Research

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.

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Rice blast is one of the main diseases of rice, causing severe economic losses to agricultural production; thus, the search for blast resistance is a top priority for rice breeding. When challenged by the blast causal fungus Magnaporthe oryzae the expression level of OsMAPKKK69 gene in rice cultivar Nipponbar was found to increase significantly. Such an induction was also found in a different genetic material, cultivar Shufanggaonuo, indicating that OsMAPKKK69 plays an important role in blast disease response. However, the function of OsMAPKKK69 remains unclear. In this study, wild type ZH11 was selected as the background material to investigate the expression and functions of OsMAPKKK69 in rice disease resistance by constructing knockout mutants. The results showed that OsMAPKKK69 is mainly expressed in four-week-old shoots and localized in cell membrane, cytoplasm, and nucleus. The two allelic knockout mutants, osmapkkk69-1 and osmapkkk69-2, were more resistant to M. oryzae and bacterial blight Xanthomonas oryzae pv. Oryzae (Xoo). Further agronomic trait analysis revealed that the osmapkkk69-1 and osmapkkk69-2 mutants had reduced plant height, smaller grain size, a significant increase in tillering number, but also a significant increase in yield per plant. Our results show that OsMAPKKK69 is involved in the immune response of rice by negatively regulating the resistance to rice blast and blight diseases, and in regulating important agronomic traits. This study lays a foundation for revealing the molecular mechanism of OsMAPKKK69 in the immune response to rice diseases and provides novel genetic resources for rice breeding.

Alternative polyadenylation (APA) generates transcript diversity by producing mRNA isoforms with distinct 3' ends. Despite the critical roles that APA plays in various biological processes, the mechanisms regulating APA in response to stresses have remained poorly understood in plants. Here, we perform comprehensive analysis of APA in tomato, and focus on a phosphate (Pi)- regulated APA gene SlSPX5, encoding a putative Pi sensor protein. SlSPX5 interacts with and sequesters the transcription factor SlPHL1 in the cytosol, thereby inhibiting the expression of Pi starvation inducible genes. We discover that a cis-natural antisense RNA (cis-NAT) is activated from SlSPX5 to promote its proximal polyadenylation under Pi-depleted conditions. The transcription of this cis-NAT induces RNA Polymerase II pausing, generating Ser2 phosphorylation signals that recruit polyadenylation machinery to the 5' end of SlSPX5. Our findings demonstrate that a cis-NAT regulates APA of its cognate gene in response to Pi starvation.