SNF1-RELATED KINASE 1 and TARGET OF RAPAMYCIN control light-responsive splicing events and developmental characteristics in etiolated Arabidopsis seedlings.

The kinases SNF1-RELATED KINASE 1 (SnRK1) and TARGET OF RAPAMYCIN (TOR) are central sensors of the energy status, linking this information via diverse regulatory mechanisms to plant development and stress responses. Despite the well-studied functions of SnRK1 and TOR under conditions of limited or ample energy availability, respectively, little is known about the extent to which the 2 sensor systems function and how they are integrated in the same molecular process or physiological context. Here, we demonstrate that both SnRK1 and TOR are required for proper skotomorphogenesis in etiolated Arabidopsis (Arabidopsis thaliana) seedlings, light-induced cotyledon opening, and regular development in light. Furthermore, we identify SnRK1 and TOR as signaling components acting upstream of light- and sugar-regulated alternative splicing events, expanding the known action spectra for these 2 key players in energy signaling. Our findings imply that concurring SnRK1 and TOR activities are required throughout various phases of plant development. Based on the current knowledge and our findings, we hypothesize that turning points in the activities of these sensor kinases, as expected to occur upon illumination of etiolated seedlings, instead of signaling thresholds reflecting the nutritional status may modulate developmental programs in response to altered energy availability.

A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis.

BACKGROUND: Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis. RESULTS: We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts-twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage. CONCLUSIONS: AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.

Subcellular Compartmentation of Alternatively Spliced Transcripts Defines SERINE/ARGININE-RICH PROTEIN30 Expression.

Alternative splicing (AS) is prevalent in higher eukaryotes, and generation of different AS variants is tightly regulated. Widespread AS occurs in response to altered light conditions and plays a critical role in seedling photomorphogenesis, but despite its frequency and effect on plant development, the functional role of AS remains unknown for most splicing variants. Here, we characterized the light-dependent AS variants of the gene encoding the splicing regulator Ser/Arg-rich protein SR30 in Arabidopsis (Arabidopsis thaliana). We demonstrated that the splicing variant SR30.2, which is predominantly produced in darkness, is enriched within the nucleus and strongly depleted from ribosomes. Light-induced AS from a downstream 3' splice site gives rise to SR30.1, which is exported to the cytosol and translated, coinciding with SR30 protein accumulation upon seedling illumination. Constitutive expression of SR30.1 and SR30.2 fused to fluorescent proteins revealed their identical subcellular localization in the nucleoplasm and nuclear speckles. Furthermore, expression of either variant shifted splicing of a genomic SR30 reporter toward SR30.2, suggesting that an autoregulatory feedback loop affects SR30 splicing. We provide evidence that SR30.2 can be further spliced and, unlike SR30.2, the resulting cassette exon variant SR30.3 is sensitive to nonsense-mediated decay. Our work delivers insight into the complex and compartmentalized RNA processing mechanisms that control the expression of the splicing regulator SR30 in a light-dependent manner.

Alternative Splicing Substantially Diversifies the Transcriptome during Early Photomorphogenesis and Correlates with the Energy Availability in Arabidopsis.

Plants use light as source of energy and information to detect diurnal rhythms and seasonal changes. Sensing changing light conditions is critical to adjust plant metabolism and to initiate developmental transitions. Here, we analyzed transcriptome-wide alterations in gene expression and alternative splicing (AS) of etiolated seedlings undergoing photomorphogenesis upon exposure to blue, red, or white light. Our analysis revealed massive transcriptome reprogramming as reflected by differential expression of ∼20% of all genes and changes in several hundred AS events. For more than 60% of all regulated AS events, light promoted the production of a presumably protein-coding variant at the expense of an mRNA with nonsense-mediated decay-triggering features. Accordingly, AS of the putative splicing factor REDUCED RED-LIGHT RESPONSES IN CRY1CRY2 BACKGROUND1, previously identified as a red light signaling component, was shifted to the functional variant under light. Downstream analyses of candidate AS events pointed at a role of photoreceptor signaling only in monochromatic but not in white light. Furthermore, we demonstrated similar AS changes upon light exposure and exogenous sugar supply, with a critical involvement of kinase signaling. We propose that AS is an integration point of signaling pathways that sense and transmit information regarding the energy availability in plants.

Phosphatidylinositol 4,5-bisphosphate influences PIN polarization by controlling clathrin-mediated membrane trafficking in Arabidopsis.

The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] during vegetative plant growth remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P2 levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux carriers PIN-FORMED (PIN1)-green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution. Furthermore, fewer brefeldin A-induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2 mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxin-dependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P2 in the regulation of clathrin-mediated endocytosis. Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for PtdIns(4,5)P2 in the control of clathrin dynamics and in auxin distribution in Arabidopsis.