Umeå University's logo

Protein–protein interactions (PPIs) are at the heart of most cellular processes but despite recent progress, their genome-wide analysis remains challenging. With this in mind, we have developed SoPPIs (sequencing of PPIs), a powerful method that facilitates parallelized PPI analyses using an innovative combination of the split-Cre/loxP system and high-throughput DNA sequencing. Sequential recombination of plasmids encodes information about pairs of interacting proteins in recombined DNA, facilitating their easy and cost-efficient identification by next-generation sequencing. Importantly, SoPPIs works with most soluble proteins, can be implemented in any cell type that can be transformed with episomal plasmids, and is in principle capable of interrogating all possible PPIs in an organism in a single experiment. To demonstrate the power of SoPPIs, we analyzed the composition of the Arabidopsis thaliana LSm/Sm ring, an evolutionarily highly conserved core component of the spliceosome and performed parallelized library screens to identify LSm/Sm-interacting proteins. Given its versatility and usability, we expect SoPPIs to quickly gain popularity and help provide insights into the PPI networks underlying complex biological systems.

Plant growth and development are regulated by many factors, including carbohydrate availability and signaling. Trehalose 6-phosphate (T6P), which is synthesized by TREHALOSE-6-PHOSPHATE SYNTHASE 1 (TPS1), is positively associated with and functions as a signal that informs the cell about the carbohydrate status. Mutations in TPS1 negatively affect the growth and development of Arabidopsis (Arabidopsis thaliana), and complete loss-of-function alleles are embryo-lethal, which can be overcome using inducible expression of TPS1 (GVG::TPS1) during embryogenesis. Using ethyl methane sulfonate mutagenesis in combination with genome re-sequencing, we have identified several alleles in the floral regulator gene HUA2 that restore flowering in tps1-2 GVG::TPS1. Genetic analyses using an HUA2 T-DNA insertion allele, hua2-4, confirmed this finding. RNA-seq analyses demonstrated that hua2-4 has widespread effects on the tps1-2 GVG::TPS1 transcriptome, including key genes and pathways involved in regulating flowering. Higher order mutants combining tps1-2 GVG::TPS1 and hua2-4 with alleles in the key flowering time regulators FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), and FLOWERING LOCUS C (FLC) were constructed to analyze the role of HUA2 during floral transition in tps1-2 in more detail. Our findings demonstrate that loss of HUA2 can restore flowering in tps1-2 GVG::TPS1, in part through activation of FT, with contributions from the upstream regulators SOC1 and FLC. Interestingly, we found that mutation of FLC is sufficient to induce flowering in tps1-2 GVG::TPS1. Furthermore, we observed that mutations in HUA2 modulate carbohydrate signaling and that this regulation might contribute to flowering in hua2-4 tps1-2 GVG::TPS1.

Plants are prone to genome duplications and tend to preserve multiple gene copies. This is also the case for the genes encoding the Sm proteins of Arabidopsis thaliana (L). The Sm proteins are best known for their roles in RNA processing such as pre-mRNA splicing and nonsense-mediated mRNA decay. In this study, we have taken a closer look at the phylogeny and differential regulation of the SmE-coding genes found in A. thaliana, PCP/SmE1, best known for its cold-sensitive phenotype, and its paralog, PCPL/SmE2. The phylogeny of the PCP homologs in the green lineage shows that SmE duplications happened multiple times independently in different plant clades and that the duplication that gave rise to PCP and PCPL occurred only in the Brassicaceae family. Our analysis revealed that A. thaliana PCP and PCPL proteins, which only differ in two amino acids, exhibit a very high level of functional conservation and can perform the same function in the cell. However, our results indicate that PCP is the prevailing copy of the two SmE genes in A. thaliana as it is more highly expressed and that the main difference between PCP and PCPL resides in their transcriptional regulation, which is strongly linked to intronic sequences. Our results provide insight into the complex mechanisms that underlie the differentiation of the paralogous gene expression as an adaptation to stress.

Background: Spliceosomes are large evolutionary conserved ribonucleoprotein complexes containing at their core heptameric rings of Sm (or LSm) proteins and U-rich snRNAs. The role of Sm proteins in animal development is well established, and recent research has begun to link mutations in these genes to growth defects in plants. One of the most studied Sm genes is SmE1/PCP, mutants of which display a temperature-dependent phenotype in Arabidopsis thaliana.

Results: This study provides a first glimpse into the function of a core splicing protein in the regulation of growth in a perennial species. Phylogenetic analysis identified two paralogous SmE genes in poplar, named SmEa and SmEb, that encode identical proteins and are orthologs of SmEs from Arabidopsis, as suggested by Y2H and in vivo experiments. CRISPR/Cas9 mutagenesis in hybrid aspen identified a role for SmEs in development in plants grown in an environment simulating seasonal photoperiod and temperature changes. Unlike in Arabidopsis, low temperatures had no or only a very minor effect on the development of sme mutants in aspen.

Conclusions: We identified specific aspects of SmE in poplar, highlighting the importance of examining the physiological and evolutionary differences that define this gene family in woody compared to herbaceous plants.

Background: Open chromatin regions host DNA regulatory motifs that are accessible to transcription factors and the transcriptional machinery. In Arabidopsis, responses to light are heavily regulated at the transcriptional level. Shade, for example, can limit photosynthesis and is rapidly perceived by phytochromes as a reduction of red to far-red light ratio (LRFR). Under shade, phytochromes become inactive, enabling PHYTOCHROME INTERACTING FACTORs (PIFs), particularly PIF7, to promote genome-wide reprogramming essential for LRFR responses. An initial strong and fast regulation of shade-responsive genes is followed by attenuation of this response under prolonged shade.

Results: To determine whether the transcriptional response to shade depends on chromatin accessibility, we use ATAC-seq to profile the chromatin of seedlings exposed to short (1 h) and long (25 h) simulated shade. We find that PIF7 binding sites are accessible for most early target genes before LRFR treatment. The transcription pattern of most acute shade-responsive genes correlates with a rapid increase in PIF levels and chromatin association at 1 h, and its decrease at 25 h of shade exposure. For a small subset of acutely responding genes, PIFs also modulate chromatin accessibility at their binding sites early and/or late in the response to shade.

Conclusions: Our results suggest that in seedlings a state of open chromatin conformation allows PIFs to easily access and recognize their binding motifs, rapidly initiating gene expression triggered by shade. This transcriptional response primarily depends on a transient increase in PIF stability and gene occupancy, accompanied by changes in chromatin accessibility in a minority of genes.

• Appropriate abiotic stress response is pivotal for plant survival and makes use of multiple signaling molecules and phytohormones to achieve specific and fast molecular adjustments. A multitude of studies has highlighted the role of alternative splicing in response to abiotic stress, including temperature, emphasizing the role of transcriptional regulation for stress response. Here we investigated the role of the core-splicing factor PORCUPINE (PCP) on temperature-dependent root development.
• We used marker lines and transcriptomic analyses to study the expression profiles of meristematic regulators and mitotic markers, and chemical treatments, as well as root hormone profiling to assess the effect of auxin signaling.
• The loss of PCP significantly alters RAM architecture in a temperature-dependent manner. Our results indicate that PCP modulates the expression of central meristematic regulators and is required to maintain appropriate levels of auxin in the RAM.
• We conclude that alternative pre-mRNA splicing is sensitive to moderate temperature fluctuations and contributes to root meristem maintenance, possibly through the regulation of phytohormone homeostasis and meristematic activity.

Many functionally distinct plant tissues have relatively low numbers of cells that are embedded within complex tissues. For example, the shoot apical meristem (SAM) consists of a small population of pluripotent stem cells surrounded by developing leaves and/or flowers at the growing tip of the plant. It is technically challenging to collect enough high-quality SAM samples for molecular analyses. Isolation of Nuclei Tagged in specific Cell Types (INTACT) is an easily reproducible method that allows the enrichment of biotin-tagged cell-type-specific nuclei from the total nuclei pool using biotin-streptavidin affinity purification. Here, we provide a detailed INTACT protocol for isolating nuclei from the Arabidopsis SAM. One can also adapt this protocol to isolate nuclei from other tissues and cell types for investigating tissue/cell-type-specific transcriptome and epigenome and their changes during developmental programs at a high spatiotemporal resolution. Furthermore, due to its low cost and simple procedures, INTACT can be conducted in any standard molecular laboratory.

Plants undergo transcriptome reprograming to adapt to daily and seasonal fluctuations in light and temperature conditions. While most efforts have focused on the role of master transcription factors, the importance of splicing factors modulating these processes is now emerging. Efficient pre-mRNA splicing depends on proper spliceosome assembly, which in plants and animals requires the methylosome complex. Ion Chloride nucleotide-sensitive protein (PICLN) is part of the methylosome complex in both humans and Arabidopsis (Arabidopsis thaliana), and we show here that the human PICLN ortholog rescues phenotypes of Arabidopsis picln mutants. Altered photomorphogenic and photoperiodic responses in Arabidopsis picln mutants are associated with changes in pre-mRNA splicing that partially overlap with those in PROTEIN ARGININE METHYL TRANSFERASE5 (prmt5) mutants. Mammalian PICLN also acts in concert with the Survival Motor Neuron (SMN) complex component GEMIN2 to modulate the late steps of UsnRNP assembly, and many alternative splicing events regulated by PICLN but not PRMT5, the main protein of the methylosome, are controlled by Arabidopsis GEMIN2. As with GEMIN2 and SM PROTEIN E1/PORCUPINE (SME1/PCP), low temperature, which increases PICLN expression, aggravates morphological and molecular defects of picln mutants. Taken together, these results establish a key role for PICLN in the regulation of pre-mRNA splicing and in mediating plant adaptation to daily and seasonal fluctuations in environmental conditions.

In temperate and boreal regions, perennials adapt their annual growth cycle to the change of seasons. These adaptations ensure survival in harsh environmental conditions, allowing growth at different latitudes and altitudes, and are therefore tightly regulated. Populus tree species cease growth and form terminal buds in autumn when photoperiod falls below a certain threshold.1 This is followed by establishment of dormancy and cold hardiness over the winter. At the center of the photoperiodic pathway in Populus is the gene FLOWERING LOCUS T2 (FT2), which is expressed during summer and harbors significant SNPs in its locus associated with timing of bud set.1–4 The paralogous gene FT1, on the other hand, is hyper-induced in chilling buds during winter.3,5 Even though its function is so far unknown, it has been suggested to be involved in the regulation of flowering and the release of winter dormancy.3,5 In this study, we employ CRISPR-Cas9-mediated gene editing to individually study the function of the FT-like genes in Populus trees. We show that while FT2 is required for vegetative growth during spring and summer and regulates the entry into dormancy, expression of FT1 is absolutely required for bud flush in spring. Gene expression profiling suggests that this function of FT1 is linked to the release of winter dormancy rather than to the regulation of bud flush per se. These data show how FT duplication and sub-functionalization have allowed Populus trees to regulate two completely different and major developmental control points during the yearly growth cycle.

Sensing carbohydrate availability is essential for plants to coordinate their growth and development. In Arabidopsis thaliana, TREHALOSE 6-PHOSPHATE SYNTHASE 1 (TPS1) and its product, trehalose 6-phosphate (T6P), are important for the metabolic control of development. tps1 mutants are embryo-lethal and unable to flower when embryogenesis is rescued. T6P regulates development in part through inhibition of SUCROSE NON-FERMENTING1 RELATED KINASE1 (SnRK1).

Here, we explored the role of SnRK1 in T6P-mediated plant growth and development using a combination of a mutant suppressor screen and genetic, cellular and transcriptomic approaches.

We report nonsynonymous amino acid substitutions in the catalytic KIN10 and regulatory SNF4 subunits of SnRK1 that can restore both embryogenesis and flowering of tps1 mutant plants. The identified SNF4 point mutations disrupt the interaction with the catalytic subunit KIN10.

Contrary to the common view that the two A. thaliana SnRK1 catalytic subunits act redundantly, we found that loss-of-function mutations in KIN11 are unable to restore embryogenesis and flowering, highlighting the important role of KIN10 in T6P signalling.