Frontiers in plant RNA research in ICAR2023: from lab to innovative agriculture.

The recent growth in global warming, soil contamination, and climate instability have widely disturbed ecosystems, and will have a significant negative impact on the growth of plants that produce grains, fruits and woody biomass. To conquer this difficult situation, we need to understand the molecular bias of plant environmental responses and promote development of new technologies for sustainable maintenance of crop production. Accumulated molecular biological data have highlighted the importance of RNA-based mechanisms for plant stress responses. Here, we report the most advanced plant RNA research presented in the 33rd International Conference on Arabidopsis Research (ICAR2023), held as a hybrid event on June 5-9, 2023 in Chiba, Japan, and focused on "Arabidopsis for Sustainable Development Goals". Six workshops/concurrent sessions in ICAR2023 targeted plant RNA biology, and many RNA-related topics could be found in other sessions. In this meeting report, we focus on the workshops/concurrent sessions targeting RNA biology, to share what is happening now at the forefront of plant RNA research.

RNA-seq analysis of alternative pre-mRNA splicing regulation mediated by photoreceptors in Physcomitrium patens.

Plants require light for carbon fixation in photosynthesis and activate a suite of signal-transducing photoreceptors that regulate plant development, ranging from seed germination to flowering and fruiting. Light perception by these photoreceptors triggers massive alterations of gene expression patterns and alternative splicing (AS) of many genes in plants. RNA sequencing (RNA-seq) is a powerful tool to study the full-length transcriptomes and AS of many model organisms, including the moss Physcomitrium patens. RNA-Seq has been applied successfully in transcriptome profiling of plants' developmental processes and responses to various environmental perturbations. Studies using this method provide valuable insights into the genetic networks of plants. Here we describe the use of a high-throughput Illumina sequencing system together with bioinformatics analysis software for transcriptome and AS analysis of Physcomitrium patens in response to red light (RL).

Highly ABA-Induced 1 (HAI1)-Interacting protein HIN1 and drought acclimation-enhanced splicing efficiency at intron retention sites.

The Highly ABA-Induced 1 (HAI1) protein phosphatase is a central component of drought-related signaling. A screen for HAI1-interacting proteins identified HAI1-Interactor 1 (HIN1), a nuclear protein of unknown function which could be dephosphorylated by HAI1 in vitro. HIN1 colocalization and interaction with serine-arginine rich (SR) splicing factors and appearance of nuclear speckle-localized HIN1 during low water potential (ψw) stress suggested a pre-mRNA splicing-related function. RNA sequencing of Arabidopsis Col-0 wild type identified more than 500 introns where moderate severity low ψw altered intron retention (IR) frequency. Surprisingly, nearly 90% of these had increased splicing efficiency (decreased IR) during stress. For one-third of these introns, ectopic HIN1 expression (35S:HIN1) in unstressed plants mimicked the increased splicing efficiency seen in stress-treated wild type. HIN1 bound to a GAA-repeat, Exonic Splicing Enhancer-like RNA motif enriched in flanking sequence around HIN1-regulated introns. Genes with stress and HIN1-affected splicing efficiency were enriched for abiotic stress and signaling-related functions. The 35S:HIN1 plants had enhanced growth maintenance during low ψw, while hin1 mutants had reduced growth, further indicating the role of HIN1 in drought response. HIN1 is annotated as an MYB/SANT domain protein but has limited homology to other MYB/SANT proteins and is not related to known yeast or metazoan RNA-binding proteins or splicing regulators. Together these data identify HIN1 as a plant-specific RNA-binding protein, show a specific effect of drought acclimation to promote splicing efficiency of IR-prone introns, and also discover HAI1-HIN1 interaction and dephosphorylation that connects stress signaling to splicing regulation.

Phosphoproteomics of Arabidopsis Highly ABA-Induced1 identifies AT-Hook-Like10 phosphorylation required for stress growth regulation.

The clade A protein phosphatase 2C Highly ABA-Induced 1 (HAI1) plays an important role in stress signaling, yet little information is available on HAI1-regulated phosphoproteins. Quantitative phosphoproteomics identified phosphopeptides of increased abundance in hai1-2 in unstressed plants and in plants exposed to low-water potential (drought) stress. The identity and localization of the phosphoproteins as well as enrichment of specific phosphorylation motifs indicated that these phosphorylation sites may be regulated directly by HAI1 or by HAI1-regulated kinases including mitogen-activated protein kinases, sucrose non-fermenting-related kinase 2, or casein kinases. One of the phosphosites putatively regulated by HAI1 was S313/S314 of AT-Hook-Like10 (AHL10), a DNA-binding protein of unclear function. HAI1 could directly dephosphorylate AHL10 in vitro, and the level of HAI1 expression affected the abundance of phosphorylated AHL10 in vivo. AHL10 S314 phosphorylation was critical for restriction of plant growth under low-water potential stress and for regulation of jasmonic acid and auxin-related gene expression as well as expression of developmental regulators including Shootmeristemless These genes were also misregulated in hai1-2 AHL10 S314 phosphorylation was required for AHL10 complexes to form foci within the nucleoplasm, suggesting that S314 phosphorylation may control AHL10 association with the nuclear matrix or with other transcriptional regulators. These data identify a set of HAI1-affected phosphorylation sites, show that HAI1-regulated phosphorylation of AHL10 S314 controls AHL10 function and localization, and indicate that HAI1-AHL10 signaling coordinates growth with stress and defense responses.

Epigenetics and RNA Processing: Connections to Drought, Salt, and ABA?

There have been great research advances in epigenetics, RNA splicing, and mRNA processing over recent years. In parallel, there have been many advances in abiotic stress and Abscisic Acid (ABA) signaling. Here we overview studies that have examined stress-induced changes in the epigenome and RNA processing as well as cases where disrupting these processes changes the plant response to abiotic stress. We also highlight some examples where specific connections of stress or ABA signaling to epigenetics or RNA processing have been found. By implication, this also points out cases where such mechanistic connections are likely to exist but are yet to be characterized. In the absence of such specific connections to stress signaling, it should be kept in mind that stress sensitivity phenotypes of some epigenetic or RNA processing mutants maybe the result of indirect, pleiotropic effects and thus may perhaps not indicate a direct function in stress acclimation.