Light regulates alternative splicing outcomes via the TOR kinase pathway

For plants, light is the source of energy and the most relevant regulator of growth and adaptations to the environment by inducing changes in gene expression at various levels, including alternative splicing. Light-triggered chloroplast retrograde signals control alternative splicing in Arabidopsis thaliana. Here, we provide evidence that light regulates the expression of a core set of splicing-related factors in roots. Alternative splicing responses in roots are not directly caused by light but are instead most likely triggered by photosynthesized sugars. The target of rapamycin (TOR) kinase plays a key role in this shoot-to-root signaling pathway. Knocking down TOR expression or pharmacologically inhibiting TOR activity disrupts the alternative splicing responses to light and exogenous sugars in roots. Consistently, splicing decisions are modulated by mitochondrial activity in roots. In conclusion, by activating the TOR pathway, sugars act as mobile signals to coordinate alternative splicing responses to light throughout the whole plant.Fil: Riegler, Stefan. Universitat Fur Bodenkultur Wien; AustriaFil: Servi, Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Scarpin, Maria Regina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of California; Estados UnidosFil: Godoy Herz, Micaela Amalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Kubaczka Zoppi, María Guillermina Jazmín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Venhuizen, Peter. Universitat Fur Bodenkultur Wien; AustriaFil: Meyer, Christian. Universite Paris-Saclay; FranciaFil: Brunkard, Jacob O.. University of California; Estados UnidosFil: Kalyna, Maria. Universitat Fur Bodenkultur Wien; AustriaFil: Barta, Andrea. Medizinische Universitat Wien; AustriaFil: Petrillo, Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Xyloglucan Remodeling Defines Auxin-Dependent Differential Tissue Expansion in Plants

Size control is a fundamental question in biology, showing incremental complexity in plants, whose cells possess a rigid cell wall. The phytohormone auxin is a vital growth regulator with central importance for differential growth control. Our results indicate that auxin-reliant growth programs affect the molecular complexity of xyloglucans, the major type of cell wall hemicellulose in eudicots. Auxin-dependent induction and repression of growth coincide with reduced and enhanced molecular complexity of xyloglucans, respectively. In agreement with a proposed function in growth control, genetic interference with xyloglucan side decorations distinctly modulates auxin-dependent differential growth rates. Our work proposes that auxin-dependent growth programs have a spatially defined effect on xyloglucan's molecular structure, which in turn affects cell wall mechanics and specifies differential, gravitropic hypocotyl growth

A high quality Arabidopsis transcriptome for accurate transcript-level analysis of alternative splicing

Alternative splicing generates multiple transcript and protein isoforms from the same gene and thus is important in gene expression regulation. To date, RNA-sequencing (RNA-seq) is the standard method for quantifying changes in alternative splicing on a genome-wide scale. Understanding the current limitations of RNA-seq is crucial for reliable analysis and the lack of high quality, comprehensive transcriptomes for most species, including model organisms such as Arabidopsis, is a major constraint in accurate quantification of transcript isoforms. To address this, we designed a novel pipeline with stringent filters and assembled a comprehensive Reference Transcript Dataset for Arabidopsis (AtRTD2) containing 82,190 non-redundant transcripts from 34 212 genes. Extensive experimental validation showed that AtRTD2 and its modified version, AtRTD2-QUASI, for use in Quantification of Alternatively Spliced Isoforms, outperform other available transcriptomes in RNA-seq analysis. This strategy can be implemented in other species to build a pipeline for transcript-level expression and alternative splicing analyses

VARIFI—Web-Based Automatic Variant Identification, Filtering and Annotation of Amplicon Sequencing Data

Fast and affordable benchtop sequencers are becoming more important in improving personalized medical treatment. Still, distinguishing genetic variants between healthy and diseased individuals from sequencing errors remains a challenge. Here we present VARIFI, a pipeline for finding reliable genetic variants (single nucleotide polymorphisms (SNPs) and insertions and deletions (indels)). We optimized parameters in VARIFI by analyzing more than 170 amplicon-sequenced cancer samples produced on the Personal Genome Machine (PGM). In contrast to existing pipelines, VARIFI combines different analysis methods and, based on their concordance, assigns a confidence score to each identified variant. Furthermore, VARIFI applies variant filters for biases associated with the sequencing technologies (e.g., incorrectly identified homopolymer-associated indels with Ion Torrent). VARIFI automatically extracts variant information from publicly available databases and incorporates methods for variant effect prediction. VARIFI requires little computational experience and no in-house compute power since the analyses are conducted on our server. VARIFI is a web-based tool available at varifi.cibiv.univie.ac.at

Experimentally increased nutrient availability at the permafrost thaw front selectively enhances biomass production of deep-rooting subarctic peatland species

Climate warming increases nitrogen (N) mineralization in superficial soil layers (the dominant rooting zone) of subarctic peatlands. Thawing and subsequent mineralization of permafrost increases plant-available N around the thaw-front. Because plant production in these peatlands is N-limited, such changes may substantially affect net primary production and species composition. We aimed to identify the potential impact of increased N-availability due to permafrost thawing on subarctic peatland plant production and species performance, relative to the impact of increased N-availability in superficial organic layers. Therefore, we investigated whether plant roots are present at the thaw-front (45 cm depth) and whether N-uptake (15N-tracer) at the thaw-front occurs during maximum thaw-depth, coinciding with the end of the growing season. Moreover, we performed a unique 3-year belowground fertilization experiment with fully factorial combinations of deep- (thaw-front) and shallow-fertilization (10 cm depth) and controls. We found that certain species are present with roots at the thaw-front (Rubus chamaemorus) and have the capacity (R. chamaemorus, Eriophorum vaginatum) for N-uptake from the thaw-front between autumn and spring when aboveground tissue is largely senescent. In response to 3-year shallow-belowground fertilization (S) both shallow- (Empetrum hermaphroditum) and deep-rooting species increased aboveground biomass and N-content, but only deep-rooting species responded positively to enhanced nutrient supply at the thaw-front (D). Moreover, the effects of shallow-fertilization and thaw-front fertilization on aboveground biomass production of the deep-rooting species were similar in magnitude (S: 71%; D: 111% increase compared to control) and additive (S + D: 181% increase). Our results show that plant-available N released from thawing permafrost can form a thus far overlooked additional N-source for deep-rooting subarctic plant species and increase their biomass production beyond the already established impact of warming-driven enhanced shallow N-mineralization. This may result in shifts in plant community composition and may partially counteract the increased carbon losses from thawing permafrost

A Collection of Pre-mRNA Splicing Mutants in Arabidopsis thaliana

To investigate factors influencing pre-mRNA splicing in plants, we conducted a forward genetic screen using an alternatively-spliced GFP reporter gene in Arabidopsis thaliana. This effort generated a collection of sixteen mutants impaired in various splicing-related proteins, many of which had not been recovered in any prior genetic screen or implicated in splicing in plants. The factors are predicted to act at different steps of the spliceosomal cycle, snRNP biogenesis pathway, transcription, and mRNA transport. We have described eleven of the mutants in recent publications. Here we present the final five mutants, which are defective, respectively, in RNA-BINDING PROTEIN 45D (rbp45d), DIGEORGE SYNDROME CRITICAL REGION 14 (dgcr14), CYCLIN-DEPENDENT KINASE G2 (cdkg2), INTERACTS WITH SPT6 (iws1) and CAP BINDING PROTEIN 80 (cbp80). We provide RNA-sequencing data and analyses of differential gene expression and alternative splicing patterns for the cbp80 mutant and for several previously published mutants, including smfa and new alleles of cwc16a, for which such information was not yet available. Sequencing of small RNAs from the cbp80 mutant highlighted the necessity of wild-type CBP80 for processing of microRNA (miRNA) precursors into mature miRNAs. Redundancy tests of paralogs encoding several of the splicing factors revealed their functional non-equivalence in the GFP reporter gene system. We discuss the cumulative findings and their implications for the regulation of pre-mRNA splicing efficiency and alternative splicing in plants. The mutant collection provides a unique resource for further studies on a coherent set of splicing factors and their roles in gene expression, alternative splicing and plant development

FRUITFULL Is a Repressor of Apical Hook Opening in Arabidopsis thaliana

Plants adjust their architecture to a constantly changing environment, requiring adaptation of differential growth. Despite their importance, molecular switches, which define growth transitions, are largely unknown. Apical hook development in dark grown Arabidopsis thaliana (A. thaliana) seedlings serves as a suitable model for differential growth transition in plants. Here, we show that the phytohormone auxin counteracts the light-induced growth transition during apical hook opening. We, subsequently, identified genes which are inversely regulated by light and auxin. We used in silico analysis of the regulatory elements in this set of genes and subsequently used natural variation in gene expression to uncover correlations between underlying transcription factors and the in silico predicted target genes. This approach uncovered that MADS box transcription factor AGAMOUS-LIKE 8 (AGL8)/FRUITFULL (FUL) modulates apical hook opening. Our data shows that transient FUL expression represses the expression of growth stimulating genes during early phases of apical hook development and therewith guards the transition to growth promotion for apical hook opening. Here, we propose a role for FUL in setting tissue identity, thereby regulating differential growth during apical hook development