sic-4 Reports in sick! Loss of SICKLE induces salicylic acid-dependent cell death in Arabidopsis

Received April 03, 2023. Accepted April 13, 2023. Advance access publication April 18, 2023 Open Access © The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. N ew s an d V ie w s

Programmed cell death (PCD) is a central defense strategy in plants that allows the plant to eliminate stress-damaged tissues and limit the systemic spread of pathogens. PCD is regulated by a complex signaling network of cell deathpromoting signals and counteracting negative regulators. Salicylic acid (SA) is a central hormonal regulator that together with reactive oxygen species (ROS) and calcium signaling drives defense-associated PCD (Van Aken and Van Breusegem 2015; Koster et al. 2022). A variety of gene regulation mechanisms, ranging from transcription to the posttranslational modification of proteins, control the process of PCD (Van Aken and Van Breusegem 2015; Huysmans et al. 2017).
SICKLE is a plant-specific single-copy gene that encodes a proline-rich protein (Zhan et al. 2012). Arabidopsis mutants deficient in SICKLE, namely sic mutants, show pleiotropic abnormalities involving alterations in plant development and hypersensitivity to abiotic stresses (Zhan et al. 2012;Marshall et al. 2016;Wu et al. 2022). Previous studies have shown that SICKLE contributes to RNA metabolism by affecting microRNA biogenesis and alternative splicing (Zhan et al. 2012;Marshall et al. 2016). Recently, Wu et al. (2022) proposed a mechanism whereby SICKLE interacts with RNA debranching enzyme 1 (DBR1) to promote the degradation of lariat intronic RNAs (lariRNAs). LariRNAs are byproducts of mRNA splicing that are either degraded or processed to form small regulatory RNAs. Thus, lariRNAs play important regulatory roles in eukaryotic cells as precursors for microRNAs, small interfering RNAs, and small nucleolar RNAs (Neil and Fairbrother 2019). Moreover, lariRNAs function as decoys for RNA-binding proteins and thus modulate mRNA stability (Li et al. 2016). Taken together, lariRNAs are emerging as important post-transcriptional regulators of gene expression (Li et al. 2016;Neil and Fairbrother 2019;Wu et al. 2022).
In this issue of Plant Physiology, Wu et al. (2023) demonstrate that loss of SICKLE affects mRNA metabolism and induces SA-dependent PCD in Arabidopsis leaves. Using histochemical staining by 3,3′-diaminobenzidine, an indicator of ROS levels, and trypan blue, an indicator of cell death, Wu et al. (2023) show that the Arabidopsis sic-4 mutant exhibits accumulation of ROS and local cell death in leaves. Moreover, the observed cell death is accompanied by changes in the expression of SA-associated genes (Wu et al. 2023). Interestingly, these hallmarks of PCD are abolished in the DBR1-OE/sic-4 mutant that overexpresses the DBR1 enzyme that catalyzes lariRNA degradation. Thus, the authors conclude that the PCD in sic-4 is caused by accumulation of lariRNAs (Wu et al. 2022(Wu et al. , 2023. By analyzing differentially expressed genes, Wu et al (2023) discovered that transcripts associated with defense and SA homeostasis and signaling are upregulated in sic-4 mutants. Interestingly, further analysis revealed that the alternatively spliced genes in sic-4 were largely different from the differentially expressed genes (Wu et al. 2023). The observed differences in the splicing of transcripts associated with PCD suggest that alternative splicing may be involved in the activation of PCD in the sic-4 mutant (Wu et al. 2023). Based on these results, Wu et al. (2023) suspected that the accumulation of lariRNAs might regulate mRNA stability by disturbing their interactions with RNA-binding proteins (Li et al. 2016). Taking advantage of the transcriptional inhibitor cordycepin and RNA sequencing, the authors analyzed the decay of mRNAs in wild-type and sic-4 mutant plants. Faster decay of transcripts characterized as negative regulators of cell death and SA levels was observed in the sic-4 mutant (Wu et al. 2023). Therefore, Wu et al. (2023) suggest that the cell death in sic-4 results from alternative splicing and changes in the mRNA decay.
Finally, Wu et al. (2023) show that SA signaling is involved in the PCD observed in the sic-4 mutant. The authors measured higher SA content and transcriptional activation of SA-responsive genes in the sic-4 mutant compared with wildtype plants (Wu et al. 2023). In line with this, the cell death phenotype of sic-4 was suppressed in sic-4/sid2-1, sic-4/npr1-1, and sic-4/pad4-1 double mutants that lack the SA biosynthesis enzyme SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2) and SA signaling activators ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and PHYTOALEXIN DEFICIENT 4 (PAD4), respectively (Wu et al. 2023). The authors conclude that SA signaling functions downstream of changes in alternative splicing and mRNA decay in the sic-4 mutant (Wu et al. 2023).
Plant stress responses and developmental processes have been extensively studied at the transcriptional level. However, our knowledge of the post-transcriptional regulation that shapes the transcriptome is deficient. Wu et al. (2022Wu et al. ( , 2023 have identified SICKLE as a post-transcriptional regulator that controls alternative splicing and mRNA stability via its interaction with DBR1. This research adds to our understanding of the factors regulating RNA metabolism in plants. In addition, the research by Wu et al. (2023) highlights the complexity of the regulatory network governing PCD in plants and introduces a novel mechanism, lariRNA hyperaccumulation, that triggers PCD (Fig. 1). Intriguingly, the authors hypothesize that PCD could be activated by a thus-far-unidentified nucleotide-binding site leucine-rich repeat protein that functions upstream of the EDS1-PAD4 signaling module and monitors the lariRNAs in plants (Wu et al. 2023). In the future, it will be interesting to study how SICKLE contributes to plant resistance to pathogen infection and if lariRNA-triggered PCD is a widespread defense mechanism in plants.

Funding
MR is financially supported by the Academy of Finland (project 349969).