Genetic and molecular analysis of trichome development in Arabis alpina.

The genetic and molecular analysis of trichome development in Arabidopsis thaliana has generated a detailed knowledge about the underlying regulatory genes and networks. However, how rapidly these mechanisms diverge during evolution is unknown. To address this problem, we used an unbiased forward genetic approach to identify most genes involved in trichome development in the related crucifer species Arabisalpina In general, we found most trichome mutant classes known in A. thaliana We identified orthologous genes of the relevant A. thaliana genes by sequence similarity and synteny and sequenced candidate genes in the A. alpina mutants. While in most cases we found a highly similar gene-phenotype relationship as known from Arabidopsis, there were also striking differences in the regulation of trichome patterning, differentiation, and morphogenesis. Our analysis of trichome patterning suggests that the formation of two classes of trichomes is regulated differentially by the homeodomain transcription factor AaGL2 Moreover, we show that overexpression of the GL3 basic helix-loop-helix transcription factor in A. alpina leads to the opposite phenotype as described in A. thaliana Mathematical modeling helps to explain how this nonintuitive behavior can be explained by different ratios of GL3 and GL1 in the two species.

The SEC6 protein is required for contractile vacuole function in Chlamydomonas reinhardtii.

Contractile vacuoles (CVs) are essential for osmoregulation in many protists. To investigate the mechanism of CV function in Chlamydomonas, we isolated novel osmoregulatory mutants. Four of the isolated mutant cell lines carried the same 33,641 base deletion, rendering the cell lines unable to grow under strong hypotonic conditions. One mutant cell line (Osmo75) was analyzed in detail. The CV morphology was variable in mutant cells, and most cells had multiple small CVs. In addition, one or two enlarged CVs or no visible CVs at all, were observed by light microscopy. These findings suggest that the mutant is impaired in homotypic vacuolar and exocytotic membrane fusion. Furthermore the mutants had long flagella. One of the affected genes is the only SEC6 homologue in Chlamydomonas (CreSEC6). The SEC6 protein is a component of the exocyst complex that is required for efficient exocytosis. Transformation of the Osmo75 mutant with a CreSEC6-GFP construct rescued the mutant completely (osmoregulation and flagellar length). Rescued strains overexpressed CreSEC6 (as a GFP-tagged protein) and displayed a modified CV activity. CVs were larger, whereas the CV contraction interval remained unchanged, leading to increased water efflux rates. Electron microscopy analysis of Osmo75 cells showed that the mutant is able to form the close contact zones between the plasma membrane and the CV membrane observed during late diastole and systole. These results indicate that CreSEC6 is essential for CV function and required for homotypic vesicle fusion during diastole and water expulsion during systole. In addition, CreSEC6 is not only necessary for CV function, but possibly influences the CV cycle in an indirect manner and flagellar length in Chlamydomonas.

Mutations in RABE1C suppress the spirrig mutant phenotype.

The plant BEACH-domain protein SPIRRIG (SPI) is involved in regulating cell morphogenesis and salt stress responses in Arabidopsis thaliana, Arabis alpina, and Marchantia polymorpha and was reported to function in the context of two unrelated cellular processes: vesicular trafficking and P-body mediated RNA metabolism. To further explore the molecular function of SPI, we isolated a second-site mutant, specifically rescuing the spi mutant trichome phenotype. The molecular analysis of the corresponding gene revealed a dominant negative mutation in RABE1C, a ras-related small GTP-binding protein that localizes to Golgi. Taken together, our data identified the genetic interaction between RABE1C and SPI, which is beneficial for further dissecting the function of SPI in vesicle trafficking-associated cell morphogenesis.

Functional analysis of the BEige and Chediak-Higashi domain gene MpSPIRRIG in Marchantia polymorpha.

BEige and Chediak-Higashi domain containing proteins (BDCPs) have been described to function in membrane-dependent processes in eukaryotes. This role was also observed for the BDCP SPIRRIG (SPI) in the model plant Arabidopsis thaliana in the context of cell morphogenesis. Additionally, AtSPI was found to control salt stress resistance by mediating mRNA stability and salt stress-dependent processing body formation. In this work, we utilize an evolutionarily comparative approach to unravel conserved, basal BDCP functions in the liverwort Marchantia polymorpha. Our phenotypic and physiological analyses show that MpSPI is involved in cell morphogenesis and salt resistance regulation, indicating that both functions are evolutionarily conserved between the two species. Co-localization was found with endosomal and P-body markers, suggesting links to membrane-dependent processes and mRNA metabolism. Finally, we present transcriptomics data showing that AtSPI and MpSPI regulate orthologous genes in A. thaliana and M. polymorpha.

Heat Stress-Dependent Association of Membrane Trafficking Proteins With mRNPs Is Selective.

The Arabidopsis AAA ATPase SKD1 is essential for ESCRT-dependent endosomal sorting by mediating the disassembly of the ESCRTIII complex in an ATP-dependent manner. In this study, we show that SKD1 localizes to messenger ribonucleoprotein complexes upon heat stress. Consistent with this, the interactome of SKD1 revealed differential interactions under normal and stress conditions and included membrane transport proteins as well as proteins associated with RNA metabolism. Localization studies with selected interactome proteins revealed that not only RNA associated proteins but also several ESCRTIII and membrane trafficking proteins were recruited to messenger ribonucleoprotein granules after heat stress.

Unravelling the molecular basis of the dominant negative effect of myosin XI tails on P-bodies.

The directional movement and positioning of organelles and macromolecules is essential for regulating and maintaining cellular functions in eukaryotic cells. In plants, these processes are actin-based and driven by class XI myosins, which transport various cargos in a directed manner. As the analysis of myosin function is challenging due to high levels of redundancy, dominant negative acting truncated myosins have frequently been used to study intracellular transport processes. A comparison of the dominant negative effect of the coiled-coil domains and the GTD domains revealed a much stronger inhibition of P-body movement by the GTD domains. In addition, we show that the GTD domain does not inhibit P-body movement when driven by a hybrid myosin in which the GTD domain was replaced by DCP2. These data suggest that the dominant negative effect of myosin tails involves a competition of the GTD domains for cargo binding sites.

Effectiveness of a psychological online training to promote physical activity among students: protocol of a randomized-controlled trial.

BACKGROUND: Many students in Germany do not meet recommended amounts of physical activity. In order to promote physical activity in students, web-based interventions are increasingly implemented. Yet, data on effectiveness of web-based interventions in university students is low. Our study aims at investigating a web-based intervention for students. The intervention is based on the Health Action Process Approach (HAPA), which discriminates between processes of intention formation (motivational processes) and processes of intention implementation (volitional processes). Primary outcome is change in physical activity; secondary outcomes are motivational and volitional variables as proposed by the HAPA as well as quality of life and depressive symptoms. METHODS: A two-armed randomized controlled trial (RCT) of parallel design is conducted. Participants are recruited via the internet platform StudiCare ( www.studicare.com ). After the baseline assessment (t1), participants are randomized to either intervention group (immediate access to web-based intervention) or control group (access only after follow-up assessment). Four weeks later, post-assessment (t2) is performed in both groups followed by a follow-up assessment (t3) 3 months later. Assessments take place online. Main outcome analyses will follow an intention-to-treat principle by including all randomized participants into the analyses. Outcomes will be analysed using a linear mixed model, assuming data are missing at random. The mixed model will include group, time, and the interaction of group and time as fixed effects and participant and university as random effect. DISCUSSION: This study is a high-quality RCT with three assessment points and intention-to-treat analysis meeting the state-of-the-art of effectiveness studies. Recruitment covers almost 20 universities in three countries, leading to high external validity. The results of this study will be of great relevance for student health campaigns, as they reflect the effectiveness of self-help interventions for young adults with regard to behaviour change as well as motivational and volitional determinants. From a lifespan perspective, it is important to help students find their way into regular physical activity. TRIAL REGISTRATION: The German clinical trials register (DRKS) DRKS00016889 . Registered on 28 February 2019.

Genetic and Molecular Analysis of Root Hair Development in Arabis alpina.

Root hair formation in Arabidopsis thaliana is a well-established model system for epidermal patterning and morphogenesis in plants. Over the last decades, many underlying regulatory genes and well-established networks have been identified by thorough genetic and molecular analysis. In this study, we used a forward genetic approach to identify genes involved in root hair development in Arabis alpina, a related crucifer species that diverged from A. thaliana approximately 26-40 million years ago. We found all root hair mutant classes known in A. thaliana and identified orthologous regulatory genes by whole-genome or candidate gene sequencing. Our findings indicate that the gene-phenotype relationships regulating root hair development are largely conserved between A. thaliana and A. alpina. Concordantly, a detailed analysis of one mutant with multiple hairs originating from one cell suggested that a mutation in the SUPERCENTIPEDE1 (SCN1) gene is causal for the phenotype and that AaSCN1 is fully functional in A. thaliana. Interestingly, we also found differences in the regulation of root hair differentiation and morphogenesis between the species, and a subset of root hair mutants could not be explained by mutations in orthologs of known genes from A. thaliana. This analysis provides insight into the conservation and divergence of root hair regulation in the Brassicaceae.

Evolutionary Comparison of the Developmental/Physiological Phenotype and the Molecular Behavior of SPIRRIG Between Arabidopsis thaliana and Arabis alpina.

Beige and Chediak Higashi (BEACH) domain proteins mediate membrane-dependent processes in eukaryotic cells. The plant BEACH domain protein SPIRRIG in A. thaliana (AtSPI) was shown to display a similar molecular behavior as its yeast and animal homologs, along with a range of cell morphological defects. In addition, AtSPI was shown to interact with the P-body component DCP1, to differentially effect RNA levels and to be involved in the regulation of RNA stability in the context of salt stress responses. To determine, whether the dual function of SPI in apparently unrelated molecular pathways and traits is evolutionary conserved, we analyzed three Aaspi alleles in Arabis alpina. We show that the molecular behavior of the SPI protein and the role in cell morphogenesis and salt stress response are similar in the two species, though we observed distinct deviations in the phenotypic spectrum.

Selection and validation of reference genes for quantitative Real-Time PCR in Arabis alpina.

Arabis alpina is a perennial arctic-alpine plant and an upcoming model organism for genetics and molecular biology for the Brassicaceae family. One essential method for most molecular approaches is the analysis of gene expression by reverse-transcription quantitative Real-Time PCR (RT-qPCR). For the normalisation of expression data in RT-qPCR experiments, it is essential to use reliable reference genes that are not affected under a wide range of conditions. In this study we establish a set of 15 A. alpina reference genes that were tested under different conditions including cold, drought, heat, salt and gibberellic acid treatments. Data analyses with geNORM, BestKeeper and NormFinder revealed the most stable reference genes for the tested conditions: RAN3, HCF and PSB33 are most suitable for cold treatments; UBQ10 and TUA5 for drought; RAN3, PSB33 and EIF4a for heat; CAC, TUA5, ACTIN 2 and PSB33 for salt and PSB33 and TUA5 for gibberellic acid treatments. CAC and ACTIN 2 showed the least variation over all tested samples. In addition, we show that two reference genes are sufficient to normalize RT-qPCR data under our treatment conditions. In future studies, these reference genes can be used for an adequate normalisation and thus help to generate high quality RT-qPCR data in A. alpina.