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Commentary

When splicing meets transgene
DNA methylation to uncover elite
alleles for plant yield

For decades, the enigma of why plant varieties demonstrate virus
recovery or display unstable and variegated traits remained
unresolved. Approximately 40 years ago, with the characterization
of expression patterns in transgenic plants, scientists began to
uncover the epigenetic intricacies behind these phenomena.
Research on transgenic plants, particularly those failing to express
introduced sequences as expected, revealed novel phenomena
where transgene loci undergo transcriptional gene silencing (TGS)
or post-transcriptional gene silencing (PTGS), even acting on
homologous endogenous genes. These processes were then linked
to either innate epigenetic defenses, naturally regulating transpo-
sable elements, duplicated genes, or viruses. They are essentially
triggered by double-stranded RNA (dsRNA) molecules, which are
converted to small interfering RNA (siRNA), the key molecules
triggering RNA degradation and/or DNA methylation (Vau-
cheret, 2022). The mechanism by which organisms detect nonself
DNA or RNA and target them for gene silencing remains
enigmatic. Nevertheless, there is a consensus that transgene-derived
aberrant RNAs initiate PTGS. First, mutations in the RNA quality
control (RQC) pathway, which degrades nonfunctional RNA,
enhance PTGS. Second, uncapped and deadenylated RNAs were
shown to be preferential substrates for RNA-dependent RNA
polymerases (RDRs), key enzymes in the generation of cellular
dsRNA. Finally, TGS in plants is linked with DNA methylation
and the inhibition of transcription through particular siRNAs,
determining initial silencing sites that can be spread out of these loci
(RNA-dependent DNA methylation (RdDM) or RdDM-linked
siRNAs). The initial indication of this form of suppression came
from observations in plants, where both transgene and viral RNAs
were found to direct DNA methylation toward particular
nucleotide sequences (Park et al., 1996). Various genetic screens
have played a crucial role in pinpointing components involved in
gene silencing. The utilization of transgenic lines that exhibit
spontaneous transgene silencing has led to the identification of
numerous mutants with impaired silencing capabilities. Alterna-
tively, mutagenesis studies carried out on nonsilenced transgenic
lines have uncovered mutations that enhance silencing. They
encompass various mutations in proteins not directly involved in
the RNA silencing machinery, such as DCLs, AGOs, or other
mRNA degradation enzymes, but revealed elements such as
splicing factors and histone-modifying epigenetic regulators. In

an article published in this issue of *New Phytologist*, Zhao *et al.* (2024; 1724-1741) explored the role of a previously uncharacterized RNA splicing factor in transcriptional transgene silencing and DNA demethylation.

‘Together, these studies highlight the importance of alternative splicing in controlling epigenetic regulations leading to phenotypic variation and contributing to plant adaptation and domestication.’

In a genetic screen looking for mutations suppressing the phenotype of the overexpression of the EIN2 ethylene receptor 35S:EIN2, the team discovered a suppressor mutant (Suppressor of Overexpression of EIN2 or SOE) that inhibited transgene expression in *trans*. This study serves as compelling evidence of the importance of not overlooking unexpected outcomes, as they often pave the way for significant discoveries. Indeed, it turned out that the gene responsible for the suppressor phenotype was not involved in ethylene signaling as the authors expected. Instead, using an elegant combination of genetic and biochemical approaches, the team demonstrated the role of a mutation in a previously uncharacterized RNA recognition motif (RRM) on the DNA methylation of viral promoters from overexpressing transgenes. Further phenotypic characterization of this SOE mutant revealed that this precise mutation in *SOE* enhances grain size and yield.

In the last few years, a number of genetic screens have identified an interplay between RNA processing and transgene silencing, both transcriptional and post-transcriptional. However, the splicing factors in RNA silencing mode of action were much less established. Recently, a screen for PTGS suppressors using post-transcriptionally silenced transgenes identified two splicing factors PRP39 and SmD1b that act synergistically in promoting PTGS in Arabidopsis. This study suggested that PRP39a and SmD1b, apart from their individual functions in splicing regulation, restrict the degradation of transgene-derived abnormal RNAs in the nucleus. This action promotes the export of abnormal RNAs to the cytoplasm, where they undergo conversion into double-stranded RNA (dsRNA), thereby initiating PTGS.

The first report showing the involvement of a spliceosome component in siRNA accumulation and RdDM in Arabidopsis came from a genetic screen looking for mutants impaired in the *de novo* methylation of the FWA transgene (Ausin *et al.*, 2012). Within this study, SR45, an RRM-containing protein previously recognized as a genuine plant-specific splicing factor, was identified. The SR45 mutant not only exhibits compromised

<footnote>This article is a Commentary on Zhao *et al.* (2024), **243**: 1724–1741.</footnote>
de novo DNA methylation capabilities at the FWA transgene but also displays reduced non-CG methylation at various endogenous RdDM targets (RdDM). Additionally, this mutant shows diminished accumulation of siRNAs associated with RdDM. Consequently, the SR45 splicing factor seems required for RdDM and likely functions within this pathway preceding siRNA production. However, no clear molecular link between the function of SR45 in splicing and its role in mediating DNA methylation could be established. The work from Zhao *et al.* elucidates this link showing that SOE, another SR protein closely related to SR45 and affecting alternative splicing in planta, binds to the mRNA of the DNA glycosylase gene DNG701 to facilitate its splicing and increases its stability. This leads to a modification of DNG701-mediated DNA demethylation and consequently trans-gene expression. Interestingly, DNG701 is the direct ortholog of Repressor Of gene Silencing 1 (ROS1) a key protein that counteracts RdDM action in Arabidopsis (Gong *et al.*, 2002). Therefore, incorrect splicing of ROS1 or other components of the fine-tuning of RdDM seems to be an emerging mechanism through which conserved splicing regulators can modulate RdDM and transgene silencing in plants.

Alternative splicing (AS) is one of the major sources of transcriptome and proteome diversity of higher organisms and has a strong impact on plant phenotypic diversity. Since AS largely contributes to proteome diversity and gene expression regulation, considerable evolutionary changes may be mediated by modifications in this process. Indeed, in mammals, a number of population-wide studies have demonstrated the prominent role of alternative splicing variations in morphological diversity, organ development, disease susceptibility, and immune adaptation, among others (Singh & Ahi, 2022).

However, little is known about its contribution to plant adaptation and domestication. In their work, Zhao *et al.* found that *SOE* mutants exhibit beneficial agronomic traits such as increased grain size and greater yield per plant. Analyzing the haplotypes of SOE from *c*. 3000 rice varieties, they discovered a haplotype in the SOE promoter that correlates with a higher weight of 1000 grains, suggesting this variant may have been selected and fixed in *japonica* accessions during domestication.

Interestingly, in maize, a population-wide transcriptome analysis revealed a substantial splicing divergence between inbred lines and identified 739 variants associated with changes in splicing of the encoded mRNA that colocalized with previous marker–trait associations (Chen *et al.*, 2018). They included DNA variants associated in cis or trans with alternative splicing variation in the population. The cis-variants may be related to the DNG701 splicing event regulated by SOE. Interestingly, a number of trans-acting variants were located within splicing regulatory protein genes. As an example, a strong association was found between a

mutation in ZmGRP1, encoding an hnRNP-like glycine-rich RNA binding protein, and the differential splicing of a cluster of genes in the maize population. Knockout of ZmGRP1 by CRISPR/Cas9 validated the role of ZmGRP1 in altering the splicing of numerous predicted downstream genes.

Together, these studies highlight the importance of AS in controlling epigenetic regulations leading to phenotypic variation and contributing to plant adaptation and domestication. This strongly suggests that genetic determinants involved in the splicing regulation of specific genes during stress responses may represent a new reservoir of breeding targets to make better cultivars. This could include more stress-resilient plants able to cope with both short- and long-term environmental changes.

ORCID

J. Bazin https://orcid.org/0000-0001-7713-5278

M. Crespi https://orcid.org/0000-0002-5698-9482

J. Bazin and M. Crespi*

Institute of Plant Sciences Paris-Saclay (IPS2), CNRS, INRAE, Universités Paris-Saclay, Evry and Paris-Cité, Bâtiment 630, Gif sur Yvette, 91190, France
(*Author for correspondence: email: martin.crespi@universite-paris-saclay.fr)

References

Ausin I, Greenberg MVC, Li CF, Jacobsen SE. 2012. The splicing factor SR45 affects the RNA-directed DNA methylation pathway in Arabidopsis. *Epigenetics* 7: 29–33.

Chen Q, Han Y, Liu H, Wang X, Sun J, Zhao B, Li W, Tian J, Liang Y, Yan J *et al.* 2018. Genome-wide association analyses reveal the importance of alternative splicing in diversifying gene function and regulating phenotypic variation in maize. *Plant Cell* 30: 1404–1423.

Gong Z, Morales-Ruiz T, Ariza RR, Roldán-Arjona T, David L, Zhu J-K. 2002. ROS1, a repressor of transcriptional gene silencing in Arabidopsis, encodes a DNA glycosylase/lyase. *Cell* 111: 803–814.

Park YD, Papp I, Moscone EA, Iglesias VA, Vaucheret H, Matzke AJ, Matzke MA. 1996. Gene silencing mediated by promoter homology occurs at the level of transcription and results in meiotically heritable alterations in methylation and gene activity. *The Plant Journal* 9: 183–194.

Singh P, Ahi EP. 2022. The importance of alternative splicing in adaptive evolution. *Molecular Ecology* 31: 1928–1938.

Vaucheret H. 2022. Epigenetic management of self and non-self: lessons from 40 years of transgenic plants. *Comptes Rendus. Biologies* 345: 149–174.

Zhao R, Wu W-A, Huang Y-H, Li X-K, Han J-Q, Jiao W, Su Y-N, Zhao H, Zhou Y, Cao W-Q *et al.* 2024. An RRM domain protein SOE suppresses transgene silencing in rice. *New Phytologist* 243: 1724–1741.

**Key words:** alternative splicing, DNA methylation, domestication, RNA binding proteins, transgene silencing.