Evolutionary Analysis of MBW Function by Phenotypic Rescue in Arabidopsis thaliana

The MBW complex consisting of the three proteins R2R3MYB, bHLH and WDR regulates five traits in Arabidopsis thaliana including trichome and root hair patterning, seed coat color, anthocyanidin production and seed coat mucilage release. The WDR gene TTG1 regulates each trait in specific combinations with different bHLH and R2R3MYB proteins. In this study we analyze to what extent the biochemical properties of the MBW proteins contribute to trait specificity by expressing them in appropriate A. thaliana mutants. We show that the rescue behavior of A. thaliana bHLH and R2R3MYB protein is sufficient to explain the function as derived previously from mutant analysis. When extending this rescue approach using MBW proteins from other species we find that proteins involved in anthocyanidin regulation typically show a rescue of the anthocyanidin phenotype but not of the other traits. Finally, we correlate the rescue abilities of MBW protein from different species with the A. thaliana proteins

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

Nuclear trapping by GL3 controls intercellular transport and redistribution of TTG1 protein in Arabidopsis

Trichome patterning on Arabidopsis leaves is one of the best-studied model systems for two-dimensional de novo patterning. In addition to an activator-inhibitor-related mechanism, we previously proposed a depletion mechanism to operate during this process such that GLABRA3 (GL3) traps the trichome-promoting factor TRANSPARENT TESTA GLABRA1 (TTG1) in trichomes that, in turn, results in a depletion of TTG1 in trichome neighbouring cells. In this manuscript we analyze the molecular basis underlying this trapping mechanism. We demonstrate the ability of GL3 to regulate TTG1 mobility by expressing TTG1 and GL3 in different tissue layers in different combinations. We further show that TTG1 trapping by GL3 is based on direct interaction between both proteins and recruitment in the nucleus

Physical, Functional and Genetic Interactions between the BEACH Domain Protein SPIRRIG and LIP5 and SKD1 and Its Role in Endosomal Trafficking to the Vacuole in Arabidopsis

Beige and Chediak Higashi (BEACH) domain-containing proteins (BDCPs) are facilitators of membrane-dependent cellular processes in eukaryotes. Mutations in BDCPs cause malfunctions of endosomal compartments in various cell types. Recently, the molecular analysis of the BDCP homolog gene SPIRRIG (SPI) has revealed a molecular function in P-bodies and the regulation of RNA stability. We therefore aimed to analyze, whether SPI has also a role in membrane-dependent processes. In this study, we show that SPI physically interacts with endosomal sorting complex required for transport associated ATPase Suppressor of K+-transport growth defect1 (SKD1) and its positive regulator, LYST Interacting Protein 5 (LIP5) and report genetic interactions between SPI and SKD1 and LIP5. We further show that the endosomal transport route of soluble proteins to the lytic vacuole is disturbed in spi lip5 double mutants but not in the single mutants. These vacuolar transport defects were suppressed by additional expression of SKD1. Our results indicate that the BEACH domain protein SPI has in addition to a role in P-bodies a function in endosomal transport routes

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

Evolutionary comparison of competitive protein-complex formation of MYB, bHLH, and WDR proteins in plants

A protein complex consisting of a MYB, basic Helix-Loop-Helix, and a WDR protein, the MBW complex, regulates five traits, namely the production of anthocyanidin, proanthocyanidin, and seed-coat mucilage, and the development of trichomes and root hairs. For complexes involved in trichome and root hair development it has been shown that the interaction of two MBW proteins can be counteracted by the respective third protein (called competitive complex formation). We examined competitive complex formation for selected MBW proteins from Arabidopsis thaliana, Arabis alpina, Gossypium hirsutum, Petunia hybrida, and Zea mays. Quantitative analyses of the competitive binding of MYBs and WDRs to bHLHs were done by pull-down assays using ProtA- and luciferase-tagged proteins expressed in human HEC cells. We found that some bHLHs show competitive complex formation whilst others do not. Competitive complex formation strongly correlated with a phylogenetic tree constructed with the bHLH proteins under investigation, suggesting a functional relevance. We demonstrate that this different behavior can be explained by changes in one amino acid and that this position is functionally relevant in trichome development but not in anthocyanidin regulation

The BEACH Domain Protein SPIRRIG Is Essential for Arabidopsis Salt Stress Tolerance and Functions as a Regulator of Transcript Stabilization and Localization

Members of the highly conserved class of BEACH domain containing proteins (BDCPs) have been established as broad facilitators of protein-protein interactions and membrane dynamics in the context of human diseases like albinism, bleeding diathesis, impaired cellular immunity, cancer predisposition, and neurological dysfunctions. Also, the Arabidopsis thaliana BDCP SPIRRIG (SPI) is important for membrane integrity, as spi mutants exhibit split vacuoles. In this work, we report a novel molecular function of the BDCP SPI in ribonucleoprotein particle formation. We show that SPI interacts with the P-body core component DECAPPING PROTEIN 1 (DCP1), associates to mRNA processing bodies (P-bodies), and regulates their assembly upon salt stress. The finding that spi mutants exhibit salt hypersensitivity suggests that the local function of SPI at P-bodies is of biological relevance. Transcriptome-wide analysis revealed qualitative differences in the salt stress-regulated transcriptional response of Col-0 and spi. We show that SPI regulates the salt stress-dependent post-transcriptional stabilization, cytoplasmic agglomeration, and localization to P-bodies of a subset of salt stress-regulated mRNAs. Finally, we show that the PH-BEACH domains of SPI and its human homolog FAN (Factor Associated with Neutral sphingomyelinase activation) interact with DCP1 isoforms from plants, mammals, and yeast, suggesting the evolutionary conservation of an association of BDCPs and P-bodies

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

Quantitative trait loci controlling leaf venation in Arabidopsis

Leaf veins provide the mechanical support and are responsible for the transport of nutrients and water to the plant. High vein density is a prerequisite for plants to have C4 photosynthesis. We investigated the genetic variation and genetic architecture of leaf venation traits within the species Arabidopsis thaliana using natural variation. Leaf venation traits, including leaf vein density (LVD) were analysed in 66 worldwide accessions and 399 lines of the multi-parent advanced generation intercross population. It was shown that there is no correlation between LVD and photosynthesis parameters within A. thaliana. Association mapping was performed for LVD and identified 16 and 17 putative quantitative trait loci (QTLs) in the multi-parent advanced generation intercross and worldwide sets, respectively. There was no overlap between the identified QTLs suggesting that many genes can affect the traits. In addition, linkage mapping was performed using two biparental recombinant inbred line populations. Combining linkage and association mapping revealed seven candidate genes. For one of the candidate genes, RCI2c, we demonstrated its function in leaf venation patterning

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