Publications

Serine/arginine-rich 45 (SR45) is a plant-specific SR-like splicing factor and an RNA-binding protein involved in Arabidopsis development and stress responses. Although SR45 has been primarily studied for its role in pre-mRNA alternative splicing (AS), whether it also regulates alternative polyadenylation (APA) remains unclear. Here, we combined poly(A) tag sequencing (PAT-seq) and RNA-seq to examine APA, AS, and gene expression in wild-type (WT) and sr45-1 plants across inflorescence, leaf, and root under normal conditions, and in leaf and root under prolonged mild salinity. Under normal conditions, SR45 loss caused widespread changes in poly(A) site usage, with distal site shifts occurring more frequently than proximal shifts across tissues. APA, AS, and differential gene expression showed only partial overlap, with significant enrichment between APA and AS but not with gene expression changes, indicating that APA represents a distinct layer of SR45-mediated transcriptome regulation. Under prolonged mild salinity conditions, pairwise analyses showed preference of distal poly(A) site usage in WT and proximal site usage in sr45-1, particularly in leaf. Replicate-aware genotype-by-treatment interaction analyses further identified genotype-dependent differences in APA and gene expression, where more genotype-treatment interacting genes detected in leaf than in root. Genes encoding polyadenylation and splicing factors also showed changes in APA, AS, or transcript abundance. Together, these results expand the known role of SR45 from splicing regulation to APA-associated transcriptome remodeling and reveal that SR45 is required for proper salt-responsive changes in transcript abundance in Arabidopsis.

Transcriptome plasticity is a key driver of proteome diversity, enabling organisms to finely tune gene expression in response to developmental cues and environmental stimuli, essential features for sessile plants facing constant challenges. Among the co-transcriptional processes contributing to this plasticity-such as alternative transcription start sites and alternative splicing-alternative polyadenylation (APA) plays a particularly prominent role, affecting 60%-70% of plant genes. Despite its prevalence, the molecular mechanisms guiding APA and its regulation remain incompletely understood. This review synthesizes recent advances in our understanding of how polyadenylation protein complexes-including Cleavage and Polyadenylation Specificity Factors (CPSF), Cleavage stimulation Factors (CstF), and Cleavage Factors (CF)-interact with cis-regulatory elements to determine poly(A) site choice. We highlight the coordinated actions of these protein families and their dynamic regulation across tissues, developmental stages, and environmental conditions. Furthermore, we propose a conceptual model of the regulatory network involving RNA-protein and protein-protein interactions that govern APA in plants. Deciphering these molecular networks offers promising avenues for targeted manipulation of APA to enhance crop resilience and productivity. Understanding the spatial and temporal regulation of polyadenylation machinery may ultimately enable precision control of gene expression in molecular breeding and agricultural biotechnology.

Alternative polyadenylation (APA) is a widespread post-transcriptional regulation that generates transcripts with variable 3' untranslated region (UTR) lengths. In this study, poly(A)-tag sequencing (PAT-seq) was performed during the process of Magnaporthe oryzae infection of rice. Genome-wide dynamic changes of APA profiles were identified during the response, with widespread shortening of gene transcripts. Shortened genes were found to function in biological processes associated with stress responses. Importantly, the changes of 3' UTR length were negatively related to the expression levels of the corresponding genes. In addition, we found that the expression levels of miRNAs that bind to the 3' UTRs of the APA genes were negatively correlated with the expression levels of the corresponding APA genes. This correlation suggests a potential regulatory strategy where immune-related genes might evade miRNA-mediated repression via 3' UTR shortening. To assess the biological impact of these APA dynamics, we functionally characterised a representative candidate, Os05g0509500, that exhibits complex and dynamic APA site switching upon infection. Knockout of Os05g0509500 via CRISPR/Cas9 revealed its positive regulatory role in rice blast, offering initial insights into the function of APA in this disease. Several core polyadenylation protein factors were significantly differentially expressed during the rice response. Our results revealed that the precise transcriptome-wide poly(A) site selection of genes during rice blast challenge, and the relationships between miRNAs and targeted APA genes, are tightly regulated. Such a mechanism provides a new perspective for designing novel strategies to control rice blast disease.

Alternative polyadenylation (APA) serves as a critical co-transcriptional regulatory mechanism that shapes mRNA fate and protein function. POLY(A) POLYMERASE 1 (PAPS1) contributes to poly(A) tail synthesis; however, its relationship to poly(A) site choice is less well characterized. Here, we profiled PAPS1-associated poly(A) site usage across Arabidopsis (Arabidopsis thaliana) tissues using UMI-quantified poly(A) tag sequencing (qPAT-seq). Across tissues, paps1-4 showed tissue-dependent shifts in poly(A) site usage, with pronounced effects in pollen. In the paps1-4 mutant, polyadenylated transcripts were biased toward longer isoforms with significantly longer 3' UTRs, and poly(A) signal usage was altered in distinct ways: a U-to-A shift immediately downstream of poly(A) sites occurred in leaves and flower buds, whereas pollen showed more complex motif combinations with reduced usage of canonical cis-elements. In pollen, DE-APAGs included multiple genes previously linked to a pollen developmental regulatory module involving AT-RICH INTERACTING DOMAIN-CONTAINING PROTEIN 1 (ARID1), RETINOBLASTOMA RELATED 1 (RBR1), and DUO POLLEN1 (DUO1), showing coordinated changes in APA and expression. Flowering-time-related terms were also enriched among genes with significantly altered APA in paps1-4, including regulators connected to FLOWERING LOCUS C (FLC) and photoperiod responses, and these changes were more consistent with poly(A) site selection differences than with widespread poly(A) tail length changes. Notably, multiple poly(A) factors in pollen switched between single- and multi-poly(A) site usage patterns in paps1-4, representing a major component of PAPS1-associated regulation. Together, these findings reveal tissue-specific APA alterations associated with PAPS1 and highlight pollen and flowering pathways as key contexts of its regulatory influence.

Extreme environments impose conditions that are lethal to most organisms, yet a limited number of evolutionary lineages have adapted to chronic stress. However, whether distinct lineages exposed to the similar stressors rely on shared adaptive mechanisms remains poorly understood. Mangroves offer a compelling system to address this question, as they thrive in shared harsh environments. Here, we examine two mangrove species, Kandelia obovata and Avicennia marina, both inhabiting intertidal zones but deploying contrasting strategies to withstand hypoxia. Phylogenomic analyses revealed that K. obovata experienced an ancestral whole-genome duplication (WGD) prior to intertidal colonization, whereas A. marina underwent two rounds of WGD that expanded its repertoire of hypoxia-responsive genes. Comparative genomic analyses showed that K. obovata underwent contraction of hypoxia-related gene families and harbours fewer gene copies, but each copy is enriched with cis-regulatory elements. In contrast, A. marina retained significantly more hypoxia-related genes derived from recent WGD. Transcriptomic profiling under controlled hypoxia gradients further showed that K. obovata relies on rapid but stable gene expression responses, while A. marina exhibits delayed but hierarchical gene regulatory networks. Together, these results demonstrate that even under identical extreme selective pressures, plant lineages can follow distinct evolutionary routes, shaped by differences in genome evolution and regulatory architecture.

Alternative polyadenylation (APA) is a widespread co-transcriptional mechanism that regulates gene expression in growth, development and environmental responses. Pollen development is essential for the reproductive success of flowering plants, yet the contribution of APA to this process remains poorly understood. Here, we combine bulk RNA-seq in multiple tissues with single-nucleus transcriptomics across pollen developmental stages to systematically characterize APA dynamics during Arabidopsis thaliana pollen development. We show that mature pollen exhibits the most tissue-specific APA profile among the examined tissues, characterized by widespread 3' untranslated region (3' UTR) shortening. At single-nucleus resolution, APA patterns display pronounced temporal and cell-type specificity, particularly during the transition from bicellular to tricellular pollen and during vegetative nucleus maturation. Sperm nuclei exhibit the most distinct poly(A) site usage patterns. Moreover, genetic analyses of representative genes showed that altered poly(A) site usage is associated with changes in transcript abundance and pollen development phenotypes. Consistent with these observations, in vivo reporter assays showed that 3' UTR configurations are sufficient to modulate gene expression at the transcript level. Together, our study establishes APA as a structured co-transcriptional regulatory layer during pollen development and provides a framework for understanding 3' end-mediated gene regulation in male gametophytes.

Rice blast, caused by Magnaporthe oryzae, severely threatens global rice production. Although the receptor-like cytoplasmic kinase OsRLCK118 positively regulates rice immunity, its downstream signaling mechanism remains unknown. To systematically identify OsRLCK118-interacting proteins, we performed immunoprecipitation-mass spectrometry (IP-MS) and a yeast library screen, yielding 781 and 287 candidates, respectively, with 35 overlapping hits. Among these, OsSAMS1, a known positive regulator of blast resistance, was selected for validation. Membrane yeast two-hybrid, split-luciferase complementation, and co-immunoprecipitation assays confirmed the physical interaction between OsRLCK118 and OsSAMS1. Furthermore, in vitro kinase assays showed that OsRLCK118 specifically phosphorylates OsSAMS1. These results uncover a novel signaling axis connecting pathogen recognition to ethylene biosynthesis via OsRLCK118-dependent phosphorylation of OsSAMS1, providing both mechanistic insight into rice immunity and potential genetic targets for resistance breeding.

Comparing closely related species with differing abiotic stress tolerances can reveal physiological and genetic mechanisms that underlie environmental adaptations. Mangroves, found primarily in tropical and subtropical coasts, are expanding poleward due to rising minimum temperatures, for which species-specific cold tolerance likely influences expansion limits. In the Indo-West Pacific, Kandelia obovata tolerates chilling temperatures below 10°C, while its sister species, Kandelia candel, is highly sensitive to chilling and restricted to tropical regions. The discrepancy in chilling tolerance offers unique materials for understanding chilling tolerance mechanisms. This study compares chilling-induced changes in photochemical efficiency, enzymatic activity and gene expression between the two species to reveal their differences in photoprotective strategies. We specifically focus on the impact of chilling on the light reactions of photosynthesis as one of the primary impacts of chilling on plant species is chilling-induced photoinhibition. Despite maintaining higher photochemical efficiency, K. obovata exhibited greater reactive oxygen species (ROS) levels and lower antioxidant enzyme activity than K. candel during chilling at 10°C. Furthermore, interspecies comparison of the expression of genes involved in photoprotection during chilling stress revealed differential expression of PGR5 and CHL, which regulate cyclic electron flow and non-photochemical quenching. The upregulation of these genes in K. obovata and their downregulation in K. candel suggest that differences in photoprotective responses may contribute to the contrasting chilling tolerances of these species. These findings highlight a potential mechanism that contributes to the cold tolerance and biogeographical distributions of subtropical and tropical plant species, emphasizing the need for further comparative studies across a broader range of taxa.

Error in Figure [...].