level</td><td>705</td></tr><tr><td>7.3. The Modulation of Alternative Splicing via Targeted Posttranslational Modification of Splicing Factors</td><td>705</td></tr><tr><td>7.4. The Directed Evolution of Splicing Regulators to Confer New Traits</td><td>706</td></tr><tr><td>8. CONCLUSIONS</td><td>707</td></tr></table>

## 1. INTRODUCTION

Plant growth and development are severely affected by adverse environmental factors, including abiotic stresses and biotic stresses, resulting in significant annual crop losses. Estimates suggest that the global production of key crops must increase to meet growing demands. This presents a significant challenge for global food security, as crops are a staple food source worldwide. Understanding plant stress responses may reveal potential solutions to enhance plant stress tolerance and
resilience to climate change (52, 132, 171, 193). Plants regulate gene expression at multiple levels in response to environmental stresses and developmental cues to adapt and survive (174). These regulations occur at epigenetic, transcriptional, posttranscriptional, and posttranslational levels (87, 106, 119, 139). Precursor mRNA (pre-mRNA) splicing is a key step in the co- and posttranscriptional regulation of gene expression (70, 126). Plants adapt to biotic and abiotic stress factors through changes in the transcriptome that enable their survival and resilience to adverse conditions. Alternative splicing (AS) plays a major role in the adaptive responses of plants to diverse abiotic and biotic stresses. Abiotic stresses, such as drought, salinity, temperature extremes, and oxidative stress, induce changes in AS patterns, leading to the production of splice variants that can enhance stress tolerance via multiple molecular mechanisms. Increased salinity has been shown to alter the splicing of genes involved in ion transport and osmotic balance, thereby enhancing salt stress adaptation (51, 80, 108). Similarly, drought conditions trigger AS events that modulate the expression of genes associated with water retention and stress signaling pathways (34, 102). Plants also respond to biotic stresses, such as pathogen infections from fungi, bacteria, and viruses, by fine-tuning AS patterns to enhance host immunity and resistance. These AS patterns can result in the differential expression of stress- and defense-related genes, improving the plant's ability to mount effective stress and immune responses (41, 99). The dynamic regulation of AS by both abiotic and biotic factors plays a crucial role in enabling plants to adapt to fluctuating environmental conditions and maintain homeostasis (140). In this review, we explore the mechanisms of RNA splicing and its role in regulating plant responses to better adapt to diverse environmental stresses.

**1.1. Pre-mRNA Splicing**

In photosynthetic eukaryotes, up to 90% of protein-coding genes contain one or more noncoding sequences (introns) (82). In splicing, the cellular machinery removes introns from the primary product of transcription (the pre-mRNA) and joins the coding sequences (exons) to create a functional transcript with an intact open reading frame (ORF) ready for translation. Pre-mRNA splicing is carried out by the spliceosome, a large ribonucleoprotein complex with high molecular weight (140) composed of 5 small nuclear RNAs (snRNAs) and more than 300 associated proteins (11, 32). The 5 snRNAs—U1, U2, U4, U5, and U6—are uridine rich and bind to their respective proteins to form distinct small nuclear ribonucleoproteins (snRNPs).

The first step in splicing is the recognition of key sites, that is, the 5' and 3' splice sites, the polypyrimidine tract within the intron, and the branch point (BP) within the pre-mRNA, by components of the spliceosome, which bind to the cis-elements in pre-mRNA transcripts. The U1 snRNP binds to the 5' splice site (5'SS) at the beginning of the intron, and U2 auxiliary factor (U2AF) binds to the 3' splice site (3'SS) at the end of the intron. U2AF has two subunits: The smaller subunit, U2AF³⁵, binds to the intron/exon border, and the larger subunit, U2AF⁶⁵, binds to the polypyrimidine tract of the pre-mRNA. The U2 snRNP binds to the BP, and a complex composed of U4, U5, and U6 snRNPs associates with the intron. In the second step, conformational changes and the dissociation of U1 and U4 snRNPs lead to two transesterification reactions and the joining of the exons (140). The major type of spliceosome is U2 dependent and includes U1, U2, U4, U5, and U6 snRNPs. It is responsible for splicing most pre-mRNA transcripts at canonical (GT-AG) splice sites. A second, minor type is the U12-dependent spliceosome, composed of U11, U12, U4atac, U5, and U6atac snRNPs. This minor spliceosome mediates the splicing of a small subset of introns, which are referred to as U12-type.

**1.2. Alternative Splicing and Its Prevalence**

Pre-mRNA splicing often generates a single mRNA from a multiexon gene. However, the spliceosome can recognize different splice sites, leading to AS and resulting in different mRNA isoforms

**Abiotic stress:**
a nonliving environmental factor, such as salinity, drought, extreme temperature, and nutrient imbalance, that negatively affects plant growth and development

**Biotic stress:**
a biological factor, such as a virus, bacterium, fungus, nematode, or herbivore, that causes damage to plants and compromises development and productivity

**Precursor mRNA splicing (pre-mRNA splicing):** a molecular process that removes introns (noncoding regions) and joins exons (coding regions) in pre-mRNA to produce mature mRNA

**Transcriptome:**
the total collection of RNA molecules, including coding and noncoding RNAs, expressed in a specific cell, tissue, or organism

**Alternative splicing (AS):** a regulated splicing mechanism that produces multiple mRNA isoforms from a single gene, leading to protein diversity
<figure><img src="image_5.png" /><figcaption>**Figure 1**</figcaption></figure>

Induction of AS by environmental factors, including abiotic and biotic stresses, and the frequency of AS events found in *Arabidopsis thaliana* (178). Abbreviations: A3′SS, alternative 3′ splice site; A5′SS, alternative 5′ splice site; AS, alternative splicing; EI, exon inclusion; ES, exon skipping; mRNA, messenger RNA; pre-mRNA, precursor messenger RNA; RI, retained intron.

from the same multiexon gene (127). High-throughput sequencing of transcriptomes (RNA-seq) suggests that about 95% of multiexon genes are alternatively spliced in humans (117) and about 79% in *Arabidopsis thaliana* (178).

Some common AS events involved in generating splice isoforms and their frequency in *Arabidopsis* (178) are shown in **Figure 1**. AS can generate transcripts characterized by a retained intron (RI), exon skipping (ES), an alternative 5' splice site (A5'SS), an alternative 3' splice site (A3'SS), or mutually exclusive exons (MXEs) (52, 127). RI events are the most prevalent type of AS in plants, and ES types are the most prevalent in animals (113, 128). Understanding plant growth, development, and stress responses affecting AS requires the generation of highly accurate transcript isoform annotation. Recent advances have generated the most comprehensive transcript dataset of *Arabidopsis*, called Reference Transcript Dataset 3 (AtRTD3), based on single-molecule full-length isoform sequencing (Iso-seq) analysis. This approach enhanced the accuracy of isoform prediction, which was usually hampered by incomplete complementary DNA (cDNA) synthesis, mRNA degradation, and/or sequencing errors (178).

AS increases transcriptome and proteome diversity and complexity (127). The different protein isoforms encoded by alternatively spliced transcripts may function similarly, distinctly, or even antagonistically. As a result, the accessibility of functional domains and regions necessary for posttranslational modification (PTM) may be altered in these isoforms. AS can create isoforms with premature termination codons (PTCs), and these mRNAs are then typically degraded via the nonsense-mediated decay (NMD) pathway. In *Arabidopsis*, AS-NMD can target 13% of genes. In addition, the different protein isoforms created by AS may have functional differences and different subcellular localizations and stabilities (140).

## 1.3. The Regulation of Alternative Splicing

Regulation of AS depends on *cis*-elements contained in the pre-mRNA and *trans*-acting splicing factors (SFs) that recognize the *cis*-regulatory elements and associate with the pre-mRNA (Figure 2). A *trans*-acting SF binds to a *cis*-element in the intronic or exonic sequences on the pre-mRNA, allowing or preventing the spliceosome from binding to the pre-mRNA and initiating AS (127, 140). The *cis*-elements are also referred to as splicing regulatory elements (SREs) and further serve as enhancers or repressors of splicing (29, 67).

**Spliceosome:** a dynamic multiprotein and RNA complex responsible for accurately splicing pre-mRNA to generate mature mRNA transcripts

**Small nuclear ribonucleoprotein (snRNP):** a key component of the spliceosome; composed of small nuclear RNAs and associated proteins

**Splice site:** a specific conserved sequence in pre-mRNA that defines the boundaries of introns and exons and is recognized by the spliceosome
<figure><img src="image_6.png" /><figcaption>**Figure 2**</figcaption></figure>

The splicing process and pre-mRNA sequence (84). The *cis*-regulatory elements include 5′SS, BP, 3′SS, ISE, ESE, ESS, and ISS. Intron borders are indicated by GU and AG. The *trans*-regulatory elements or snRNP assemble and bind *cis*-elements on the pre-mRNA. The binding of SR proteins or hnRNP at the SRE sequences affects the splicing process, allowing the production of differentially spliced isoforms. Abbreviations: 3′SS, 3′ splice site; 5′SS, 5′ splice site; BP, branch point; ESE, exonic splicing enhancer; ESS, exonic splicing silencer; hnRNP, heterogenous ribonucleoproteins; ISE, intronic splicing enhancer; ISS, intronic splicing silencer; mRNA, messenger RNA; pre-mRNA, precursor messenger RNA; snRNP, small nuclear ribonucleoprotein; SR, serine/arginine-rich; SRE, splicing regulatory element; (Y)ₙ, polypyrimidine tract. Figure adapted from images created with BioRender.com.

SREs are short conserved sequences 5-10 nucleotides in length and are mainly found in clusters in the genome (29, 103). Several techniques have been successfully used to identify transcripts associated with RNA-binding proteins (RBPs), including selective evolution of ligands through exponential enrichment (SELEX) (38), RNA immunoprecipitation (RIP) (10, 184), and cross-linking immunoprecipitation (CLIP) (158). Many SREs have been identified in animals (9, 29, 58, 92) and have been comprehensively analyzed, enabling computational prediction of ES events (9). Only a limited number of studies have conducted RIP or CLIP assays on plant RBPs (107, 160, 192). Unfortunately, because few plant SREs have been discovered computationally (118) or experimentally (8, 134, 135, 147, 168), it is difficult to develop computational tools to predict the RNA-protein interactions of the splicing machinery in plants (127).

The secondary structure of the RNA transcript also plays a role in the regulation of AS. Splicing can be prevented (or favored) by specific structural elements in the mRNA through masking (or exposing) the available splice sites (73). For example, a 5S ribosomal RNA (rRNA) mimic (P5SM) that binds RBPs has been shown to control AS of the transcription factor (TF) TFIIIA (59).

Another structural element regulating AS is a riboswitch that binds to metabolites and changes their configuration to regulate gene expression. For example, thiamine metabolism is regulated by a plant-conserved riboswitch found in the 3' untranslated region (UTR) of *THIAMINE C SYNTHASE (THIC)* (12, 155). The *Arabidopsis thic* mutant, which has a mutated thiamine

**Splice isoform:**

a variant mRNA produced through alternative splicing, encoding distinct protein products with potentially unique biological functions or contributing to gene regulation

**Exon skipping (ES):**

a specific type of alternative splicing where an exon is excluded from the final mRNA, altering the encoded protein sequence
**Proteome:** the entire
set of proteins
expressed by a
genome, cell, tissue, or
organism under
specific conditions

**Posttranslational modification (PTM):**
a covalent chemical modification of proteins, such as phosphorylation or ubiquitination, that alters their activity, localization, or stability

**Nonsense-mediated decay (NMD):**
a cellular
quality-control
mechanism that
degrades mRNA
transcripts containing
premature termination
codons to prevent the
production of
truncated proteins

**Splicing regulatory element (SRE):**
a cis-acting sequence in pre-mRNA that binds splicing factors to modulate the inclusion or exclusion of specific exons or introns

**Splicing factor (SF):**
a protein that regulates splicing by interacting with spliceosomal components and specific sequences in the pre-mRNA transcript

pyrophosphate riboswitch region, exhibited phenotypic changes such as delayed flowering, growth retardation, and chlorosis, suggesting the important role of the riboswitch in regulating plant physiology (13).

Because structural elements within the RNA transcript can serve as binding sites for molecules such as ions and osmolytes, and cellular concentrations of these molecules can be affected by abiotic stresses, structural RNA elements are likely to be involved in AS regulation during abiotic stress responses (127). Multiple transgenic plant species, including *Arabidopsis*, rice, and maize, expressing a global bacterial RNA chaperone—responsible for regulating RNA folding—have exhibited enhanced stress tolerance, suggesting that RNA structures play a crucial role in the stress response (25).

Certain long noncoding RNAs (lncRNAs; transcripts without coding potential and larger than 200 bp) can also modulate AS by interacting with SFs or pre-mRNAs or influencing chromatin remodeling, further increasing the complexity of splicing regulation (133). For example, *ALTERNATIVE SPLICING COMPETITOR (ASCO)*, a lncRNA that binds to several SFs, influences the AS of many genes, participates in lateral root formation, and changes the response of *Arabidopsis* roots to bacterial flagellin. Knockdown of *ASCO* increases *Arabidopsis* sensitivity to flagellin (131). Additionally, circular RNAs (circRNAs) are stable single-stranded lncRNAs generated by backsplicing the exon-intron junction. These circRNAs contain at least an exon and intron in circular form and can be recognized by SFs in competition with pre-mRNAs to regulate AS output. In addition, they can also bind to their own parent DNA by forming an R-loop (i.e., an RNA-DNA hybrid), adding several additional layers of complexity to the regulation of splicing and gene expression (133). For instance, *SEPALLATA 3* (*SEP3*), which encodes a DNA-binding protein that functions in flower development, has three mRNA isoforms (*SEP3.1*, *SEP3.2*, and *SEP3.3*). When a circRNA was produced from exon 6, the level of the *SEP3.3* isoform (in which exon 6 is skipped) increased, and the resulting transgenic plants showed an altered floral phenotype. Also, the overexpressed *SEP3* circRNA formed an RNA-DNA hybrid with the *SEP3* locus in *trans*, which induced the production of the *SEP3.3* isoform (7, 36).

These examples highlight the complex interplay among *cis*- and *trans*-acting factors, including RNA sequence and structure, protein SFs, and lncRNAs, demonstrating the vast challenge required to unravel the molecular regulation of AS in stress responses. Next, we highlight selected studies that shed light on the role of AS in stress responses.

## 2. THE ROLE OF ALTERNATIVE SPLICING IN ABIOTIC STRESS RESPONSES

Abiotic stresses are major challenges to plant growth and development and include salinity, drought, low and high temperature, low and high light, and lack or excess of nutrients (52). The regulation of gene expression by AS has gained increased attention for its role in adaptive plant stress responses. Therefore, manipulating AS has the potential to create plants with enhanced agronomic traits, including stress tolerance (52).

Abscisic acid (ABA) is an important phytohormone that regulates a plethora of responses during abiotic stresses, notably drought stress. Following stress perception and ABA induction, signals are transduced to downstream TFs that regulate stress-related genes (84). The biosynthesis of stress-induced ABA, followed by the interaction between ABA and its receptors, results in the inhibition of HYPERSENSITIVE TO ABA 1 (HAB1), a protein phosphatase 2C (PP2C). HAB1 functions as a negative regulator of ABA by dephosphorylating sucrose nonfermenting 1-related protein kinase 2.6 (SnRK2.6). This kinase phosphorylates downstream regulators, triggering ABA signaling. Interestingly, the SF RNA-binding motif protein RBM25 regulates AS of the *HAB1*
pre-mRNA, which produces *HAB1.1*, a functional isoform that accumulates at low ABA levels, and *HAB1.2*, a nonfunctional truncated isoform resulting from RI triggered by increased ABA levels. RBM25 binds to and induces AS of *HAB1* pre-mRNA to produce the functional HAB1.1 isoform. *Arabidopsis rbm25* mutant seedlings are hypersensitive to exogenous ABA, and this phenotype can be rescued by overexpression of the *HAB1.1* isoform in *rbm25*, confirming the role of SFs in regulating ABA sensitivity (84, 157).

## 2.1. Alternative Splicing Rep Programs Gene Expression of Critical Regulators in Response to Cold Stress and Low Temperature

Severe cold can negatively affect plant survival, and splicing patterns change in response to cold stress. Exploration of AS variants in various plant species has shed light on the intricate regulatory mechanisms involved in plant responses to cold stress. Transcriptomic studies in crops have identified cold-induced splice variants in a variety of plant species in addition to model plants such as *Arabidopsis* and rice (17, 20, 89, 170, 187). For example, cassava (*Manihot esculenta*) responds to cold stress through intensive quantitative and qualitative modulation of gene expression via AS pathways, revealing the high complexity and specificity of gene regulation in abiotic stress responses (182). Maize (*Zea mays*) showed genome-wide changes in AS during low-temperature stress, affecting genes associated with hormone and oxidoreductase activity (175).

Studies of AS in the orchid *Dendrobium catenatum* suggested that AS has a regulatory role in response to cold stress, with genes involved in amine metabolism and spliceosomes being differentially modulated (187). The cold-resistant CRQ64 variety of quinoa (*Chenopodium quinoa*) showed a more pronounced response to cold stress in terms of differentially expressed genes (DEGs) and AS events compared to the cold-sensitive CSQ5 variety (186). Similarly, the rubber tree *Hevea brasiliensis* exhibited distinct chilling tolerance mechanisms, as highlighted by DEGs and AS events identified in the RRIM600 and GT1 genotypes during cold treatments (21).

Plants' biological processes align with daily changes in light and temperature through the circadian clock. A genetic screen in *Arabidopsis* identified the *SICKLE* (*SIC*) gene, which influences circadian clock phenotypes under ambient low-temperature cycles. *sic-3* mutants display altered expression of core circadian clock genes and accumulate splice variants of *LHY* and *CCA1*. The role of *SIC* in pre-mRNA metabolism, along with its impact on circadian clock responses to cold stress, reveals a complex relationship between AS, clock genes, and temperature regulation (101).

The *Arabidopsis* clock gene *CCA1* undergoes dynamic self-regulation through a splice variant, *CCA1β*, which links the circadian clock to cold acclimation. Interestingly, low temperatures suppress the AS of *CCA1*, reducing the production of *CCA1β*. Transgenic plants overexpressing *CCA1α* display enhanced freezing tolerance, whereas those overexpressing *CCA1β* are more sensitive to freezing (136). However, it was later suggested that the ORF of the *CCA1* transcripts may have been erroneously annotated (15).

Several TFs, including those involved in regulating the circadian cycle, undergo AS under low-temperature and cold-stress conditions in *Arabidopsis*. This splicing mechanism plays a role in the timing of floral transitions in response to ambient temperature changes. Among these, the MADS-box TFs FLOWERING LOCUS M (FLM) and SHORT VEGETATIVE PHASE (SVP) are essential regulators of flowering. The proteins encoded by two primary splice variants of FLM, FLM-β and FLM-δ, compete for interaction with the floral repressor SVP in a temperature-dependent manner. At low temperatures, the SVP-FLM-β complex predominates, preventing premature flowering. By contrast, at higher temperatures, the competing SVP-FLM-δ complex, which has reduced DNA-binding capacity, acts as a dominant activator of flowering (120). Subsequent studies have confirmed the functional importance of
the FLM-β isoform in regulating flowering time, with FLM-δ playing a more minor role (22, 97).

## 2.2. Pre-mRNAs of Genes Other Than Transcription Factors Showed Alterations in Splicing Patterns During Cold Stress

AS is a key mechanism that modulates the expression of other genes, including those encoding nucleic acid-binding proteins and crucial enzymes in the complex response of plants to cold stress. DEAD-box RNA helicase in *Chorispora bungeana* (*CbDRH*), for instance, generates two transcript variants, *CbDRH.1* and *CbDRH.2*, through AS, and cold stress upregulates the *CbDRH.2* splice isoform. *CbDRH.2* interacts with the cold-responsive RBP *CbGRP*, providing a glimpse into the molecular interactions that occur during cold adaptation (165).

Plant cold responses affect photosynthesis through AS. For example, in *Medicago sativa* ssp. *falcata*, MfEF2, which encodes eukaryotic translation elongation factor 2, orchestrates cold tolerance by regulating hundreds of proteins, affecting photosystem subunits, and promoting AS under low temperatures by increasing the levels of spliceosomal proteins (137). *Lolium perenne* produces increased levels of splicing variants of Rubisco activase during prehardening and cold acclimation, contributing to increased Rubisco activity and photosynthetic acclimation to cold (75). Moreover, cold stress can involve oxidative stress. Indeed, AS of the reactive oxygen species (ROS)-detoxifying enzyme gene iron-superoxide dismutase (*OsFe-SOD*) in rice is sensitive to low temperatures (49).

*High expression of osmotically responsive genes 1 (HOS1)*, which encodes an *Arabidopsis* E3 ubiquitin ligase that is the key regulator of low-temperature response and flowering time, exhibited AS, with one particular splice variant (*HOS1-L*) influencing flowering time (86).

AS can also affect product quality; for example, in potato (*Solanum tuberosum*), cold stress induces AS events in genes coding for invertase (14). The vacuolar invertase inhibitor *INH2* RNA is alternatively spliced under cold conditions, and this is important for preventing cold-induced sweetening of potatoes. When potatoes are stored at low temperatures, sweetening is induced as starch is catabolized into sucrose and then to fructose and glucose by invertase. Investigation of cold-induced sweetening-resistant lines showed an increased presence of two splice isoforms of *INH2* (16).

Collectively, these studies not only underscore the pervasive influence of AS on the functions of genes involved in the complex responses of plants to cold stress but also reveal a sophisticated layer of molecular regulation that shapes adaptation and survival strategies in low-temperature environments.

## 2.3. Salt Stress Generates Novel Splice Variants

AS of RNAs encoding TFs also plays a pivotal role in orchestrating the intricate molecular responses of plants to salt stress. For example, barley *Dehydration-Responsive Factor 1* (*HvDRF1*) is upregulated in leaves and roots in response to drought, salt, or ABA treatment. The AS of *HvDRF1* generates three transcript isoforms, two of which encode AP2 proteins. These AP2 proteins act as transcriptional activators, exemplifying how AS contributes to the functional diversity of TFs in stress-related gene regulation (163).

The endoplasmic reticulum (ER) is a cellular compartment essential for protein synthesis and transport. During ER stress, the unfolded protein response (UPR) is activated to manage the accumulation of misfolded proteins, ensuring ER protein homeostasis. TFs of the bZIP family, such as bZIP17 and bZIP60, exhibit distinct roles in the UPR under salt stress conditions. The differential accumulation of transcripts for chaperones such as BiP3 and the modulation of bZIP17
target genes highlight the specificity of AS in regulating stress-related gene expression. The nu-
anced roles of spliced and unspliced forms of bZIP60 further underscore the complex regulatory
networks shaped by AS in plant physiology (61).

Several other genes play important roles in plants during salt stress, functioning in diverse cel-
lular processes such as ion transport and antioxidant activity. Rice OsIM1, predicted to encode a
chloroplast-localized terminal oxidase, generates two AS isoforms that were differentially regu-
lated by salt stress in salt-tolerant and salt-sensitive rice varieties, highlighting the intricate splicing
dynamics in salt tolerance (80).

A dehydrin-encoding gene in Bermudagrass (Cynodon spp.), *CdDHN4*, produces two transcripts through AS. Heterologous overexpression of either isoform in *Arabidopsis* enhanced the scavenging capacity of ROS, leading to improved salt and drought tolerance compared with wild-type (WT) plants (173). Transgenic *Arabidopsis* plants expressing the *CdDHN4-s* isoform exhibited significantly lower electrolyte leakage and enhanced photosynthetic capacity, suggesting its functional relevance in drought tolerance.

SALT-RESPONSIVE ALTERNATIVELY SPLICED 1 (SRAS1) produces two splice isoforms (*At-SRAS1.1* and *At-SRAS1.2*) in *Arabidopsis*. At-SRAS1.1 functions as an E3 ubiquitin ligase, but this function is lost in the truncated At-SRAS1.2 isoform. Under high salinity, the At-SRAS1.1 isoform accumulates, and the amount of the At-SRAS1.2 isoform decreases, indicating a differential response to the stress. Interestingly, higher salt tolerance was seen in the lines overexpressing *At-SRAS1.1* compared to the lines overexpressing *At-SRAS1.2* (189).

Another example is the vacuolar Na⁺/H⁺ antiporter gene OsNHX1. In high-salt conditions, vacuolar antiporters transport excess sodium ions to the vacuole to maintain cellular ion homeostasis. Pre-mRNA splicing of the OsNHX1 produces three isoforms, including a truncated isoform that lacks about 150 bp in the 3'UTR. Overexpressing the isoforms with normal 5' and 3'UTRs resulted in enhanced salt tolerance compared to the lines overexpressing the isoform with the truncated 3'UTR. Interactions with proteins and microRNAs at the 3'UTR can stabilize or localize mRNAs, suggesting that the 3'UTR may play a crucial role in stress (6). Also, RNA for OsMAPK5, a mitogen-activated protein kinase involved in stress response, undergoes AS and produces an OsMAPK5a isoform that maintains kinase activity. The OsMAPK5b isoform is about 104 amino acid residues shorter and, as a result, lacks the kinase function. Overexpressing OsMAPK5a results in enhanced tolerance to multiple abiotic stresses including salinity and drought, whereas RNAi-silenced seedlings exhibit reduced stress tolerance (161).

## 2.4. The Impact of Drought Stress on Alternative Splicing

The intricate regulation of AS in genes encoding DREB2-type TFs is conserved across the plant kingdom. Qin et al. (122) found that maize *ZmDREB2A* produced two forms of transcripts, and quantitative real-time polymerase chain reaction (PCR) analyses demonstrated that only the functional transcript of *ZmDREB2A* was significantly induced by stress (122). A similar conserved regulatory mechanism also exists in bread wheat (*Triticum aestivum*) and its diploid ancestors, where different isoforms of *DREB2* are regulated in opposite directions by dehydration stress (146). The wheat DREB gene *TaDREB3* (also known as *WDREB2*) undergoes dynamic AS regulation, generating three transcript forms, two of which exhibit transient increases under drought and salt stresses, while the level of the third isoform remains relatively stable (46). Further study revealed that transcripts I and II can produce DREB proteins, whereas transcript III does not generate a DREB protein. Only transcript I proves to be instrumental in enhancing resistance to drought, salt, and heat stresses in transgenic *Arabidopsis* (116).
**Serine/arginine-rich (SR) proteins:**

a conserved family of
splicing factors that
mediate interactions
with RNA and
proteins during
splicing via their RNA
recognition motif and
RS domain

**Nuclear speckle:**

a dynamic
biomolecular
condensate within the
nucleus that is
enriched in
pre-mRNA splicing
factors and plays a
crucial role in splicing
regulation

AS regulation was recently reported for the bZIP TF FD of citrus; its two isoforms, CiFDα and CiFDβ, exhibit differential induction by low temperature and drought, respectively. CiFDα (the full-length form) forms a floral activation complex (FAC) to regulate drought-induced flowering through AS, while CiFDβ (the truncated form) operates independently of the FAC, highlighting the versatility of AS in modulating responses to diverse stressors (166). Moreover, AS of the sugarcane TF *Myeloblastosis MYBAS1* RNA produces four variants with changes in their MYB domains that translate into functional differences in drought tolerance. Rice transgenic plants overexpressing *Sc-MYBAS1-2* and *Sc-MYBAS1-3* showed increased water content compared to the WT. In addition, overexpressing *Sc-MYBAS1-3* enhanced plant biomass, whereas overexpressing *Sc-MYBAS1-2* decreased the biomass compared to the WT (48).

The Highly ABA-Induced 1 (HAI1) protein phosphatase is a central component of drought-related signaling. A HAI1-interacting protein, HAI1-Interactor 1 (HIN1), interacts with serine/arginine-rich (SR) SFs and localizes in nuclear speckles during drought stress. Plants ectopically expressing *HIN1* show drought stress-mimicking splicing patterns for hundreds of genes (34). Maize *ZmPP2C26* undergoes AS, generating *ZmPP2C26L* and *ZmPP2C26S* isoforms. ZmPP2C26S has 71 fewer amino acid residues, includes a MAPK interaction motif, and shows higher phosphatase activity than ZmPP2C26L. Drought stress inhibits the expression of these isoforms, and a *Zmpp2c26* mutant exhibits enhanced drought tolerance. Conversely, overexpression of *ZmPP2C26L* and *ZmPP2C26S* decreases drought tolerance in *Arabidopsis* and rice, resulting in shorter roots, decreased chlorophyll content, and lower photosynthetic rates (150). These findings collectively highlight the multifaceted role of AS in shaping the dynamics of protein kinases and PP2Cs, providing plants with a complex regulatory mechanism to address the challenges posed by drought stress.

In addition to TFs and signaling genes, drought stress affects the AS of many other genes, shedding light on the molecular mechanisms underlying stress adaptation. Δ¹-*PYRROLINE-5-CARBOXYLATE SYNTHETASE 1 (P5CS1)* in *Arabidopsis* has polymorphisms in introns that cause AS, leading to the production of a nonfunctional splice variant lacking exon 3. The protein encoded by this AS variant, referred to as exon 3-skip *P5CS1*, plays a crucial role in determining proline accumulation under conditions of low water potential, contributing to natural variation in proline metabolism among *Arabidopsis* accessions and suggesting a role in environmental adaptation (79). Another instance is found in the *ZINC-INDUCED FACILITATOR-LIKE 1 (ZIFL1)*, a major facilitator superfamily (MFS) transporter gene in *Arabidopsis*. The full-length ZIFL1.1 and a truncated isoform produced by AS, ZIFL1.3, exhibit distinct subcellular localizations and functions. ZIFL1.1 regulates auxin-related processes in roots, while ZIFL1.3 mediates drought tolerance by regulating stomatal closure (130).

Recently, vegetative growth to reproductive growth transition factor 1 (VRF1) was identified as a key player in stress-induced early flowering, a strategy that allows plants to escape environmental stress. AS of *VRF1* produces two functional isoforms: VRF1-AS1, which regulates flowering time, and VRF1-AS3, which enhances drought tolerance. Thus, the AS of *VRF1* functions as a molecular switch, modulating isoform ratios in response to environmental cues (30).

These studies underscore the versatility of AS in regulating key processes associated with not only transcription but also ion homeostasis, enzyme activity, and abiotic stress tolerance. Understanding these intricate AS regulatory networks provides valuable insights into the molecular mechanisms governing plant adaptation to challenging environmental conditions and highlights the role of AS in shaping the transcriptional responses of plants to salt stress. The dynamic and context-dependent nature of AS indicates its significance as a regulatory mechanism, offering plants the flexibility to fine-tune their stress responses based on environmental cues. This intricate interplay between transcriptional and posttranscriptional regulation, as exemplified by the diverse
TFs discussed, contributes to the adaptability and resilience of plants in the face of challenging salt stress conditions.

3. ALTERNATIVE SPLICING IN RESPONSE TO BIOTIC STRESSES

AS is a key mechanism in plant responses to virus infections, revealing a complex interplay be-
tween host genes and pathogens. Disease resistance (R) genes, which often undergo AS to produce
truncated isoforms, play prominent roles in plant immunity by suppressing negative regulation,
initiating immunity, or directly participating in effector-triggered signaling, emphasizing AS's
multifaceted roles in defense (164).

3.1. Alternative Splicing in Response to Viruses Confers Resistance

In virus infections, *Brachypodium distachyon* infected with *Panicum* mosaic virus (PMV) and its satellite virus (SPMV) exhibit AS in about 42% of multiexon genes, including immune-related genes such as receptor-like kinases; nucleotide-binding site, leucine-rich repeat (NB-LRR) resistance proteins; and splicing-associated proteins (99). A transcriptomic study in pepper (*Capsicum annuum*) leaves infected with cucumber mosaic virus (CMV) identified 73,597 AS events in 12,615 genes, reflecting extensive transcriptome reprogramming (191).

R genes are crucial for pathogen detection and mitigation in biotic stress tolerance (111). For example, in tobacco, the *N* gene, an *R* gene, is alternatively spliced to produce two isoforms: a short isoform, *N*<sub>s</sub>, and a long isoform, *N*<sub>L</sub>. The *N*<sub>L</sub> isoform includes a PTC that truncates the LRR. Before infection, the *N*<sub>s</sub> isoform prevails, but after TMV infection, *N*<sub>L</sub> becomes dominant. Transgenic plants expressing only *N*<sub>s</sub> or both isoforms equally exhibit low resistance, indicating that AS regulation is key to full resistance to TMV (41).

AS also regulates various immune-related genes. The gene encoding acidic chitinase in *Cit-rus clementina* undergoes AS in response to *Tetranychus urticae* infestation and methyl jasmonate treatment, resulting in truncated proteins with potential chitinase activity (40).

Interaction between the viral Replication Initiator Protein (Rep; required for viral nucleic acid replication) of tomato yellow leaf curl virus (TYLCV) and a protein complex that controls RNA splicing and mRNA export was observed in tomato (100). Similarly, potato virus A (PVA) infection altered ribosomes and splicing-related proteins in potato leaf cells (124). Virus-induced alterations in transcriptomic reprogramming, AS, and RNA methylation during potato virus Y (PVY) infection underscore AS's regulatory role in host-virus interactions (53). *Rosa chinensis* RcCPR5, which encodes a constitutive expressor of pathogenesis-related genes, has two splicing variants: RcCPR5-2, with a large C-terminal deletion, was quickly upregulated during disease development and coordinated with RcCPR5-1 to resist powdery mildew (96).

3.2. Fungal, Oomycete, Bacterial, and Nematode Infection Regulates
Alternative Splicing

In the dynamic interplay between plants and microbial counterparts, AS is crucial in host–pathogen interactions. In rice, two splice variants are produced from the rice *R* gene *RGA5*. Isoform *RGA5-B* contains an *RI* that disrupts the *ORF*, while isoform *RGA5-A* confers resistance against *Magnaporthe oryzae*. The susceptibility of a transgenic plant expressing *RGA5-B* and its interaction with the *AVR1-CO39* effector (26) suggest a role for *RGA5-B* in response to infection. *Pi-ta*, another *R* gene providing resistance to rice blast, undergoes AS to generate splice variants with *RI*, shorter *UTRs*, or longer C-terminal regions. The transcripts with longer C-terminal regions are most prevalent in the blast-resistant cultivar, suggesting involvement in pathogen response (37).
NONEXPRESSOR OF PATHOGENICITY-RELATED 3 (OsNPR3) generates three isoforms by AS. The OsNPR3.3 isoform was highly expressed in a cultivar resistant to Xanthomonas oryzae pv. oryzae (Xoo), while the expression of other isoforms either decreased or did not change following Xoo infection (72). Similarly, OsWRKY62 and OsWRKY67 are alternatively spliced, regulating resistance to M. oryzae and Xoo. The truncated isoforms OsWRKY62.2 and OsWRKY76.2 positively regulate rice resistance, while the full isoforms enhance susceptibility by causing transcriptional repression (94).

OsDR11, encoding a LAMMER kinase (a kinase with the EHLAMMERILG motif, important for kinase activity), has two antagonistic splice variants. In rice, overexpression of OsDR11s or knockdown of OsDR11L improved resistance to Xoo. OsDR11L inhibits jasmonic acid signaling and resistance, while OsDR11s prevents the transcription and kinase activity of OsDR11L, resulting in resistance (45).

A recent example showed how an oomycete pathogen suppresses plant immunity by controlling host pre-mRNA splicing. The *Phytophthora sojae* effector PsAvr3c interacts with serine/lysine/arginine-rich proteins from soybean (GmSKRPs), which are negative regulators of plant immunity. Overexpressing GmSKRP1 or PsAvr3c resulted in differentially spliced genes, including defense-related genes such as *NAC* and *WRKY* TFs (66). In addition, AtSKRP, a splicing repressor, was shown to regulate the pre-mRNA splicing of positive immune regulatory genes, suppressing immunity in *Arabidopsis* (28).

Cellular RBPs, essential for RNA stability and splicing, regulate immune-responsive tran-
scripts. Mutants with defects in AS of resistance genes exhibit compromised resistance (141). The
SFs SUA and RSN2 are implicated in the proper splicing of RNAs for the RLKs SNC4 and
CERK1, which are required for pathogen recognition and defense responses (183). SWP73A,
a chromatin-remodeling protein, suppresses nucleotide-binding domain and leucine-rich-repeat
receptor (NLR) expression by influencing the AS of NLRs, adding another layer to plant defense
regulation (65).

The response of wheat plants to powdery mildew caused by *Blumeria graminis* f. sp. *tritici* involves RLK-V, which undergoes AS through RI to produce RLK-V1.1 and RLK-V1.2. While both are upregulated during infection, only RLK-V1.1 confers resistance (62).

In wheat, *Puccinia striiformis* f. sp. *tritici* (*Pst*) infection leads to TabZIP74 mRNA splicing in seedling leaves; both spliced and unspliced forms are detected in roots, showing that TabZIP74 is critical for stripe rust resistance, drought tolerance, and lateral root development (156). In *M. oryzae*, 4,270 AS isoforms from 2,413 genes were identified, with 499 genes regulated by infection-specific AS, indicating sophisticated adaptive strategies by the fungus (71).

Nematode infections also interfere with AS. Root-knot nematodes (RKNs) are among the most damaging pests of crops. EFFECTOR 18 (EFF18) protein from *Meloidogyne incognita* interacts with spliceosomal protein SmD1 in *Arabidopsis* and tomato, disrupting its function in AS regulation. Specific SmD1 alleles unable to interact with EFF18 confer increased resistance to nematodes, suggesting spliceosomal components as new targets for broad RKN resistance (104, 105). These findings underscore the multifaceted role of AS in fine-tuning plant immune responses and its significance in the molecular arms race between plants and pathogens.

4. INVOLVEMENT OF SPLICING FACTORS AND REGULATORS
IN STRESS RESPONSES

Among the commonly studied non-snRNP SFs are the SR proteins and the heterogeneous
nuclear ribonucleoproteins (hnRNPs). Members of these protein families bind the pre-mRNA
through their RNA recognition motif (RRM) to regulate splice site selection (126). Members of
the SR protein family function in both constitutive splicing and AS and are characterized by an
N-terminal RRM (or two RRMs) that mediates interactions with the RNA and a C-terminal SR domain that mediates interactions with the spliceosome (129). Similarly, hnRNPs participate in mRNA metabolism, possessing domains that may further broaden their activities or RNA-binding capabilities (154). Under different stresses, SFs such as the SR proteins undergo transcriptional changes, posttranscriptional changes such as stress-induced AS or altered phosphorylation, or show altered subcellular localizations (81, 126, 129, 140).

Many functional studies involving mutagenesis and overexpression have been done to elucidate the role of SFs in stress responses (reviewed in 52, 81, 84, 127, 140, 148). For example, loss-of-function mutants of the SR family, such as *rs40* and *rs41*, showed hypersensitivity to salt stress and ABA in *Arabidopsis* (31). The SR-like protein SR45 interacts with spliceosomal components, including U1-70K and U2AF³⁵b (39). Similarly, the loss-of-function mutant *sr45-1* had a defective response to glucose and ABA and was hypersensitive to salt stress (2, 23, 24). Moreover, the loss-of-function mutant *scl30a-1* was hypersensitive to ABA and salt stress, whereas *SCL30a* overexpression reduced the sensitivity to ABA and enhanced salt stress tolerance during seed germination (83). In rice, ABA and abiotic stresses such as NaCl, heat, and cold induce AS of SR transcripts (177). SR proteins are involved in maintaining nutrient homeostasis, confirmed by the roles of SR40, SCL25, and SCL57 in phosphate remobilization in rice shoots (42).

Similarly, another study showed the importance of OsSCL26 in controlling rice phosphate homeostasis via regulating the transcription and splicing of phosphate transporter genes. In comparison to the WT, the *osscl26* mutant plants showed higher phosphate concentration in the shoot, as well as increased expression of phosphate-related genes, including four members of *OsPHT*, two members of *OsSPX-MFS*, and *OsNLA2*. Moreover, levels of the isoforms *OsSPX-MFS2.2*, *OsNLA2.1*, and *OsNLA 2.2* were highly increased in the *osscl26* mutant (95). In *Arabidopsis*, the *sr34b* mutant displayed a shorter root phenotype in response to cadmium treatment, with a higher accumulation of cadmium in the root of *sr34b* compared to that of the WT (179). The *rs33* loss-of-function rice mutant was hypersensitive to salt and low-temperature stresses, and RS33 regulates pre-mRNA splicing in response to abiotic stresses (17, 19). Also, we recently showed that the SR-related gene *OsSCR106* regulates gene expression and pre-mRNA splicing of abiotic stress-related genes and that it is essential for rice tolerance to multiple abiotic stresses (4). Single and multiple mutants of SR genes have been created using CRISPR-Cas engineering and can help us to understand the role of SR proteins in plant development and stress responses (17, 19).

Overexpression of an SR protein from *Arabidopsis*, rice, cassava, or poplar can enhance or decrease stress tolerance. For example, *AtSCL30a* overexpression enhanced *Arabidopsis* seed germination under ABA and salt stress (83). The overexpression of SR45 improved *Arabidopsis* tolerance to ABA (3), and overexpression of cassava *MeSR34* or *MeRS40* promoted salt tolerance in *Arabidopsis* (56, 98). In addition, overexpressing *MeSCL30* or *MeRSZ21b* enhanced drought tolerance in *Arabidopsis* (33, 159). Transgenic *Arabidopsis* overexpressing *PtSC27* showed improved tolerance to salt and ABA during seed germination, seedling growth, and adult plant stages (85). By contrast, *OsSCL30* overexpression decreases rice tolerance to multiple abiotic stresses (176). Overexpression of *MeRS40* decreased cassava salt tolerance (98). *Arabidopsis* lines overexpressing *PtSCL30* showed an enhanced freezing-sensitive phenotype and were hypersensitive to salt stress (185), and *Arabidopsis* lines overexpressing *MeSCL30A* were hypersensitive to salt stress (63).

SR protein family members may also participate in biotic stress responses. For example, a recent report suggested the role of the SR-like protein SR45 in regulating innate immunity in *Arabidopsis*. RNA-seq analysis comparing the WT and *sr45-1* mutant revealed that defense response genes were highly enriched. Many genes implicated in defense response, such as SA pathway genes, LRR receptor-like protein genes, and defense marker genes, were highly expressed in the *sr45-1* mutant compared to the WT. After treatment with pathogens such as *Pseudomonas syringae*

the application of Cas
enzymes to precisely
edit, regulate, or
modify genetic
material, including
DNA or RNA, for
targeted genome,
epigenome, or
transcriptome editing
*PmaDG3* bacteria or the oomycete *Hyaloperonospora parasitica* Noco2, the *sr45-1* mutant displayed high resistance compared to the WT *Arabidopsis*. Moreover, the *sr45-1* mutant showed an increased host response against the bacterial flagellin22 (flg22) and had increased callose deposition in response to flg22 compared to Col-0. In addition, the total salicylic acid concentration was highly increased in the *sr45-1* mutant compared to the WT (181). Moreover, the SR-related SF OsSCR106 regulates the expression of some genes implicated in defense responses to fungi and bacteria (4). In addition, other protein families implicated in splicing, such as hnRNP-like SF, also mediate resistance to pathogens (57, 114).

## 5. THE INTERPLAY BETWEEN ALTERNATIVE SPLICING MODULATION AND OTHER TYPES OF REGULATION

The intricate interplay between AS and epigenetic regulation in plants, particularly in response to stress, unveils a complex regulatory network. In *Arabidopsis*, DNA methylation is established through the RNA-directed DNA methylation (RdDM) pathway. *RDM16* encodes pre-mRNA SF3, a component of the U4/U6 snRNP, indicating the interaction between pre-mRNA AS regulation and DNA methylation, which is confirmed by the observation that 308 RI events occurred in the *rdm16* mutant (64). In addition, studies have revealed characteristic patterns of DNA methylation and nucleosome occupancy in the vicinity of intron-exon boundaries in *Arabidopsis*. Perturbations in these patterns, occurring under normal or cold conditions, have been shown to change AS patterns in *Arabidopsis* (27, 68, 69). By contrast, Gnan et al. (54) demonstrated that GC nucleotide content, not nucleosome positioning, directly contributes to intron-splicing efficiency in the unicellular animal *Paramecium tetraurelia* (54). In rice and *Arabidopsis*, RI is positively correlated with open chromatin (151). A recent study in humans provided evidence for the role of specific transcription in regulating RI (152). Similar studies have not been performed in plants.

Because the exposure of plants to abiotic and biotic stresses can affect DNA integrity, plants have evolved different mechanisms for the detection, signaling, and repair of DNA damage. Pre-mRNAs of many plant DNA repair genes are alternatively spliced. Recent reports suggest that AS may be implicated in the DNA damage response (DDR) (115). Collectively, these examples highlight the role of AS in epigenetic regulation or in regulating DNA repair genes and, therefore, AS likely plays a pivotal role in modulating the DDR in plants.

## 6. SPLICING REPORTERS FOR IN VIVO DETECTION OF SPLICE ISOFORM EXPRESSION AND IDENTIFICATION OF SPLICING REGULATORS

Several methods to detect and visualize pre-mRNA splicing in vivo have been developed, including single or dual reporter genes using fluorescent proteins (FPs) or bioluminescent proteins. These reporters can be highly specific and sensitive and linked to the target gene, allowing noninvasive and dynamic monitoring of in vivo splicing in real time. In animals, these reporters were applied during screenings of splicing inhibitors, splicing sites, and splicing regulators (recently reviewed in 138). In plants, several splicing reporters were created to analyze RNA splicing (described in 128). A recently developed dual FP reporter showed a differential tissue-specific expression of alternatively spliced isoforms in vivo and allowed researchers to follow the action of external stimuli such as hormones (78). Inserting the green fluorescent protein (GFP) and red fluorescent protein (RFP) together around the spliced coding region of the brassinosteroid synthesis gene (*DWF4*) allowed the production of tagged splice isoforms, each with a single reporter for simultaneous visualization. Isoform 4.1 (GFP) was detected throughout the root tip transition zone to the proximal region, whereas isoform 4.2 (RFP) was detected mainly in the root meristem. Treatment
with brassinolide, an inhibitor of brassinosteroid synthesis, for 4 h abolished the FP signals from both isoforms (78).

Moreover, single FP reporters were also developed to monitor the activity of *PIN7* isoforms simultaneously, revealing their functional significance in regulating auxin-mediated plant development (77, 78). Such approaches could be applied to stress-responsive targets to visually identify the influence of abiotic and biotic stresses on splice isoform expression, localization, and dynamics at temporal and spatial levels in planta. In another study, an assay was developed to examine the capacity of splicing regulator-translated isoforms in intron splicing. The assay consisted of two coexpressed constructs, one that carried Rluc in addition to the target gene—including an intron flanked by two exons—followed by luciferase and another construct expressing different isoforms of SR45a SF. Intron splicing was assessed by creating constructs carrying introns from three different genes, including *Waxy* (*Wx*), *HSFTF9*, and *SR45a*. The coexpression of the two constructs in different combinations in maize protoplasts showed that different isoforms have different efficiencies in intron splicing. The study also used this system to assess the influence of SREs on splicing efficiency by expressing reporters with mutated SREs (90). Recently, a new dual-color splicing reporter called splicing regulation uncovered by steric hindrance (SpliceRUSH) was created to map SREs in a gene of interest based on a CRISPR-dCas13d high-throughput screening platform. The systems expressed enhanced GFP (eGFP) constitutively, followed by the target region, including exons and introns (*SMN2* exons 6–8), and ended with tdTomato. The binding of the coexpressed dCas13d/guide RNA to the SREs in the region of the reporter system near the gene of interest modulated the splicing of exon 7, allowing the inclusion or exclusion of exon 7, changing the reading frame, and affecting the expression of tdTomato (125).

Recently, a visual splicing reporter based on Ruby (60) was developed (121) **(Figure 3)**. The production of a red color and its intensity can be used as a proxy for splicing. This splicing reporter can be expressed transiently and stably in transgenic plants **(Figure 3)**. Plants expressing this or similar splicing reporters will be highly useful in forward visual genetic screens to identify splicing regulators under normal and diverse stress conditions and also for rapid high-throughput

<figure><img src="image_7.png" /><figcaption>**Figure 3**</figcaption></figure>

A genetically encoded visual splicing reporter. Three genes encode enzymes that convert tyrosine to betalain (Ruby), a visible red color compound. This schematic diagram depicts a *Ruby* construct with an intron and its expression in transient assays and stable lines (121). Abbreviations: 35S promoter, cauliflower mosaic virus 35S promoter; CYP76AD1, a P450 oxygenase; DODA, an L-DOPA 4,5 dioxygenase; T, *Arabidopsis heat shock protein 18.2 T* transcription terminator.
screening of chemical libraries for splicing inhibitors for potential use in treating diseases, as many
human diseases are caused by aberrant splicing (123, 143, 167).

7. ENGINEERING ALTERNATIVE SPLICING FOR TRAIT IMPROVEMENT

Based on the studies described above, the development of stress-resilient crops requires manipu-
lation of the levels of specific splice isoforms, which requires precise modulation of AS. Using the
CRISPR-Cas tools, AS can be regulated at the DNA, RNA, or protein levels (Figure 4).

## 7.1. Alternative Splicing Modulation at the DNA Level

T-DNA insertional mutations are tools used to disrupt gene function by randomly integrating transferred DNA into the genome, facilitating functional gene studies. T-DNA insertional muta-tions and CRISPR-Cas9 mutagenesis systems have been used to edit the genes involved in the splicing process, which can result in global changes in pre-mRNA splicing (Figure 4a). Mutat-ing a particular SF often affects many downstream genes that regulate different processes. Hence, this approach may lead to major defects in splicing, allowing fewer controlled changes that are required for engineering specific traits. For example, RNA-seq analysis comparing the WT to an rs33 loss-of-function rice mutant generated by CRISPR-Cas9 showed a huge influence of RS33 on global gene expression and pre-mRNA splicing in response to abiotic stresses, observing at least 2,058 DEG and 1,116 differentially alternatively spliced (DAS) events in the mutant un-der salt stress and 1,230 DEG and 625 DAS events under cold stress. Notably, the rs33 mutant was hypersensitive to salt and low-temperature stresses (17). Similarly, an RNA-seq analysis com-paring the WT to scr106, a loss-of-function rice mutant generated by CRISPR-Cas9, revealed a major role of the SR-like protein SCR106 in genome-wide gene expression and RNA splicing changes in response to salt stress. At least 5,843 DEGs and 1,043 DAS genes were found in the mutant under salt stress, while defense-related genes were differentially expressed under control conditions. The scr106 was hypersensitive to salt, ABA, and low temperature (4). Knocking out IMMUNOREGULATORY RNA-BINDING-PROTEIN (IRR), an SF with an RRM and an SR-rich region enhanced Arabidopsis and maize immunity. RNA-seq analysis comparing the WT to the irr mutant revealed a role for IRR in gene expression and RNA splicing, with at least 600 DEGs, including upregulated defense- and immunity-related genes, and about 181 DAS events in the mu-tant line, including negative regulators of immunity. IRR induces the splicing of the active variant of a negative regulator of plant immunity, CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28). Under plant elicitor peptide (Pep)-induced dephosphorylation, the interaction of IRR with CPK28 transcripts is abolished, generating the RI CPK28 variant, which encodes an inactive truncated CPK28 protein. The knockout mutant irr plants show increased levels of IR-CPK28, and improved Pep-induced immunity (43). Therefore, the mutation of SFs is likely to be too pleiotropic to be useful in improving agronomic traits, except for knocking out negative regulators of the stress response.

A more precise approach for engineering AS is editing the splice sites where the spliceosomal
components bind to the pre-mRNA (Figure 4a). Creating specific changes at splice sites using
CRISPR systems may disrupt the recruitment of the spliceosome to the pre-mRNA, thereby al-
tering the splicing outcomes. This targeted approach may allow more precise control over gene
expression for engineering traits. For example, the betaine aldehyde dehydrogenase (BADH)
encoded by the rice gene OsBADH2 inhibits the production of the naturally aromatic 2-acetyl-1-
pyrroline (2AP). A badh2 mutant rice plant showed increased 2AP levels and hence was fragrant.
Modulating the pre-mRNA splicing of OsBADH2 via CRISPR-Cas9 resulted in an isoform that
skipped exon 2, which harbored a PTC and thus was destined for degradation, subsequently el-
evating levels of 2AP and producing fragrant grains. The exonic nucleotide preceding the splice
donor site (GT) of intron 2 in *OsBADH2* was targeted for mutagenesis to cause the missplicing
(exon skipping), probably by disrupting accurate spliceosomal recognition (145).

Another interesting case involves eukaryotic initiation factor 4E (eIF4E), a regulator of resistance to cassava brown streak disease caused by the RNA viruses Ugandan cassava brown streak virus (UCBSV), and cassava brown streak virus (CBSV). Members of the eIF4E protein family are exploited by potyviruses for viral movement, genome stability, and/or translation initiation. The

<figure><img src="image_8.png" /></figure>

(Caption appears on following page)
**Figure 4** (*Figure appears on preceding page*)

Modulation of AS at multiple levels. (a) AS modulation at the DNA level can be achieved by using Cas9 to knock out genes encoding SFs or NRs, inducing global expression and transcriptomic changes or inducing a specific isoform, respectively. Also, nCas9 can be fused to a base editor targeted for DNA base conversions to induce differentially spliced isoforms. (b) AS modulation at the RNA level can be achieved by using Cas13 to bind specific isoforms for isoform-targeted degradation. Also, dCas13 can be fused to the SF domain to induce specific splicing events such as exon inclusion or skipping. (c) Modulation of AS at the protein level can be achieved by using Cas proteins fused to modifiers, while attracting RBP to bind SREs impeded in the gRNA sequence. Once the RBP is bound to the gRNA-SRE, the modifier then can induce modifications such as phosphorylation, dephosphorylation, or ubiquitylation. (d) CDE of splicing regulators can prevent splicing inhibitors from binding to them. The binding site encoded in the SF where the inhibitor binds can be mutated using Cas9-mediated mutagenesis (18). Abbreviations: A3′SS, alternative 3' splice site; A5′SS, alternative 5' splice site; AS, alternative splicing; CDE, CRISPR-Cas-directed evolution; CS, constitutive splicing; gRNA, guide RNA; M1, mutant 1; M2, mutant 2; M3, mutant 3; nCas9, Cas9-nickase; NR, negative regulator; P, phosphorylation; PTC, premature termination codon; PTM, posttranslational modification; RBP, RNA-binding protein; RI, retained intron; RRM, RNA recognition motif; SF, splicing factor; sgRNA, single-guide RNA; SRE, splicing regulatory element; SS, splice site; Ub, ubiquitylation; WT, wild type. Figure adapted from images created with BioRender.com.

CRISPR-Cas9 mutagenesis system was used to simultaneously target the exon-intron junction boundary of two eIF4E genes, nCBP-1 and nCBP-2. The *ncbP-1 ncbP-2* lines produced out-of-frame isoforms or isoforms with PTCs. Following inoculation with a CBSV Naliendele isolate, the *ncbP-1 ncbP-2* double mutant showed delayed symptoms and decreased CBSV severity compared to the WT. Additionally, the roots of the *ncbP-1 ncbP-2* lines experienced decreased symptom severity and had less viral RNA in comparison to the WT (55).

Moreover, RNA from *Arabidopsis FLM* undergoes AS in response to temperature. At elevated temperatures, the level of the FLM-β isoform known for floral repression activity decreases, inducing flowering. To determine whether flowering is promoted by the reduction of *FLM-β* expression or by the antagonist activity of the FLM-δ isoform, the CRISPR-Cas9 system was used to target genomic sequences of *FLM*, producing isoforms that resemble the original *FLM-β* and *FLM-δ* isoforms in sequence. Using two single-guide RNAs (sgRNAs), *FLM* exon 2 or 3 was individually targeted for deletion. The edited lines lack either the second exon, in this case producing the *FLM-δ* isoform, or the third exon, producing the *FLM-β* isoform. On one hand, when the *FLM-β* isoform, but not the *FLM-δ* isoform, was induced, the edited lines exhibited delayed flowering. On the other hand, expressing the *FLM-δ* isoform, but not the *FLM-β* isoform, in the edited lines promoted early flowering. However, flowering of the *FLM-δ*-engineered lines was later than in the loss-of-function mutant *flm-3*, an indication that FLM-δ activity in regulating flowering time in Col-0 *Arabidopsis* is less important (22).

In yet another study, the CRISPR-Cas9 system was used to target the intron splicing site in the 5'UTR of alleles of rice Wx, which encodes a granule-bound NDP-glucose-starch glucosyltransferase (172). The quality, in terms of cooking, eating, and appearance, of the rice grain depends on the gel consistency, amylose content, and pasting viscosity of grain starch, which differ according to changes within the Wx alleles. Grains with increased gel consistency, but mid-range amylose content are preferred, as they have improved taste. To enhance the grain quality of the high-yielding rice TianFengB, its Wxa allele was targeted to reduce the amylose content. Different mutants were recovered and confirmed to have mutations at the intron GT splice site, with corresponding reduced mRNA levels and reduced amylose content, yet the 1,000-grain weight was marginally reduced. Rather than editing the splice sites around the coding sequences, which would induce PTCs and cause loss-of-function mutants, editing the intron splicing site at the 5'UTR may hold greater potential for crop improvements, as it allows quantitative regulation of gene expression and, hence, phenotypic variations (172).

Further advances have demonstrated the use of base-editing approaches in regulating AS (Figure 4a). An adenine base editor guided by Cas9-nickase (nCas9) to the targeted sites induced

**Base editing:** A type of CRISPR-Cas system fused to a base-modifying enzyme that can convert one DNA base to another; can be used to induce specific point mutations to change splice sites and control which isoform is expressed
A-to-G conversion at the intron-exon junction of phytoene desaturase 3 (PDS3), resulting in the missplicing of PDS3 with mosaic albino and dwarf *Arabidopsis* mutants (76). A similar approach was used with *Arabidopsis* mRNA adenosine methylase (MTA) and rice *Glossy1-Homologous gene-1* (GL1-1) and *NARROW LEAF1 (NAL1)*, targeting donor GT or acceptor AG sites with a cytosine base editor for C-to-T conversions. This results in the misplacing of these genes, creating dwarf *Arabidopsis* mutants and rice mutants with hydrophilic leaf surfaces or narrow leaves (91). Another study targeted *Arabidopsis HAB1.1* and the *RS31A* intron 5' splice site to produce a G-to-A mutation. The *hab1.1* mutants had an RI event with an included PTC and displayed enhanced sensitivity to ABA, while the *rs31a* mutant expressed only two of the three isoforms normally expressed in the WT. In addition, the *rs31a* mutant displayed reduced sensitivity to genotoxic stress compared to the WT (162).

## 7.2. The Modulation of Specific Splice Isoforms at the RNA Level

Altering cis-regulatory elements in RNA and SRE via CRISPR-mediated RNA editing systems can target specific isoforms for degradation while allowing others to be produced. Different variants of CRISPR-Cas13 (149) could be used to study functional isoforms, for example, by targeting an AS isoform with an RI during certain environmental or developmental conditions (Figure 4b). Moreover, analysis of the regulation of splicing could be further extended by fusing functional domains to dCas13 variants to induce targeted splicing or change splice outcomes (109). For example, the CPK28 RI isoform could be produced while preserving the IRR SF, which may participate in the splicing of other genes important for cellular processes. Unlike DNA editing via CRISPR-Cas9, RNA editing will always require a transgene to be present in the genome, as the T-DNA encodes the editing machinery.

The recently developed CRISPR Artificial SF (CasFx) system leverages an RNA-guided RNA-targeting system fused to regulatory domains of splicing to promote exon inclusion and exclusion. The system can affect two distant targets simultaneously. Additionally, the activity of the system can be induced by small molecules. The activity was also demonstrated on an endogenous target (44). A similar system using plant SF domains could be developed to modulate pre-mRNA splicing of endogenous genes targeted for trait improvements and induce the expression of isoforms that positively regulate desirable traits (Figure 4b). The alternatively produced truncated isoforms *OsWRKY62.2* and *OsWRKY76.2* accumulate in disease-resistant rice lines and improve resistance to pathogen infection by regulating the production of their full-length immunity repressor isoforms (94). Moreover, the shorter isoform of *OsDr11* improves rice resistance against *Xoo*, perhaps by regulating the longer isoform transcriptionally and posttranslationally (45). A recent study revealed for the first time that CRISPR-mediated RNA breaks could be repaired in human cells by excision, providing another window for modulating the RNA, which may potentially translate into therapeutic application. The results were confirmed by removing stop codons designed in single- and dual-reporter systems, in addition to editing endogenous transcripts of human cells (112). Such an approach could be tested in plant cells for various applications, such as the removal of splice sites or SREs for AS modulation, as well as deleting RNA motifs bound by negative regulators of immunity, including pathogen effectors (144).

## 7.3. The Modulation of Alternative Splicing via Targeted Posttranslational
Modification of Splicing Factors

PTM, including phosphorylation and dephosphorylation of SFs, may serve as another layer at which AS can be modulated. However, to induce protein modification, an induction system or a trigger will be needed. This could be an external stimulus such as an environmental factor or a
system expressed conditionally to allow the modification of the desired protein to help study the effect of PTM on pre-mRNA splicing. Here, an RBP of interest could be targeted for PTM using a modified CRISPR-Cas system. A dCas9 or dCas13 could be fused to a modifier, and the 3' region of the sgRNA scaffold could be extended to contain an extra RNA sequence that recognizes the targeted RBP. As the dCas-sgRNA(extra) modifier assembles, the RNA recognition site on the 3' extra region of the sgRNA scaffold is free to attract the targeted RBP, bringing it in close proximity to the modifier for targeted modification (Figure 4c). For example, a report indicated that changes to the phosphorylation status of the *Arabidopsis* SR45 SF influence its mobility and localization (5).

Furthermore, the phosphorylation or dephosphorylation of SR proteins, implemented by specific kinases (188, 190), may be detrimental to their splicing activity (153). SR proteins also undergo ubiquitylation/deubiquitylation (74, 110). Petal development in *Arabidopsis* is influenced by the phosphorylation status at T218 of SR45.1 (180). A recently described mechanism reveals how ABA-mediated dephosphorylation at T264 stabilizes SR45 to maintain plant growth under ABA stress (3). Recently, arginine methylation of the spliceosome component SM-LIKE PROTEIN 4 (LSM4) was shown to be essential for plant growth under abiotic stress (1). Clearly, further studies should be conducted to determine how PTMs of SFs influence stress tolerance. However, the approach proposed above requires proof-of-concept validation and could currently target RBPs with predicted target SRE sequences, such as SR45, SCL33, GRP7, Tra2, and SRP38 (35, 47, 50, 88, 142, 160, 181). Changes to this approach may further enhance the targeting capacity, modifying more than one target protein simultaneously by multiplexing.

Apart from induced modulation of pre-mRNA splicing, nature has found ways of diversifying traits via sequence variations that affect pre-mRNA splicing. This may have created a pool of traits induced by DNA changes yet to be discovered and could be incorporated into our cultivars via overexpression or recreated by gene editing. The MADS-box TF 1 (OsMADS1), encoded by the *Oryza sativa japonica* rice quantitative trait locus (QTL) OsLG3B regulates grain length and weight. Natural variation in OsLG3B has created several single-nucleotide polymorphisms (SNPs) (exonic and intronic), subsequently inducing A3'SS of intron 7 and resulting in a shorter exon 8. This event produced a PTC-containing isoform encoding a truncated protein that is associated with longer grain, as validated from different tropical *japonica* varieties. For further validation, the truncated isoform was expressed in a short-grain variety, increasing the grain length in the transgenic plants (169). Another example illustrates how variations at splice sites affect the splicing outcome and, subsequently, disease resistance. Maize ZmBGLU17 encodes a β-glucosidase 17 that is involved in the biosynthesis of defense compounds such as lignin and DIMBOA. Genetic analysis revealed that inbred lines ZmBGLU17GEMS10 and ZmBGLU17TY4 differ in the base at the fourth position of the fifth intron. An A-to-T mutation in ZmBGLU17TY4 produces three splice variants, with nonfunctional variants being predominant, and is more susceptible to Pythium aphanidermatum, a causal agent of corn stalk rot, compared to ZmBGLU17GEMS10, which encodes a single functional variant (93).

## 7.4. The Directed Evolution of Splicing Regulators to Confer New Traits

Ensuring efficient pre-mRNA splicing can also be achieved by protecting spliceosomal components from splicing inhibitors. A CRISPR-Cas-directed evolution (CDE) system was utilized for the directed evolution of the spliceosomal protein SF3B1 to resist herbicides (18) (Figure 4d). Therefore, modulation of splicing to improve plant traits could be achieved by using CRISPR-Cas systems to knock out or knock down SFs or negative regulators of stress tolerance and immunity. Moreover, the CRISPR-Cas systems could be utilized for splice isoform-targeted degradation,
editing of splice sites and SRE, induced posttranslational modifications, and protein-directed evolution. Additionally, overexpressing natural isoforms may confer desired traits.

## 8. CONCLUSIONS

AS helps plants adapt to ever-changing environments. The intricate regulation of gene expression through AS allows plants to produce and diversify the transcriptome outputs, enhancing their ability to respond to unfavorable conditions. Numerous studies in recent years have shown that alteration in isoform ratios and/or generation of novel isoforms plays a crucial role in regulating many developmental processes and conferring tolerance to diverse stresses. The dynamic nature of AS underscores its significance in plant stress biology, offering insights into how plants maintain homeostasis and resilience under adverse conditions. Advances in our understanding of AS under different environmental and developmental cues have increased. However, a deeper understanding of the molecular interplay between stress and splicing regulation is necessary to breed and engineer plants resistant to climate-changing conditions. Technical advances have been made to engineer, report, quantify, assess, and phenotype the consequences of AS events in plants. These technologies will enhance our understanding of the molecular processes that plants employ to adapt to environmental changes and will enable us to engineer plants to resist climate change conditions, thereby improving food security.

Future research should focus on unraveling the molecular mechanisms governing AS regulation and its interaction with other posttranscriptional and posttranslational modifications. Understanding the crosstalk between AS and other regulatory pathways will provide a holistic view of adaptive plant stress responses and facilitate the development of crops with improved stress tolerance. Advances in high-throughput sequencing; computational biology; and gene, epigenome, and transcriptome editing will enable the identification of novel AS events and their functional implications. Ultimately, leveraging the knowledge of AS regulation in plants holds the potential to utilize technological advances to engineer and breed crops that enhance agricultural productivity and sustainability in the face of global climate challenges and food security concerns.

## SUMMARY POINTS

1. Increasing global population and escalating environmental challenges are compounding threats to food security, emphasizing the urgent need for resilient agricultural systems.

2. Plants are subjected to diverse environmental stresses, including abiotic factors such as salinity, drought, and temperature extremes, as well as biotic threats such as pathogens, which significantly impair growth, development, and productivity.

3. Plants have evolved intricate cellular mechanisms, such as alternative splicing, to dynamically regulate gene expression and respond effectively to environmental cues.

4. Alternative splicing enhances transcriptome and proteome diversity by generating multiple messenger RNA (mRNA) isoforms from a single gene, each potentially contributing unique functional roles critical for stress adaptation.

5. Alternative splicing is a prominent regulatory mechanism under stress conditions, such as salinity, cold, and drought, highlighting its central role in plant resilience to environmental challenges.
6. Recent innovations have led to the development of advanced tools to visualize precursor mRNA (pre-mRNA) splicing in real time, opening new avenues for identifying splicing regulatory proteins, discovering novel splicing inhibitors, studying splicing dynamics, and teaching the intricacies of the central dogma in an engaging way.

7. The advent of CRISPR-Cas technologies offers precise tools to modulate alternative splicing outcomes, paving the way for novel applications in plant biotechnology and crop improvement.

8. Manipulating alternative splicing pathways presents a transformative strategy for developing crop varieties with enhanced stress tolerance and improved agronomic traits.

## DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.

## ACKNOWLEDGMENTS

This research was supported by KAUST baseline funding awarded to M.M. Splicing research in the A.S.N.R. laboratory was supported by the National Science Foundation (MCB-2014542). We thank the members of the Genome Engineering and Synthetic Biology Laboratory at KAUST for their invaluable feedback and critical review of the manuscript.

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