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The number, diversity and significance of peptides as regulators of cellular differentiation, growth, development and defence of plants has long been underestimated. Peptides have now emerged as an important class of signals for cell‐to‐cell communication over short distances, and also for long‐range signalling. We refer to these signalling molecules as peptide growth factors and peptide hormones, respectively. As compared to remarkable progress with respect to the mechanisms of peptide perception and signal transduction, the biogenesis of signalling peptides is still in its infancy. This review focuses on the biogenesis and activity of small post‐translationally modified peptides. These peptides are derived from inactive pre‐pro‐peptides of approximately 70–120 amino acids. Multiple post‐translational modifications (PTMs) may be required for peptide maturation and activation, including proteolytic processing, tyrosine sulfation, proline hydroxylation and hydroxyproline glycosylation. While many of the enzymes responsible for these modifications have been identified, their impact on peptide activity and signalling is not fully understood. These PTMs may or may not be required for bioactivity, they may inactivate the peptide or modify its signalling specificity, they may affect peptide stability or targeting, or its binding affinity with the receptor. In the present review, we will first introduce the peptides that undergo PTMs and for which these PTMs were shown to be functionally relevant. We will then discuss the different types of PTMs and the impact they have on peptide activity and plant growth and development. We conclude with an outlook on the open questions that need to be addressed in future research. [ABSTRACT FROM AUTHOR]

Soil water‐logging and flooding are common environmental stress conditions that can impair plant fitness. Roots are the first organs to be confronted with reduced oxygen tension as a result of flooding. While anatomical and morphological adaptations of roots are extensively studied, the root system architecture is only now becoming a focus of flooding research. Adventitious root (AR) formation shifts the root system higher up the plant, thereby facilitating supply with oxygen, and thus improving root and plant survival.We used Arabidopsis knockout mutants and overexpressors of ERFVII transcription factors to study their role in AR formation under hypoxic conditions and in response to ethylene.Results show that ethylene inhibits AR formation. Hypoxia mainly promotes AR elongation rather than formation mediated by ERFVII transcription factors, as indicated by reduced AR elongation in erfVII seedlings. Overexpression of HRE2 induces AR elongation to the same degree as hypoxia, while ethylene overrides HRE2‐induced AR elongation.The ERFVII transcription factors promote establishment of an AR system that is under negative control by ethylene. Inhibition of growth of the main root system and promotion of AR elongation under hypoxia strengthens the root system in upper soil layers where oxygen shortage may last for shorter time periods. [ABSTRACT FROM AUTHOR]

The emission of volatiles is a common, but mostly neglected, ability of bacteria that is important for inter‐ and intraspecific interactions. Currently, limited information is available on how the bacterial volatile (mVOC) signal is integrated into a plant's life at the physiological, transcriptional and metabolic level. Previous results provided evidence for volatile‐dependent regulation of WRKY18, a pathogen‐responsive transcription factor of Arabidopsis thaliana in co‐culture with two rhizobacteria, Serratia plymuthica HRO‐C48 and Stenotrophomonas maltophilia R3089.Dual cultures of these bacteria and A. thaliana; application of the common mVOC 2‐phenyl‐ethanol; extraction of metabolites of A. thaliana after exposure to bacterial volatiles; and analysis of the metabolomes (GC‐TOF/MS) were carried out.The prominent microbial aromatic compound 2‐phenyl‐ethanol, emitted by both bacteria, negatively affects growth of A. thaliana wild type, whereas WRKY18 T‐DNA insertion mutants were significantly more tolerant than wild‐type seedlings. This paper also demonstrates for the first time the impact of the rhizobacterial volatiles on the metabolome of A. thaliana. Upon mVOC exposure the plants rearrange their metabolism by accumulation of e.g. amino acids and TCA intermediates that potentially allow plants to cope with and survive this stress.Our findings illustrate the high degree of complexity of metabolic rearrangements underlying the interactions of bacterial volatile elicitors and resulting plant responses. Furthermore, the impact of the volatile 2‐phenyl‐ethanol as a signal in the WRKY18‐dependent pathway highlights this compound as an important molecular player. [ABSTRACT FROM AUTHOR]

The green microalga Lobosphaera incisa is an oleaginous eukaryotic alga that is rich in arachidonic acid (20:4). Being rich in this polyunsaturated fatty acid (PUFA), however, makes it sensitive to oxidation. In plants, lipoxygenases (LOXs) are the major enzymes that oxidise these molecules.Here, we describe, to our best knowledge, the first characterisation of a cDNA encoding a LOX (LiLOX) from a green alga. To obtain first insights into its function, we expressed it in E. coli, purified the recombinant enzyme and analysed its enzyme activity.The protein sequence suggests that LiLOX and plastidic LOXs from bryophytes and flowering plants may share a common ancestor. The fact that LiLOX oxidises all PUFAs tested with a consistent oxidation on the carbon n‐6, suggests that PUFAs enter the substrate channel through their methyl group first (tail first). Additionally, LiLOX form the fatty acid hydroperoxide in strict S configuration.LiLOX may represent a good model to study plastid LOX, because it is stable after heterologous expression in E. coli and highly active in vitro. Moreover, as the first characterised LOX from green microalgae, it opens the possibility to study endogenous LOX pathways in these organisms. [ABSTRACT FROM AUTHOR]

Alanine and aspartate are essential transfer metabolites for C4 species of the NAD‐malic enzyme and phosphoenolpyruvate carboxykinase subtype. To some degree both amino acids are also part of the metabolite shuttle in NADP‐malic enzyme plants. In comparison with C3 species, the majority of C4 species are therefore characterised by enhanced expression and activity of alanine and aspartate aminotransferases (AT) in the photosynthetically active tissue. Both enzymes exist in multiple copies and have been found in different subcellular compartments. We tested whether different C4 species show preferential recruitment of enzymes from specific lineages and subcellular compartments.Phylogenetic analysis of alanine and aspartate ATs from a variety of monocot and eudicot C4 species and their C3 relatives was combined with subcellular prediction tools and analysis of the subsequent transcript amounts in mature leaves.Recruitment of aspartate AT from a specific subcellular compartment was strongly connected to the biochemical subtype. Deviation from the main model was however observed in Gynandropsis gynandra. The configuration of alanine AT generally differed in monocot and eudicot species. C4 monocots recruited an alanine AT from a specific cytosolic branch, but eudicots use alanine AT copies from a mitochondrial branch.Generally, plants display high plasticity in the setup of the C4 pathway. Beside the common models for the different C4 subtypes, individual solutions were found for plant groups or lineages. [ABSTRACT FROM AUTHOR]

Ascorbic acid (AsA) biosynthesis in plants predominantly occurs via a pathway with d‐mannose and l‐galactose as intermediates. One alternative pathway for AsA synthesis, which is similar to the biosynthesis route in mammals, is controversially discussed for plants. Here, myo‐inositol is cleaved to glucuronic acid and then converted via l‐gulonate to AsA. In contrast to animals, plants have an effective recycling pathway for glucuronic acid, being a competitor for the metabolic rate. Recycling involves a phosphorylation at C1 by the enzyme glucuronokinase.Two previously described T‐DNA insertion lines in the gene coding for glucuronokinase1 show wild type‐like expression levels of the mRNA in our experiments and do not accumulate glucuronic acid in labelling experiments disproving that these lines are true knockouts. As suitable T‐DNA insertion lines were not available, we generated frameshift mutations in the major expressed isoform glucuronokinase1 (At3g01640) to potentially redirect metabolites to AsA.However, radiotracer experiments with 3H‐myo‐inositol revealed that the mutants in glucuronokinase1 accumulate only glucuronic acid and incorporate less metabolite into cell wall polymers. AsA was not labelled, suggesting that Arabidopsis cannot efficiently use glucuronic acid for AsA biosynthesis.All four mutants in glucuronokinase as well as the wild type have the same level of AsA in leaves. [ABSTRACT FROM AUTHOR]

Low temperature is one of the most important environmental factors that affect global survival of humans and animals and equally importantly the distribution of plants and crop productivity. Survival of metazoan cells under cold stress requires regulation of the sensor‐kinase Target Of Rapamycin (TOR). TOR controls growth of eukaryotic cells by adjusting anabolic and catabolic metabolism. Previous studies identified the Thyroid Adenoma Associated (THADA) gene as the major effect locus by positive selection in the evolution of modern human adapted to cold. Here we investigate the role of THADA in TOR signaling and cold acclimation of plants.We applied BLAST searches and homology modeling to identify the AtTHADA (AT3G55160) in Arabidopsis thaliana as the highly probable orthologue protein. Reverse genetics approaches were combined with immunological detection of TOR activity and metabolite profiling to address the role of the TOR and THADA for growth regulation and cold acclimation.Depletion of the AtTHADA gene caused complete or partial loss of full‐length mRNA, respectively, and significant retardation of growth under non‐stressed conditions. Furthermore, depletion of AtTHADA caused hypersensitivity towards low‐temperatures. Atthada displayed a lowered energy charge. This went along with decreased TOR activity, which offers a molecular explanation for the slow growth phenotype of Atthada. Finally, we used TOR RNAi lines to identify the de‐regulation of TOR activity as one determinant for sensitivity towards low‐temperatures.Taken together our results provide evidence for a conserved function of THADA in cold acclimation of eukaryotes and suggest that cold acclimation in plants requires regulation of TOR. [ABSTRACT FROM AUTHOR]

Physcomitrella patens is a monoecious moss that is predominantly selfing in the wild. Laboratory crossing techniques have been established and crosses between the sequenced Gransden ecotype and the genetically divergent Villersexel ecotype were used for genetic mapping. The recently introduced ecotype Reute has a high fertility rate and is genetically more closely related to the Gransden ecotype than the Villersexel ecotype. Reute sexual reproduction phenology is similar to Gransden, which should allow successful crossing.Using the Reute ecotype and an existing Gransden mutant as a test case, we applied a normalised crossing approach to demonstrate crossing potential between these ecotypes. Also, using a standard transformation approach, we generated Reute fluorescent strains expressing mCherry that allow an easy detection of crossed offspring (sporophyte).We show that Reute can be successfully crossed with a self‐infertile DR5:DsRed2 mutant generated in the Gransden background. Using newly established Reute fluorescent strains, we show that they can efficiently fertilise Reute as well as Gransden wild type. The resulting progeny display Mendelian 1:1 segregation of the fluorescent marker(s), demonstrating the suitability of such strains for genetic crossing.Overall our results demonstrate that Reute is highly suitable for genetic crossing. The Reute mCherry strain can be used as a suitable background for offspring selection after crossing. [ABSTRACT FROM AUTHOR]

Context: Infants born at very low birth weight (VLBW) require high levels of nursing intensity. The role of nursing in outcomes for these infants in the United States is not known.Objective: To examine the relationships between hospital recognition for nursing excellence (RNE) and VLBW infant outcomes.Design, Setting, and Patients: Cohort study of 72,235 inborn VLBW infants weighing 501 to 1500 g born in 558 Vermont Oxford Network hospital neonatal intensive care units between January 1, 2007, and December 31, 2008. Hospital RNE was determined from the American Nurses Credentialing Center. The RNE designation is awarded when nursing care achieves exemplary practice or leadership in 5 areas.Main Outcome Measures: Seven-day, 28-day, and hospital stay mortality; nosocomial infection, defined as an infection in blood or cerebrospinal fluid culture occurring more than 3 days after birth; and severe (grade 3 or 4) intraventricular hemorrhage.Results: Overall, the outcome rates were as follows: for 7-day mortality, 7.3% (5258/71,955); 28-day mortality, 10.4% (7450/71,953); hospital stay mortality, 12.9% (9278/71,936); severe intraventricular hemorrhage, 7.6% (4842/63,525); and infection, 17.9% (11,915/66,496). The 7-day mortality was 7.0% in RNE hospitals and 7.4% in non-RNE hospitals (adjusted odds ratio [OR], 0.87; 95% CI, 0.76-0.99; P = .04). The 28-day mortality was 10.0% in RNE hospitals and 10.5% in non-RNE hospitals (adjusted OR, 0.90; 95% CI, 0.80-1.01; P = .08). Hospital stay mortality was 12.4% in RNE hospitals and 13.1% in non-RNE hospitals (adjusted OR, 0.90; 95% CI, 0.81-1.01; P = .06). Severe intraventricular hemorrhage was 7.2% in RNE hospitals and 7.8% in non-RNE hospitals (adjusted OR, 0.88; 95% CI, 0.77-1.00; P = .045). Infection was 16.7% in RNE hospitals and 18.3% in non-RNE hospitals (adjusted OR, 0.86; 95% CI, 0.75-0.99; P = .04). Compared with RNE hospitals, the adjusted absolute decrease in risk of outcomes in RNE hospitals ranged from 0.9% to 2.1%. All 5 outcomes were jointly significant (P < .001). The mean effect across all 5 outcomes was OR, 0.88 (95% CI, 0.83-0.94; P < .001). In a subgroup of 68,253 infants with gestational age of 24 weeks or older, the ORs for RNE for all 3 mortality outcomes and infection were statistically significant.Conclusion: Among VLBW infants born in RNE hospitals compared with non-RNE hospitals, there was a significantly lower risk-adjusted rate of 7-day mortality, nosocomial infection, and severe intraventricular hemorrhage but not of 28-day mortality or hospital stay mortality. [ABSTRACT FROM AUTHOR]

Background: Previous studies have documented racial disparities in treatment of acute myocardial infarction (AMI) among Medicare beneficiaries. However, the extent to which unobserved differences between hospitals explains some of these differences is unknown.Objective: The objective of this study was to determine whether the observed racial treatment disparities for AMI narrow when analyses account for differences in where blacks and whites are hospitalized.Research Design: Retrospective observational cohort study using Medicare claims and medical record review.Subjects: This study included 130,709 white and 8286 black Medicare patients treated in 4690 hospitals in 50 US states for confirmed AMI in 1994 and 1995.Measures: Measures in this study were receipt of reperfusion, aspirin, and smoking cessation counseling during hospitalization; prescription of aspirin, angiotensin-converting enzyme inhibitor, and beta-blocker at hospital discharge; receipt of cardiac catheterization, percutaneous coronary intervention (PCI), or bypass surgery (CABG) within 30 days of AMI; and 30-day and 1-year mortality.Results: Within-hospital analyses narrowed or erased black-white disparities for medical treatments received during the acute hospitalization, widened black-white disparities for follow-up surgical treatments, and augmented the survival advantage among blacks. These findings indicate that, on average, blacks went to hospitals that had lower rates of evidence-based medical treatments, higher rates of cardiac procedures, and worse risk-adjusted mortality after AMI.Conclusions: Incorporating the hospital effect altered the findings of racial disparity analyses in AMI and explained more of the disparities than race. A policy of targeted hospital-level interventions may be required for success of national efforts to reduce disparities. [ABSTRACT FROM AUTHOR]

Objectives: To identify recent national trends in the employment and earnings of nursing personnel and determine whether managed care is associated with changes in the employment and wage growth of nursing personnel.Design: Retrospective analysis of trends in data on employment and earnings of nursing personnel based on monthly US Bureau of the Census Current Population Surveys between 1983 and 1994, and comparison of trends between states with high and low rates of enrollment in health maintenance organizations (HMOs).Population: Registered nurses (RNs), licensed practical nurses (LPNs), and nurse aides/assistants, orderlies, and attendants (referred to collectively as aides) between the ages of 21 and 64 years.Outcome Measures: Full- and part-time employment, unemployment, percentage of nursing personnel employed in key sectors of the nurse labor market, and inflation-adjusted hourly wages.Results: From 1983 through 1994, there was strong overall growth in both RN employment (37%) and inflation-adjusted wages (22%). Beginning in the early 1990s, however, RNs experienced stagnant wages and a small but steady shift toward employment in lower-paying nonhospital settings, particularly in home health care. In states with high HMO enrollment, RN and LPN employment has grown more slowly since 1990, and the shift of RN employment out of the hospital was strongest. For aides and LPNs, the shift out of hospital employment occurred years before that of RNs and at a much greater rate. Since 1990 the employment of aides has grown rapidly in nursing homes and in home health care settings, whereas employment of LPNs has shifted primarily into physician offices and nursing homes. Overall, the movement toward nonhospital employment has had a modest negative impact on wages for all nursing personnel.Conclusions: Managed care is associated with slower employment growth for RNs in hospitals and a shift toward employment in nonhospital settings, but its effect on earnings has been overshadowed by other forces impacting nurse wages. [ABSTRACT FROM AUTHOR]

Alternative splicing is essential for plants, enabling a single gene to produce multiple transcript variants to boost functional diversity and fine-tune responses to environmental and developmental cues. At-RS31, a plant-specific splicing factor in the Serine/Arginine (SR)-rich protein family, responds to light and the Target of Rapamycin (TOR) signaling pathway, yet its downstream targets and regulatory impact remain unknown.To identify At-RS31 targets, we applied individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) and RNAcompete assays. Transcriptomic analyses of At-RS31 mutant and overexpressing plants further revealed its effects on alternative splicing.iCLIP identified 4,034 At-RS31 binding sites across 1,421 genes, enriched in CU-rich and CAGA RNA motifs. Comparative iCLIP and RNAcompete data indicate that the RS domain of At-RS31 may influence its binding specificity in planta , underscoring the value of combining in vivo and in vitro approaches. Transcriptomic analysis showed that At-RS31 modulates diverse splicing events, particularly intron retention and exitron splicing, and influences other splicing modulators, acting as a hierarchical regulator.By regulating stress-response genes and genes in both TOR and abscisic acid (ABA) signaling pathways, At-RS31 may help integrate these signals, balancing plant growth with environmental adaptability through alternative splicing., Competing Interests: COMPETING INTERESTS None declared.

A bacterial strain was isolated from pathogenic lesions of Acer campestre tree leaves from the Teutoburg Forest in North Rhine-Westphalia, Germany, by culture on non-selective agar plates. 16S rRNA sequencing revealed 100% similarity to Sanguibacter keddieii and Sanguibacter inulinus , as well as 99% similarity to Sanguibacter gelidistatuariae and Sanguibacter antarcticus . Here, we used genome-based taxonomy with the Type (Strain) Genome Server (TYGS), which suggests the isolation of a novel prokaryotic strain. According to TYGS-analysis, using whole genome digital DNA-DNA hybridization, only 65.5% similarity to the closest relative S. inulinus was revealed, suggesting a novel species. Growth was observed at both aerobic and anaerobic conditions. Bacterial cells depicted coryneform motile rods, with a length of 1.1-3.3 µm and a constant diameter of 0.5 µm. Cells did not form spores under the tested conditions and stain Gram-positive. Growth occurred between 0.5 and 4% NaCl (optimal: 1%), at pH 5.5-9.5 (optimal: 8.0-9.0). The strain was mesophilic with an optimal growth at 25   °C. Major cellular fatty acids of the novel strain were anteiso-C 15 : 0 and C 16 : 0 .

N6-methyladenosine (m 6 A) exerts many of its regulatory effects on eukaryotic mRNAs by recruiting cytoplasmic YT521-B homology-domain family (YTHDF) proteins. Here, we show that in Arabidopsis thaliana, the interaction between m 6 A and the major YTHDF protein ECT2 also involves the mRNA-binding ALBA protein family. ALBA and YTHDF proteins physically associate via a deeply conserved short linear motif in the intrinsically disordered region of YTHDF proteins and their mRNA target sets overlap, with ALBA4 binding sites being juxtaposed to m 6 A sites. These binding sites correspond to pyrimidine-rich elements previously found to be important for m 6 A binding to ECT2. Accordingly, both the biological functions of ECT2, and its binding to m 6 A targets in vivo, require ALBA association. Our results introduce the YTHDF-ALBA complex as the functional cytoplasmic m 6 A-reader in Arabidopsis, and define a molecular foundation for the concept of facilitated m 6 A reading, which increases the potential for combinatorial control of biological m 6 A effects., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

Background: The pandemic profoundly stressed practicing nurses and could have thereby affected trends in nursing-sensitive quality indicators (NSIs), measures that detect changes in patient health status directly affected by nursing care., Objectives: The aim of the study was to determine if NSIs have worsened in response to the pandemic and then returned to prepandemic levels using data from 2019 through 2022., Methods: We conducted a cross-sectional descriptive study of annual trends, examining unit data from the National Database of Nursing Quality Indicators (NDNQI) from 2019 through 2022 for five indicators: rates of falls, central line-associated bloodstream infections (CLABSI), catheter-associated urinary tract infections (CAUTI), hospital-acquired pressure injuries (HAPI), and ventilator-associated events (VAE). The NDNQI is the largest repository of nursing quality indicators, which are derived from patient-level events, reported at the nursing unit level, and submitted quarterly by over 2,000 member hospitals. Adult medical-surgical or critical care inpatient nursing units with complete data for the 4 years were included, with samples ranging from 456 to 5,818 nursing units in 2,346 hospitals. Analysis of variance was conducted by comparing the 2019 rates to each subsequent year., Results: In decreasing order of prevalence, the mean prepandemic rates were 6.58 VAE per 1,000 ventilator days (critical care only), 2.41 HAPI per 1,000 device days, 2.20 falls per 1,000 patient days, 0.96 CAUTI per 1,000 catheter days, and 0.68 CLABSI per 1,000 central line days for medical-surgical and critical care units combined. The rates for all five nurse-sensitive indicators increased significantly beginning in 2020 and have begun to decline but have not returned to baseline by 2022. The maximum rate was observed in 2020 for falls and 2021 for the remaining indicators. These increases to the maximum ranged from a 12% increase in CAUTI to 49% for CLABSI., Discussion: NSIs increased during the pandemic and are now returning to baseline. The pandemic underscored the importance of nursing practice. The pandemic's enduring negative effects on the nursing workforce must be addressed to preserve patient safety., Competing Interests: Drs. Pascal and Warshawsky are employed by Press Ganey but did not receive any special incentives for this project. The remaining authors have no conflicts of interest to report., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

PROTEIN ARGININE METHYLTRANSFERASES (PRMTs) catalyze arginine (R) methylation that is critical for transcriptional and post-transcriptional gene regulation. In Arabidopsis, PRMT5 that catalyzes symmetric R dimethylation is best characterized. PRMT5 mutants are late-flowering and show altered responses to environmental stress. Among PRMT5 targets are Arabidopsis thaliana GLYCINE RICH RNA BINDING PROTEIN 7 ( At GRP7) and At GRP8 that promote the transition to flowering. At GRP7 R141 has been shown to be modified by PRMT5. Here, we tested whether this symmetric dimethylation of R141 is important for At GRP7's physiological role in flowering time control. We constructed At GRP7 mutant variants with non-methylable R141 (R141A, R141K). Genomic clones containing these variants complemented the late-flowering phenotype of the grp7-1 mutant to the same extent as wild-type At GRP7. Furthermore, overexpression of At GRP7 R141A or R141K promoted flowering similar to overexpression of the wild-type protein. Thus, flowering time does not depend on R141 and its modification. However, germination experiments showed that R141 contributes to the activity of At GRP7 in response to abiotic stress reactions mediated by abscisic acid during early development. Immunoprecipitation of At GRP7-GFP in the prmt5 background revealed that antibodies against dimethylated arginine still recognized At GRP7, suggesting that additional methyltransferases may be responsible for modification of At GRP7.

Background: The interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA processing. RNA interactome capture (RIC) techniques have been implemented to catalogue RNA- binding proteins in the cell. In RIC, RNA-protein complexes are stabilized by UV crosslinking in vivo. Polyadenylated RNAs and associated proteins are pulled down from cell lysates using oligo(dT) beads and the RNA-binding proteome is identified by quantitative mass spectrometry. However, insights into the RNA-binding proteome of a single RNA that would yield mechanistic information on how RNA expression patterns are orchestrated, are scarce., Results: Here, we explored RIC in Arabidopsis to identify proteins interacting with a single mRNA, using the circadian clock-regulated Arabidopsis thaliana GLYCINE-RICH RNA-BINDING PROTEIN 7 (AtGRP7) transcript, one of the most abundant transcripts in Arabidopsis, as a showcase. Seedlings were treated with UV light to covalently crosslink RNA and proteins. The AtGRP7 transcript was captured from cell lysates with antisense oligonucleotides directed against the 5'untranslated region (UTR). The efficiency of RNA capture was greatly improved by using locked nucleic acid (LNA)/DNA oligonucleotides, as done in the enhanced RIC protocol. Furthermore, performing a tandem capture with two rounds of pulldown with the 5'UTR oligonucleotide increased the yield. In total, we identified 356 proteins enriched relative to a pulldown from atgrp7 mutant plants. These were benchmarked against proteins pulled down from nuclear lysates by AtGRP7 in vitro transcripts immobilized on beads. Among the proteins validated by in vitro interaction we found the family of Acetylation Lowers Binding Affinity (ALBA) proteins. Interaction of ALBA4 with the AtGRP7 RNA was independently validated via individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP). The expression of the AtGRP7 transcript in an alba loss-of-function mutant was slightly changed compared to wild-type, demonstrating the functional relevance of the interaction., Conclusion: We adapted specific RNA interactome capture with LNA/DNA oligonucleotides for use in plants using AtGRP7 as a showcase. We anticipate that with further optimization and up scaling the protocol should be applicable for less abundant transcripts., (© 2024. The Author(s).)

Arabidopsis (Arabidopsis thaliana) PROTEIN ARGININE METHYLTRANSFERASE5 (PRMT5) post-translationally modifies RNA-binding proteins by arginine (R) methylation. However, the impact of this modification on the regulation of RNA processing is largely unknown. We used the spliceosome component, SM-LIKE PROTEIN 4 (LSM4), as a paradigm to study the role of R-methylation in RNA processing. We found that LSM4 regulates alternative splicing (AS) of a suite of its in vivo targets identified here. The lsm4 and prmt5 mutants show a considerable overlap of genes with altered AS raising the possibility that splicing of those genes could be regulated by PRMT5-dependent LSM4 methylation. Indeed, LSM4 methylation impacts AS, particularly of genes linked with stress response. Wild-type LSM4 and an unmethylable version complement the lsm4-1 mutant, suggesting that methylation is not critical for growth in normal environments. However, LSM4 methylation increases with abscisic acid and is necessary for plants to grow under abiotic stress. Conversely, bacterial infection reduces LSM4 methylation, and plants that express unmethylable-LSM4 are more resistant to Pseudomonas than those expressing wild-type LSM4. This tolerance correlates with decreased intron retention of immune-response genes upon infection. Taken together, this provides direct evidence that R-methylation adjusts LSM4 function on pre-mRNA splicing in an antagonistic manner in response to biotic and abiotic stress., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Despite crucial roles of RNA-binding proteins (RBPs) in plant physiology and development, methods for determining their transcriptome-wide binding landscape are less developed than those used in other model organisms. Cross-linking and immunoprecipitation (CLIP) methods (based on UV-mediated generation of covalent bonds between RNAs and cognate RBPs in vivo, purification of the cross-linked complexes and identification of the co-purified RNAs by high-throughput sequencing) have been applied mainly in mammalian cells growing in monolayers or in translucent tissue. We have developed plant iCLIP2, an efficient protocol for performing individual-nucleotide-resolution CLIP (iCLIP) in plants, tailored to overcome the experimental hurdles posed by plant tissue. We optimized the UV dosage to efficiently cross-link RNA and proteins in plants and expressed epitope-tagged RBPs under the control of their native promoters in loss-of-function mutants. We select epitopes for which nanobodies are available, allowing stringent conditions for immunopurification of the RNA-protein complexes to be established. To overcome the inherently high RNase content of plant cells, RNase inhibitors are added and the limited RNA fragmentation step is modified. We combine the optimized isolation of RBP-bound RNAs with iCLIP2, a streamlined protocol that greatly enhances the efficiency of library preparation for high-throughput sequencing. Plant researchers with experience in molecular biology and handling of RNA can complete this iCLIP2 protocol in ~5 d. Finally, we describe a bioinformatics workflow to determine targets of Arabidopsis RBPs from iCLIP data, covering all steps from downloading sequencing reads to identifying cross-linking events ( https://github.com/malewins/Plant-iCLIPseq ), and present the R/Bioconductor package BindingSiteFinder to extract reproducible binding sites ( https://bioconductor.org/packages/release/bioc/html/BindingSiteFinder.html )., (© 2024. Springer Nature Limited.)

The importance of RNA-binding proteins (RBPs) for plant responses to environmental stimuli and development is well documented. Insights into the portfolio of RNAs they recognize, however, clearly lack behind the understanding gathered in non-plant model organisms. Here, we characterize binding of the circadian clock-regulated Arabidopsis thaliana GLYCINE-RICH RNA-BINDING PROTEIN 7 (AtGRP7) to its target transcripts. We identified novel RNA targets from individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) data using an improved bioinformatics pipeline that will be broadly applicable to plant RBP iCLIP data. 2705 transcripts with binding sites were identified in plants expressing AtGRP7-GFP that were not recovered in plants expressing an RNA-binding dead variant or GFP alone. A conserved RNA motif enriched in uridine residues was identified at the AtGRP7 binding sites. NMR titrations confirmed the preference of AtGRP7 for RNAs with a central U-rich motif. Among the bound RNAs, circadian clock-regulated transcripts were overrepresented. Peak abundance of the LHCB1.1 transcript encoding a chlorophyll-binding protein was reduced in plants overexpressing AtGRP7 whereas it was elevated in atgrp7 mutants, indicating that LHCB1.1 was regulated by AtGRP7 in a dose-dependent manner. In plants overexpressing AtGRP7, the LHCB1.1 half-life was shorter compared to wild-type plants whereas in atgrp7 mutant plants, the half-life was significantly longer. Thus, AtGRP7 modulates circadian oscillations of its in vivo binding target LHCB1.1 by affecting RNA stability., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)