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Review

Pre-mRNA Splicing Functions in Plant Sexual Reproduction Development

by
Dongjie Shao
1,
Xinqi Gao
2,* and
Yiming Wei
1,*
1
College of Life Sciences, Zaozhuang University, Zaozhuang 277160, China
2
State Key Laboratory of Wheat Improvement, College of Life Sciences, Shandong Agricultural University, Taian 271018, China
*
Authors to whom correspondence should be addressed.
Plants 2025, 14(10), 1472; https://doi.org/10.3390/plants14101472
Submission received: 9 April 2025 / Revised: 11 May 2025 / Accepted: 12 May 2025 / Published: 14 May 2025
(This article belongs to the Special Issue Plant Reproduction and Embryonic Development)
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Abstract

:
Precursor messenger RNA (pre-mRNA) splicing is a critical post-transcriptional regulatory mechanism in gene expression. The precise splicing of pre-mRNAs is essential for plant development and responding to genetic and environmental signals. In plant sexual reproduction, gene expression regulation relies on the accurate processing of pre-mRNAs, which is fundamental for coordinating developmental programs. The alternation of generations in plants involves two key phases: gametophyte development, which produces gametes, and fertilization, which leads to the formation of a diploid sporophyte. Gametophyte and embryo development represent essential processes in plant sexual reproduction. This review focuses on summarizing and analyzing the current evidence regarding the role of pre-mRNA splicing in plant sexual reproduction, with an emphasis on its involvement in gametophyte formation and embryo development. Future challenges in understanding RNA splicing regulation in plant sexual reproduction are also discussed, particularly in modulating splicing factor levels and activities and identifying target mRNAs and non-coding RNAs regulated by these factors. This review provides crucial insights into the molecular mechanisms of plant reproductive development and offers a theoretical basis for improving plant fertility and adaptability via RNA splicing regulation.

1. Introduction

Higher plants undergo two distinct multicellular stages in their life cycle: the haploid gametophyte (pollen and embryo sac) and the diploid sporophyte (embryo and seedling). Gametophytes generate gametes, and their fusion gives rise to the diploid sporophyte, marking two critical phases in the alternation of generations. Gene expression regulation plays a key role in gametophyte formation and embryo development. Precursor messenger RNA (pre-mRNA) splicing is a post-transcriptional regulatory mechanism that involves the removal of introns and the joining of exons to produce mature mRNAs for translation. Since plant growth and development rely on precise gene expression, accurate pre-mRNA processing is essential for maintaining normal cellular functions and responding to genetic and environmental signals [1,2,3,4,5,6]. Studies have shown that pre-mRNA splicing influences plant sexual reproduction. It is noteworthy that many genes involved in plant pre-mRNA splicing are essential genes, and their homozygous mutations often result in embryonic lethality, while defects in heterozygous mutants are particularly pronounced in the haploid gametophyte and early embryogenesis. Therefore, the haploid generation and early embryogenesis stage are ideal for analyzing the role of pre-mRNA splicing in plant sexual reproduction. This review summarizes the current findings on the role of pre-mRNA splicing in gametophyte formation and embryo development and discusses future challenges in understanding its regulatory impact on plant reproduction.

2. Pre-mRNA Splicing

Introns are non-coding sequences within genes that are removed from precursor RNA during splicing. Based on their splicing mechanisms, introns are classified into four major groups: self-splicing group I and group II introns, tRNA and/or archaeal introns, and spliceosome-catalyzed introns [7,8,9,10]. The excision of group I introns occurs through a two-step transesterification reaction that requires exogenous guanosine (exoG) as a cofactor (Figure 1A). On the other hand, group II introns undergo splicing via a different mechanism, forming a lariat structure during intron removal (Figure 1B). The removal of introns from tRNA involves splicing endonuclease and ligase, which process the precursor to generate a functional tRNA (Figure 1C). Spliceosomal introns in nuclear pre-mRNA are excised by the spliceosome, a large ribonucleoprotein complex [11], and their removal follows a mechanism similar to that of group II introns (Figure 1D).
Studies in metazoans have shown that the spliceosome can be classified into two types based on the introns they process: the U2-type (major) spliceosome and the U12-type (minor) spliceosome [12]. The major spliceosome comprises five small nuclear ribonucleoproteins (snRNPs)—U1, U2, U4/U6, and U5—along with various non-snRNP factors. Spliceosome-mediated splicing is a complex and dynamic process involving the sequential assembly and disassembly of different snRNPs [13]. This process can be broadly divided into two phases, spliceosome assembly and catalysis, ultimately leading to the formation of a mature transcript (Figure 2). During spliceosome assembly, the process begins with U1 snRNP binding to the 5′ splice site through RNA/RNA base pairing. Simultaneously, splicing factor 1 (SF1) and U2 auxiliary factor (U2AF) recognize and bind to the branch point sequence (BPS) and the polypyrimidine tract (PPT), respectively. U2AF consists of two subunits: U2AF65, which binds the PPT, and U2AF35, which associates with the 3′ splice site. This initial assembly forms the E (early) complex. Next, complex A is established as U2 snRNP binds to the BPS. The U4/U5/U6 tri-snRNP then joins, leading to the formation of complex B. The later dissociation of U1 and U4 triggers structural rearrangements, resulting in an activated complex (Bact), which then transitions into the catalytically active B* complex. This final complex facilitates intron lariat removal and exon ligation [14]. Furthermore, various non-snRNP protein factors contribute to the splicing process, including the NineTeen Complex (NTC), NTC-associated proteins, serine/arginine (SR)-rich proteins, the REtention and Splicing (RES) complex, and ATPases [15].

3. Role of Splicing in Pollen Development

Alternative splicing increases functional complexity in plants by producing multiple transcript variants from a single gene, thus improving protein diversity [16,17,18,19]. Emerging evidence indicates that alternative splicing isoforms play a role in male gametophyte development (Figure 3, Table 1). AUXIN RESPONSE FACTOR 8 (ARF8) produces multiple functionally distinct transcripts through alternative splicing, including ARF8.1, ARF8.2, and ARF8.4, each contributing to different stages and processes of pollen development. The full-length isoform, ARF8.1, regulates pollen cell wall formation by directly controlling the expression of key transcription factors—DEFECTIVE IN TAPETAL DEVELOPMENT (TDF1), ABORTED MICROSPORE (AMS), and MALE STERILITY 188 (MS188)—in tapetum cells, which are essential for post-meiotic pollen and tapetum development [20]. Anther dehiscence, a crucial step for pollen release and pollination, can be prematurely triggered by the combined action of ARF8.2 and ARF8.4. ARF8.2 facilitates anther dehiscence by modulating jasmonic acid (JA) biosynthesis through the regulation of DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1). Meanwhile, ARF8.4 accelerates anther dehiscence by regulating MYB26, which influences anther wall lignification. The ARF8.4 promotes filament elongation by controlling the expression of INDOLE-3-ACETIC ACID INDUCIBLE19 (AUX/IAA19), ensuring optimal pollen positioning for successful pollination [21,22]. Lily 135-ABP is a villin superfamily protein that plays a role in actin cytoskeleton rearrangement. Overexpression of ABP29, a splicing variant of 135-ABP, in lily pollen significantly disrupts actin filament elongation and depolymerization, leading to inhibited pollen germination and pollen tube growth [23]. In Brassica juncea var. tumida, T1170 and T1243 are alternative transcripts of the mitochondrial T gene, which is associated with cytoplasmic male sterility. While these isoforms display distinct expression patterns, only T1243 contributes to the male sterility phenotype [24]. In Arabidopsis thaliana, the Jasmonate ZIM-domain 10 (JAZ10) protein functions as a repressor of jasmonate signaling and contains both a Jas domain and a ZIM domain [25]. JAZ10.4 is a splice variant of JAZ10 that encodes a protein lacking the Jas domain. Transgenic plants overexpressing JAZ10.4 show strong JA insensitivity and male sterility due to impaired filament elongation and anther dehiscence [26]. Reactive oxygen species (ROS) signaling plays a crucial role in plant reproduction [27,28]. In Arabidopsis, the respiratory-burst oxidase homolog E (RBOHE) is a tapetum-specific NADPH oxidase involved in ROS production, essential for tapetal function and pollen development. RBOHE has three splice variants, RBOHE.1–3. Overexpression of the RBOHE.3 splice variant disrupts tapetal ROS homeostasis, accelerates tapetal programmed cell death (PCD), and leads to male sterility [29].
Numerous studies have highlighted the critical role of splicing factors in regulating male gametophyte development (Figure 3, Table 1). U2AF65, a component of U2AF, is essential for recruiting the U2 snRNP complex. It plays a key role in defining 3′ splice sites and regulating alternative splicing [30]. In Arabidopsis, two isoforms of U2AF65, U2AF65a and U2AF65b, have been identified. AtU2AF65b is involved in the splicing of FLOWERING LOCUS C and ABSCISIC ACID-INSENSITIVE 5, influencing flowering [31]. The atu2af65a atu2af65b double mutants show reduced male transmission and defective pollen tube growth [32]. However, the AtU2AF65a/b-spliced genes essential for male gametophyte function and pollen tube growth remain unidentified. Furthermore, a mutation in GAMETOPHYTIC FACTOR 1 (GFA1), which encodes a U5 snRNP-associated protein, disrupts pollen tube growth toward the female gametophyte (FG) [33]. The target genes of GFA1 that regulate pollen tube growth, however, remain unknown.
Table 1. RNA splicing factors involved in male gamete formation.
Table 1. RNA splicing factors involved in male gamete formation.
SpeciesBiological ProcessType of Splicing FactorSplicing FactorTargeted GenesReferences
Arabidopsis thalianaMale gametophyte transmission, pollen tube growthNon-snRNP, one subunit of U2AFU2AF65 Unknown [32]
A. thalianaMale gametophyte transmissionDEAH-box RNA-dependent ATPase Prp16CUVGenes involved in auxin biosynthesis, polar auxin transport, and auxin perception and signaling[34]
A. thalianaPollen tube growthU5 snRNP componentGFA1Unknown[33,35,36]
A. thalianaPollen wall formationCyclin-dependent protein kinases (SR protein)CDKG1CalS5[37]
A. thalianaMale fertility at modestly ET (elevated temperature)A major component of the UPR signaling pathwayIRE1bZIP60[38]
Lilium longiflorumPollen germinationSR proteinLlSR28AtVLN1[39]
A. thalianaPollen germinationSR proteinAtSR45AtVLN1[39]
A. thalianaPollen germination, pollen tube growthSR proteinatRSZ33Unknown[40]
A. thalianaPollen development A U2 snRNP-associated protein AtSAP130QRT1 and QRT3[41]
A. thalianaMale gametophyte transmissionA U4/U6 snRNP associated proteinRDM16Unknown[42]
A. thalianaMale gametophyte developmentU5 snRNP component PRP8A, PRP8B Unknown [43]
CYCLIN-DEPENDENT KINASE G1 (CDKG1), an SR motif-containing protein, is recruited to U1 snRNP through its interaction with an arginine/serine-rich splicing factor, facilitating the efficient splicing of Callose synthase 5 (CalS5) [37]. CalS5 encodes the primary enzyme responsible for synthesizing the callose layer during microspore development [44]. As a result, the Arabidopsis CDKG1 loss-of-function mutant shows impaired male fertility, characterized by defective pollen wall formation at the tetrad stage [37]. Further studies have established the role of CDKG1 in meiosis, particularly under high-temperature conditions, where it is essential for homologous chromosome pairing and recombination during male meiosis. Specifically, CDKG1, in complex with CYCLINL, maintains chromosome pairing stability and crossover events, ensuring proper meiotic progression. While cdkg1 mutants display normal vegetative growth, they become sterile under high temperatures [45]. The findings of a recent study indicate that the long isoform of CDKG1 (CDKG1L) is required for fertility maintenance under high-temperature conditions, whereas the short isoform (CDKG1S) cannot rescue fertility in the cdkg1 mutant [46]. This suggests that the CDKG1 function is determined not only by its kinase activity but also by the expression and role of its splice isoforms.
Arabidopsis SR45 (AtSR45) is an SR-rich protein that regulates the alternative splicing of multiple SR gene mRNAs [47]. In the atsr45 mutant, the full-length transcript of VILLIN1 increases, while the short transcript decreases, resulting in reduced F-actin levels in hydrated pollen [39]. The lily LlSR28 shares structural similarities with AtSR45. In atsr45 mutants, pollen germination occurs earlier than in the wild type, whereas LlSR28 overexpression in Arabidopsis completely inhibits pollen germination. These findings suggest that LlSR28 and AtSR45 regulate filamentous actin dynamics by modulating the alternative splicing of VILLIN1, therefore influencing pollen germination [39]. Similarly, Arabidopsis RSZ33 (AtRSZ33) is an SR protein involved in the splicing of AtSRp30 and AtSRp34/SR1. The overexpression of AtRSZ33 leads to impaired pollen germination [40]. However, the specific target genes regulated by AtRSZ33 remain unidentified.

4. Splicing and Female Gametogenesis

Splicing factors also play a crucial role in the normal development of female gametophytes. Mutations in spliceosome-associated genes frequently lead to defects in female gametophyte development (Figure 4, Table 2). For example, GFA1 directly interacts with two U5 snRNP components, AtBRR2 and AtPRP8, as well as the RNA helicase ROOT INITIATION DEFECTIVE 1 (RID1), a homolog of the yeast splicing factor Prp22 in Arabidopsis [33,35,36]. GFA1 and RID1 are essential for embryo sac development, as they participate in the pre-mRNA splicing of key genes required for female gametophyte formation, including EMBRYO SAC DEVELOPMENT ARREST 26 and 4, FOLYLPOLYGLUTAMATE SYNTHETASE ISOFORM 3, and GAST1 PROTEIN HOMOLOG 4 [36,48]. ATROPOS (ATO), a homolog of the yeast pre-mRNA processing factor 9 (PRP9), is a splicing factor associated with U2 snRNP-binding pre-mRNA. Mutation in ATO leads to fertility defects, resulting in the production of supernumerary egg cells during female gametophyte development in Arabidopsis [49]. LACHESIS (LIS) encodes an Arabidopsis homolog of yeast PRP4, an RNA splicing factor with seven WD40 repeats essential for early spliceosome assembly. In lis mutants, female gametophytes show impaired pollen tube attraction, and many accessory cells differentiate into extra egg cells [50,51]. CWC15 is a potential component of the NTC, and its loss of function leads to widespread alterations in splice sites, primarily characterized by increased intron retention. The cwc15-2 T-DNA insertion mutant displays defective double fertilization, mainly due to significantly reduced female gametophyte transmission efficiency [52]. Arabidopsis RRC1 encodes a protein similar to the human splicing factor SR140, which is involved in splicing regulation. The null allele rrc1-4 results in developmental defects in the female gametophyte; however, the specific target genes responsible for this phenotype remain unknown [53].
Many splicing factors are constitutively expressed in plants, and their mutations often lead to defects in the development and function of male and female reproductive organs. Arabidopsis SPLICEOSOME-ASSOCIATED PROTEIN 130 (AtSAP130) is a subunit of splicing factor 3b (SF3b), which is essential for pre-spliceosome assembly and pre-mRNA splicing [54]. The RNAi-mediated knockdown of AtSAP130 results in functional impairments in female reproductive organs and disrupts pollen transition from the microspore to the bicellular stage. AtSAP130 is likely involved in the splicing of QRT1 and QRT3, genes associated with post-tetrad pollen development [41,55]. Arabidopsis RDM16 is a U4/U6 snRNP-associated component essential for pre-mRNA splicing. Loss-of-function mutants of RDM16 show increased intron retention events, reduced viability in both male and female gametophytes, and decreased transmission efficiency through gametes [42]. PRP8 is a core component of the snRNP complex, contributing to the formation of the larger catalytically active spliceosomal B complex [56]. The Arabidopsis genome encodes two copies of PRP8, PRP8A and PRP8B, which are involved in the splicing of spliceosome factors and key functional genes required for embryo sac development, including EMBRYO SAC DEVELOPMENT ARREST 9 (EAD9), EAD30, EAD35, and MATERNAL EFFECT EMBRYO ARREST 29 (MEE29). In the prp8a prp8b double mutant, ovules fail to attract pollen tubes, and pollen tube perception of ovular attraction signals is impaired [43].

5. Splicing-Regulated Embryo Development

Research has demonstrated that post-transcriptional RNA processing plays a crucial role in embryo development, with mutations affecting splicing leading to abnormal embryogenesis. This highlights the necessity of accurate pre-mRNA splicing for the proper transmission of genetic information during normal plant development (Table 3). Arabidopsis DEBRANCHING ENZYME 1 (AtDBR1) encodes an RNA lariat debranching enzyme, which facilitates the excision of intron lariats during splicing. The mutation of AtDBR1 causes embryo development to arrest at an early stage [57]. AtBUD13 encodes an Arabidopsis homolog of yeast Bud13, a subunit of the RES complex. Mutations in AtBUD13 result in early embryonic lethality due to the retention of shorter introns in genes essential for early embryo development [58]. In addition to AtBUD13, GROWTH, DEVELOPMENT AND SPLICING 1 (GDS1), and DAWDLE (DDL) are key components of the RES complex in Arabidopsis. Mutations in GDS1 and DDL disrupt the proper splicing of multiple genes involved in cell proliferation and early embryogenesis, leading to developmental defects [59]. Maize ROUGH ENDOSPERM3 encodes a cofactor of the U2AF35-related protein, influencing alternative splicing and playing a role in embryo and endosperm development as well as their interaction [60]. In Arabidopsis, the core splicing factor REPLICATION TERMINATION FACTOR 2 (AtRTF2) is a RING finger-containing protein essential for genome-wide pre-mRNA splicing. Loss of AtRTF2 function leads to increased intron retention and embryo abortion [61]. LEFKOTHEA (LEFKO), a nuclear-encoded protein, regulates both nuclear and chloroplast pre-mRNA splicing and is crucial for embryogenesis. A LEFKO knockout mutation results in embryo lethality due to splicing defects in nuclear and chloroplast gene mRNAs [62]. The direct target genes of LEFKO involved in embryo development regulation remain unidentified. In Arabidopsis, AtPRP17, a homolog of the yeast splicing factor PRP17, plays a vital role in embryonic pattern formation. The embryonic lethality observed in atprp17 mutants is likely due to its involvement in the splicing of genes associated with embryo development [63]. Similarly, JANUS, a homolog of the conserved U2 snRNP assembly factor in yeast and humans, is essential for embryonic pattern formation [64,65]. Arabidopsis SM-like (LSM) proteins form two distinct heptameric complexes: LSM1-7 in the cytoplasm and LSM2-8 in the nucleus. The LSM2-8 complex selectively regulates the splicing of specific embryogenesis-related genes, such as EMBRYO DEFECTIVE 2785 (EMB2785) and EMBRYO DEFECTIVE 2016 (EMB2016), by stabilizing U6 snRNA. Splicing defects in lsm8 mutants have been linked to shorter siliques, reduced seed numbers, and seed abortion [66]. SmD3, a core component of the snRNP complex required for pre-mRNA splicing, is encoded by two genes in Arabidopsis (SmD3-a and SmD3-b). The smd3-b mutant represents pleiotropic defects, including 10% ovule abortion, while the inability to generate smd3-a/smd3-b double mutants suggests that SmD3 is essential for embryogenesis. However, the specific target genes responsible for this phenotype remain unidentified [67].
In higher plants, the splicing of introns in organelle genes relies on a large family of pentatricopeptide repeat (PPR) proteins [78]. These proteins contain 2–30 tandem repeats of a degenerate 31- to 36-amino acid motif, which plays a role in post-transcriptional regulation, including RNA splicing in plant organelles [79]. PPR proteins are classified into two types: the P-type, which contains PPR (P) motifs, and the PLS-type, which includes P motifs along with longer (L) or shorter (S) variants. A subset of P-type PPR proteins has been identified as essential for the splicing of group II introns in organelle genes [80]. MITOCHONDRION-MEDIATED GROWTH DEFECT 1 (MID1) is a P-type PPR protein in Arabidopsis that facilitates the splicing of intron 1 in the mitochondrial NADH dehydrogenase 2 gene by interacting with MITOCHONDRIAL INTRON SF1. Loss-of-function mutations in MID1 result in arrested embryogenesis [73]. Several P-type PPR proteins in Arabidopsis, maize, and rice, including DEFECTIVE KERNELS and EMPTY PERICARPS, are involved in the intron splicing of NADH dehydrogenase genes encoding subunits of the mitochondrial respiratory chain complex. Mutations in these proteins often lead to mitochondrial dysfunction, adversely affecting embryo and pollen development [69,70,71,72,74,75,76,77,81,82].
Embryo development begins with egg cell fertilization; therefore, defects in female gametophyte development often compromise embryogenesis. For instance, a partial loss-of-function mutation in GFA1 not only disrupts female gametophyte development but also arrests embryo development before the octant stage [35]. In the cwc15-2 mutant, impaired female gametophyte function significantly reduces fertilization success. Even when fertilization occurs, abnormal gene expression in cwc15-2 leads to early embryonic arrest, resulting in an embryonic lethal phenotype [52]. Similarly, the rrc1-4 mutant shows reduced female gametophyte transmission efficiency while maintaining normal male transmission. The low frequency of rrc1-4 homozygotes in progeny from self-fertilized rrc1-4 heterozygotes suggests additional defects in embryogenesis following fertilization [53]. CLUMSY VEIN (CUV) encodes an Arabidopsis ortholog of yeast Prp16, an RNA splicing factor involved in the second-step transesterification reaction during splicing. CUV regulates the splicing of genes associated with auxin biosynthesis, transport, perception, and signaling. Phenotypic analysis of cuv mutants revealed developmental defects typically linked to auxin, including altered leaf venation, defective gynoecium and stamen development, and arrested embryogenesis [34]. As previously mentioned, the prp8a prp8b double mutant disrupts embryo sac attraction for the pollen tube [43], while the prp8a mutant alone is embryo-lethal [61,68].

6. Conclusions and Future Prospects

This review explores the critical role of pre-mRNA splicing in plant reproductive development, summarizing the functional diversity of various transcripts during reproductive processes and highlighting the essential roles of splicing factors in both female and male gametophytes, as well as embryonic development. These studies demonstrate that the precise regulation of splicing is crucial for the proper progression of plant reproductive development. As a model organism, Arabidopsis has laid a crucial foundation for studying pre-mRNA splicing regulation in plant reproductive development. From an evolutionary perspective, the core mechanisms of pre-mRNA splicing, such as the basic composition of the spliceosome, the removal of introns, and splice site recognition, are relatively conserved in higher plants [19,83]. Nevertheless, pre-mRNA splicing regulation networks may vary among different plant species, and this diversity could enable plants to adapt to different environmental conditions and influence their reproductive strategies [19]. Therefore, future research should focus on investigating the role of pre-mRNA splicing in plant reproductive development across more species, exploring potential species-specific pre-mRNA splicing regulatory networks to provide a more comprehensive understanding of plant reproductive development.
Beyond the classic splicing factors, an increasing number of studies show that non-splicing factors also significantly impact plant reproductive development by influencing splicing. For example, Arabidopsis INOSITOL-REQUIRING ENZYME 1 (IRE1), a dual-function protein kinase/ribonuclease [84,85], plays a vital role in the splicing of bZIP60 mRNA at elevated temperatures, which results in reduced male fertility due to interference with pollen coat deposition and normal pollen grain dispersal. Furthermore, the overexpression of SEC31A, a gene transcriptionally activated by bZIP60, rescues the temperature-sensitive sterility in ire1a ire1b mutants [38]. SHOOT REDIFFERENTIATION DEFECTIVE 2 (SRD2), which encodes a nuclear protein homologous to human SNAP50 that activates snRNA transcription, also influences splicing. Loss of SRD2 function reduces snRNA levels, impairing splicing and causing reproductive defects, such as failed pollen tube attraction and embryo arrest before the globular stage [86,87,88].
In conclusion, the precise regulation of pre-mRNA splicing, involving both classical and non-classical splicing factors, is essential for plant reproductive development, with these factors influencing splicing through various mechanisms. RNA splicing regulation serves as a crucial mechanism for post-transcriptional gene expression control in plants, offering a faster response to developmental signals compared to the slower processes of transcriptional activation and pre-mRNA accumulation. Many splicing factors play a role in the splicing of key genes involved in plant gametophyte (Table 1 and Table 2) and embryo development (Table 3). However, the mechanisms by which developmental signals—such as hormones and environmental cues—regulate the levels and activities of these splicing factors remain unclear. Investigating how splicing factor levels and activities are modulated by these signals could provide valuable insights into the regulation of splicing during gametophyte and embryo development in plants. Studies have suggested that hormones, epigenetic modifications, and environmental signals influence mRNA splicing [89,90,91,92,93,94,95], but the specific regulation of splicing factors in plant gametophyte and embryo development is yet to be fully explored. Another challenge is identifying the direct mRNA targets of splicing factors involved in these processes. Recent advances in techniques can aid in this task. For example, RNA immunoprecipitation sequencing (RIP-seq) can detect the mRNA targets of splicing factors with RNA-recognition motifs, although it does not reveal binding site information. On the other hand, cross-linking immunoprecipitation (CLIP) coupled with high-throughput sequencing and its variants—individual nucleotide resolution CLIP (iCLIP) and photoactivatable-ribonucleoside-enhanced CLIP (PAR-CLIP)—not only identify the mRNA targets of splicing factors but also provide insights into their binding sites on target mRNAs [96]. Non-coding RNAs (ncRNAs), including microRNA (miRNA), small interfering RNA (siRNA), and long non-coding RNA (lncRNA), play a key role in regulating gene expression both at the transcriptional and post-transcriptional levels [97,98,99]. Multiple studies have shown that ncRNAs are direct targets of splicing factors [100,101,102,103]. Moreover, ncRNAs are involved in plant sexual reproduction and plants’ responses to environmental changes [104,105,106,107,108,109,110,111,112]. Identifying the ncRNA targets of splicing factors could improve our understanding of the role of splicing in plant gametophyte and embryo development.

Author Contributions

Conceptualization, Y.W. and X.G.; writing—original draft preparation, D.S.; writing—review and editing, Y.W., X.G. and D.S.; visualization, D.S.; supervision, Y.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Natural Science Foundation of Shandong (grant number ZR2024QC152 and ZR2023QC162) and National Natural Science Foundation of China (32370343 and 31970190).

Conflicts of Interest

The authors declare no conflicts of interest.

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Plants 14 01472 g001
Figure 1. Schematic representation of four pre-mRNA splicing mechanisms. (A) In group I introns, splicing is initiated when a cofactor attacks the 5′ splice site (5′ ss) and attaches to the intron, releasing the upstream exon. The 3′ end of the released exon then attacks the 3′ splice site (3′ ss), resulting in exon ligation and intron release. (B) Group II introns undergo a similar process, where the 2′-OH of adenosine at the branch site attacks the 5′ ss, leading to cleavage of the 5′ exon and formation of a lariat-3′exon intermediate. The 3′-OH of the cleaved 5′ exon then attacks the 3′ ss, producing ligated exons and a lariat intron. (C) tRNA introns are removed by a splicing endonuclease, generating exons with 2′,3′-cyclic phosphate and 5′-OH ends. The exon halves fold into a tRNA-like structure, which is then sealed by a ligase. (D) Spliceosome introns follow a two-step transesterification reaction akin to group II introns, but the process is catalyzed by the spliceosome complex.
Figure 1. Schematic representation of four pre-mRNA splicing mechanisms. (A) In group I introns, splicing is initiated when a cofactor attacks the 5′ splice site (5′ ss) and attaches to the intron, releasing the upstream exon. The 3′ end of the released exon then attacks the 3′ splice site (3′ ss), resulting in exon ligation and intron release. (B) Group II introns undergo a similar process, where the 2′-OH of adenosine at the branch site attacks the 5′ ss, leading to cleavage of the 5′ exon and formation of a lariat-3′exon intermediate. The 3′-OH of the cleaved 5′ exon then attacks the 3′ ss, producing ligated exons and a lariat intron. (C) tRNA introns are removed by a splicing endonuclease, generating exons with 2′,3′-cyclic phosphate and 5′-OH ends. The exon halves fold into a tRNA-like structure, which is then sealed by a ligase. (D) Spliceosome introns follow a two-step transesterification reaction akin to group II introns, but the process is catalyzed by the spliceosome complex.
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Figure 2. A basic model of the RNA splicing mechanism. The schematic diagram represents the spliceosome assembly and catalysis during transcript maturation. The process begins with U1 snRNP binding to the 5′ splice site of the intron in the pre-mRNA, forming complex E, while U2 snRNP binds to the branch site to form complex A. The U4/U5/U6 tri-snRNP then joins complex A to form the B complex. After the dissociation of U1 and U4, the spliceosome undergoes rearrangements, resulting in the activated Bact complex. The catalytically active B* complex is formed, facilitating the two transesterification reactions that remove the intron lariat and ligate the two exons. E: early complex, the early spliceosome. A: A complex, pre-spliceosome. B: B complex, precatalytic spliceosome. B*: B*complex, catalytically activated spliceosome. C: C complex, catalytic step I spliceosome. C*: C* complex, step Ⅱ catalytically activated spliceosome. P: P complex, post-splicing complex. ILS: intron-lariat spliceosome.
Figure 2. A basic model of the RNA splicing mechanism. The schematic diagram represents the spliceosome assembly and catalysis during transcript maturation. The process begins with U1 snRNP binding to the 5′ splice site of the intron in the pre-mRNA, forming complex E, while U2 snRNP binds to the branch site to form complex A. The U4/U5/U6 tri-snRNP then joins complex A to form the B complex. After the dissociation of U1 and U4, the spliceosome undergoes rearrangements, resulting in the activated Bact complex. The catalytically active B* complex is formed, facilitating the two transesterification reactions that remove the intron lariat and ligate the two exons. E: early complex, the early spliceosome. A: A complex, pre-spliceosome. B: B complex, precatalytic spliceosome. B*: B*complex, catalytically activated spliceosome. C: C complex, catalytic step I spliceosome. C*: C* complex, step Ⅱ catalytically activated spliceosome. P: P complex, post-splicing complex. ILS: intron-lariat spliceosome.
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Figure 3. A role of splicing factors in pollen development, germination, and tube growth in Arabidopsis. Schematic overview of pollen development, germination, and tube growth in Arabidopsis, emphasizing splicing factors crucial for these processes. Pollen development begins within the anther locules, where microsporocytes undergo two meiotic divisions, producing tetrads of four haploid microspores enclosed by callosic cell walls. The tetrads separate as callose is degraded, releasing individual microspores, which then enlarge, polarize, and undergo asymmetric division to form a large vegetative cell and a small generative cell. The generative cell then divides once more to produce two sperm cells, resulting in mature tricellular pollen. Upon germination, pollen grains extend pollen tubes for fertilization. The genes labeled in the figure indicate their functional stages, with red for splicing factors and blue for splicing-related genes.
Figure 3. A role of splicing factors in pollen development, germination, and tube growth in Arabidopsis. Schematic overview of pollen development, germination, and tube growth in Arabidopsis, emphasizing splicing factors crucial for these processes. Pollen development begins within the anther locules, where microsporocytes undergo two meiotic divisions, producing tetrads of four haploid microspores enclosed by callosic cell walls. The tetrads separate as callose is degraded, releasing individual microspores, which then enlarge, polarize, and undergo asymmetric division to form a large vegetative cell and a small generative cell. The generative cell then divides once more to produce two sperm cells, resulting in mature tricellular pollen. Upon germination, pollen grains extend pollen tubes for fertilization. The genes labeled in the figure indicate their functional stages, with red for splicing factors and blue for splicing-related genes.
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Figure 4. Arabidopsis female gametophyte (FG) development highlighting splicing-related factors essential for FG formation. The figure shows Arabidopsis female gametophyte (FG) development, highlighting splicing-related factors involved in this process. Arabidopsis FG development is categorized into eight stages based on morphological changes. The archesporial cell differentiates into the megaspore mother cell (MMC), which undergoes meiosis to produce a tetrad of haploid megaspores, of which only one survives as the functional megaspore (FM) at stage FG1. The FM undergoes three rounds of mitosis, forming an eight-nucleate coenocyte with four nuclei at each pole separated by a central vacuole. By late FG5, the female gametophyte develops into a seven-celled, eight-nucleate embryo sac with unfused polar nuclei, which fuse at FG6. At FG7, three antipodal cells degenerate, forming a four-celled female gametophyte. At FG8, one synergid cell degrades, resulting in a three-celled female gametophyte. Red-labeled genes represent splicing factors that play roles in different stages of FG development.
Figure 4. Arabidopsis female gametophyte (FG) development highlighting splicing-related factors essential for FG formation. The figure shows Arabidopsis female gametophyte (FG) development, highlighting splicing-related factors involved in this process. Arabidopsis FG development is categorized into eight stages based on morphological changes. The archesporial cell differentiates into the megaspore mother cell (MMC), which undergoes meiosis to produce a tetrad of haploid megaspores, of which only one survives as the functional megaspore (FM) at stage FG1. The FM undergoes three rounds of mitosis, forming an eight-nucleate coenocyte with four nuclei at each pole separated by a central vacuole. By late FG5, the female gametophyte develops into a seven-celled, eight-nucleate embryo sac with unfused polar nuclei, which fuse at FG6. At FG7, three antipodal cells degenerate, forming a four-celled female gametophyte. At FG8, one synergid cell degrades, resulting in a three-celled female gametophyte. Red-labeled genes represent splicing factors that play roles in different stages of FG development.
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Table 2. RNA splicing factors involved in female gamete formation.
Table 2. RNA splicing factors involved in female gamete formation.
SpeciesBiological ProcessType of Splicing FactorSplicing FactorTargeted GenesReferences
A. thalianaFemale gametophyte development DEAH-box RNA-dependent ATPase Prp22 RID1EDA26, EDA4, FGPS3, GASA4[36,48]
A. thalianaMegagametogenesisU5 snRNP componentGFA1EDA26, EDA4, FGPS3, GASA4[33,35,36,49]
A. thalianaFemale gametophyte developmentU2 snRNP-associated proteinATOUnknown[49]
A. thalianaSupernumerary egg cells, female gametophytes developmentU4/U6 snRNP-associated proteinLISUnknown[50,51]
A. thalianaFemale gametophyte development A U2 snRNP-associated protein AtSAP130Unknown[41]
A. thalianaFemale gametophyte transmissionA U4/U6 snRNP associated proteinRDM16Unknown[42]
A. thalianaA functional redundancy between PRP8A and PRP8B in female gametophyte development U5 snRNP componentPRP8A; PRP8BEDA9, EDA30, EDA35, MEE29[43]
A. thalianaFemale gametophyte transmissionPotential component of the NTC CWC15 Unknown[52]
A. thalianaFemale gametophyte transmissionRS domain (SR-like) protein RRC1 Unknown[53]
Table 3. RNA splicing factors involved in embryo development.
Table 3. RNA splicing factors involved in embryo development.
SpeciesBiological ProcessType of Splicing FactorSplicing FactorTargeted GenesReferences
Zea maysKernel developmentRNA lariat debranching enzymeAtDBR1Unknown[57]
A. thalianaEmbryogenesis Nucleus-encoded RNA-binding protein LEFKO Unknown [62]
A. thalianaEarly embryogenesisOne composition of the RES complex AtBUD13Early embryo developmental genes, e.g., ZYG1/APC11, PFI and ATML1[58]
A. thalianaEarly embryogenesisOne composition of the RES complexDDLGenes associated with cell proliferation and early embryo development
e.g., AGO10/ZLL, ATML1		[59]
A. thalianaEarly embryogenesisOne composition of the RES complexGDS1Genes associated with cell proliferation and early embryo development
e.g., AGO10/ZLL, ATML1		[59]
A. thalianaEmbryogenesisU5 snRNP component PRP8A Unknown [61,68]
Z. maysKernel developmentU2AF35-related proteinRgh3Unknown[60]
Z. maysKernel developmentMitochondrion-targeted P-type PRP proteinDEK43/DEK41Mitochondrial nad4 introns 1 and 3[69,70]
Z. maysKernel developmentMitochondria-targeted P-type PPR proteinEMP8nad1 intron 4, nad4 intron 1 and nad2 intron 1 [71]
Z. maysKernel developmentMitochondria-targeted P-type PPR proteinDEK35Mitochondrial nad4 intron 1[72]
A. thalianaEmbryogenesisMitochondria-targeted P-type PPR proteinMID1Mitochondrial nad2 intron 1[73]
A. thalianaSeed developmentMitochondria-targeted P-type PPR proteinOTP43Mitochondrial nad1 Intron 1[74]
A. thalianaSeed developmentMitochondria-targeted P-type PPR proteinSLO3Mitochondrial nad7 Intron 2[75]
A. thalianaEmbryogenesisRtf2-domain splicing-related proteinAtRTF2Unknown[61]
Oryza sativaGrain developmentMitochondrion-targeted P-type PRP proteinRL1Mitochondrial nad4 intron 1[76]
O. sativaGrain developmentNucleolus-localized PPR proteinFLO14/OsNPPR3nad 1–2 and nad 2[77]
A. thalianaEmbryogenesisDEAH-box RNA-dependent ATPase Prp16 CUVGenes involved in auxin biosynthesis, polar auxin transport, and auxin perception and signaling[34]
A. thalianaEmbryogenesisU5 snRNP component GFA1Unknown[35]
A. thalianaEmbryogenesisSR proteinAtRSZ33Unknown[40]
A. thalianaEmbryogenesisA homolog of the yeast splicing factor PRP17AtPRP17Unknown[63]
A. thalianaEmbryonic pattern formationU2 snRNP assembly factor JANUSUnknown[64,65]
A. thalianaEmbryogenesisU6 SnRNP component (SM-like protein)LSMGenes involved in embryo development
e.g., EMB2785, EMB2016		[66]
A. thalianaEmbryogenesisRS domain (SR-like) proteinRRC1Unknown[53]
A. thalianaEmbryogenesisSnRNP core protein SmD3Unknown[67]
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Shao, D.; Gao, X.; Wei, Y. Pre-mRNA Splicing Functions in Plant Sexual Reproduction Development. Plants 2025, 14, 1472. https://doi.org/10.3390/plants14101472

AMA Style

Shao D, Gao X, Wei Y. Pre-mRNA Splicing Functions in Plant Sexual Reproduction Development. Plants. 2025; 14(10):1472. https://doi.org/10.3390/plants14101472

Chicago/Turabian Style

Shao, Dongjie, Xinqi Gao, and Yiming Wei. 2025. "Pre-mRNA Splicing Functions in Plant Sexual Reproduction Development" Plants 14, no. 10: 1472. https://doi.org/10.3390/plants14101472

APA Style

Shao, D., Gao, X., & Wei, Y. (2025). Pre-mRNA Splicing Functions in Plant Sexual Reproduction Development. Plants, 14(10), 1472. https://doi.org/10.3390/plants14101472

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