The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P 2 ] 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)P 2 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 auxindependent 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)P 2 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)P 2 in the control of clathrin dynamics and in auxin distribution 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 reprograming 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 BACKGROUND 1 (RRC1), 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 Plant Cell Advance Publication. Published on November 1, 2016November 1, , doi:10.1105 ©2016 American Society of Plant Biologists. All Rights Reserved 2 integration point of signaling pathways that sense and transmit information regarding the energy availability in plants.

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 39 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.Maturation of eukaryotic mRNAs involves intricate co-and posttranscriptional RNA processing, which has critical functions in regulating gene expression and diversifying the transcriptome. Among several mechanisms, alternative precursor mRNA splicing (AS) in particular generates many transcript variants by removing distinct intronic regions and joining the resulting exons. Deep analysis of transcriptomes via high-throughput RNA sequencing (RNA-seq) has revealed that a major fraction of all intron-containing genes from higher eukaryotes generates AS variants. In humans, more than 95% of multiexon genes display AS (Pan et al., 2008). The prevalence of AS has also been demonstrated for other eukaryotes including plants (Reddy et al., 2013;Staiger and Brown, 2013), with current estimates of ;61% and ;42% of intron-containing genes giving rise to AS variants in the model plants Arabidopsis (Arabidopsis thaliana;Marquez et al., 2012) and Brachypodium distachyon (Mandadi and Scholthof, 2015), respectively.Besides its pivotal role in increasing the coding and regulatory capacity of the transcriptome, AS finetunes gene expression by varying the output ratios of splicing variants. The full extent of AS regulation likely exceeds the current estimates, as the production of many transcript variants can be specifically controlled under certain conditions, such as cell and tissue types, developmental stages, and in response to stresses and other environmental factors (Reddy et al., 2013;Staige...