Ammonium is a prominent source of inorganic nitrogen for plant nutrition, but excessive amounts can be toxic for many species. However, most conifers are tolerant to ammonium, a relevant physiological feature of this ancient evolutionary lineage. For a better understanding of the molecular basis of this trait, ammonium‐induced changes in the transcriptome of maritime pine (Pinus pinaster Ait.) root apex have been determined by laser capture microdissection and RNA sequencing. Ammonium promoted changes in the transcriptional profiles of multiple transcription factors, such as SHORT‐ROOT, and phytohormone‐related transcripts, such as ACO, involved in the development of the root meristem. Nano‐PALDI‐MSI and transcriptomic analyses showed that the distributions of IAA and CKs were altered in the root apex in response to ammonium nutrition. Taken together, the data suggest that this early response is involved in the increased lateral root branching and principal root growth, which characterize the long‐term response to ammonium supply in pine. All these results suggest that ammonium induces changes in the root system architecture through the IAA‐CK‐ET phytohormone crosstalk and transcriptional regulation.

Background: Alternative Splicing (AS) is a post-transcriptional process that allows a single RNA to produce different mRNA variants and, in some cases, multiple proteins. Various processes, many yet to be discovered, regulate AS. This study focuses on regulation by RNA-binding proteins (RBPs), which are not only crucial for splicing regulation but also linked to cancer prognosis and are emerging as therapeutic targets for cancer treatment. CLIP-seq experiments help identify where RBPs bind on nascent transcripts, potentially revealing changes in splicing status that suggest causal relationships. Selecting specific RBPs for CLIP-seq experiments is often driven by a priori hypotheses. Results: We developed an algorithm to detect RBPs likely related to splicing changes between conditions by integrating several CLIP-seq databases and a differential splicing detection algorithm. This work refines a previous study by improving splicing event prediction, testing different enrichment statistics, and performing additional validation experiments. The new method provides more accurate predictions and is included in the Bioconductor package EventPointer 3.14. We tested the algorithm in four experiments involving knockdowns of seven different RBPs. The algorithm accurately assessed the statistical significance of these RBPs using only splicing alterations. Additionally, we applied the algorithm to study sixteen cancer types from The Cancer Genome Atlas (TCGA) and three from TARGET. We identified relationships between RBPs and various cancer types, including alterations in CREBBP and MBNL2 in adenocarcinomas of the lung, liver, prostate, rectum, stomach, and colon. Some of these findings are validated in the literature, while others are novel. Conclusions: The developed algorithm enhances the ability to predict and understand RBP-related splicing changes, offering more accurate predictions and novel insights into cancer-related splicing alterations. This work highlights the potential of RBPs as therapeutic targets and contributes to the broader understanding of their roles in cancer biology.

Epitranscriptomic modifications constitute a gene expression checkpoint in all living organisms. As nitrogen is an essential element for plant growth and development, a reasonable hypothesis is that changes in the epitranscriptome may regulate nitrogen acquisition and metabolism. In this study, epitranscriptomic modifications caused by ammonium nutrition were monitored in maritime pine roots through direct RNA sequencing using Oxford Nanopore Technology. Transcriptomic responses mainly affected transcripts involved in nitrogen and carbon metabolism, defense, hormone synthesis/signaling, and translation. Global detection of epitranscriptomic marks was performed to evaluate this posttranscriptional mechanism in untreated and ammonium-treated seedlings. Increased m ⁶ A deposition in the 3’-UTR was observed in response to ammonium, which seems to be correlated with poly(A) lengths and changes in the relative abundance of the corresponding proteins. The results showed that m ⁶ A deposition and its dynamics seem to be important regulators of translation under ammonium nutrition. These findings suggest that protein translation is finely regulated through epitranscriptomic marks likely by changes in mRNA poly(A) length, transcript abundance and ribosome protein composition. An integration of multiomics data suggests that the epitranscriptome modulates responses to developmental and environmental changes, including ammonium nutrition, through buffering, filtering, and focusing the final products of gene expression.

Ammonium is a prominent source of inorganic nitrogen for plant nutrition, but excessive amounts can be toxic for many species. However, most conifers are tolerant to ammonium, a relevant physiological feature of this ancient evolutionary lineage. For a better understanding of the molecular basis of this trait, ammonium-induced changes in the transcriptome of maritime pine ( Pinus pinaster Ait.) root apex have been determined by laser capture microdissection and RNA sequencing. Ammonium promoted changes in the transcriptional profiles of multiple transcription factors, such as SHORT-ROOT , and phytohormone-related transcripts, such as ACO , involved in the development of the root meristem. Nano-PALDI-MSI and transcriptomic analyses showed that the distributions of IAA and CKs were altered in the root apex in response to ammonium nutrition. Taken together, the data suggest that this early response is involved in the increased lateral root branching and principal root growth, which characterize the long-term response to ammonium supply in pine. All these results suggest that ammonium induces changes in the root system architecture through the IAA-CK-ET phytohormone crosstalk and transcriptional regulation.