Vascular tissues provide both the mechanical support to the plant body and the conducting cells for the transport of water, mineral solutes, hormones and other signaling molecules, amino acids, and sugars. To identify genes that may regulate vascular tissue-specific functions, we isolated xylem, phloem-cambium, and nonvascular tissues from the Arabidopsis root-hypocotyl, performed a genome-wide comparative analysis of tissue-specific transcripts using the 24K Affymetrix Arabidopsis ATH1 Genome Array (24K GeneChip), and identified potential genes that are required for xylem and phloem differentiation or tissue-specific functions. Based on this comparative analysis, two phloem-specific G2-like transcription factors, MYR1 and MYR2, and a xylem-specific NAC domain family member, XND1, were selected for further characterization. Under continuous light, myr2 plants flowered early, while myr1 plants did not differ significantly from wild type controls. However, double mutant myr1myr2 plants exhibited a novel phenotype characterized by elongated petioles, semi-erect leaf orientation, and suppression of lateral shoot outgrowth. These characteristics are reminiscent of yucca, a dominant Arabidopsis mutant with elevated levels of free auxin. Preliminary results indicated that like yucca, myr1myr2 plants were more resistant than wt plants to 5-mT, a toxic tryptophan analog, suggesting that MYR1 and MYR2 may be involved in regulating tryptophan-dependent auxin biosynthesis. Overexpression of any one of MYR1 isoforms resulted in a phenotype that in some cases resembled that observed in the double mutant, indicating that the regulation mediated by MYR1 and MYR2 may depend on formation of specific heterodimers consisting of isoforms of MYR1 and/or MYR2, and that the dimerization was susceptible to disruption both by overexpression and loss-of-function of MYR1/MYR2. Overexpression of XND1 resulted in the absence of TEs as determined from the absence of both secondary cell wall deposition and TE death. Using 3 tissue-specific promoter-GUS lines as genetic backgrounds, we demonstrated that overexpression of XND1 suppressed only TE-specific GUS expression but not phloem-specific GUS expression. Three T-DNA/transposon insertion lines, xnd1-1, -2, and -3, were identified. Under normal conditions, xnd1 did not exhibit significantly different growth and development compared to wild type plants. However, preliminary data indicated that xnd1 plants were ABA and cold hypersensitive. Yeast-two hybrid screening using the N-terminal portion of XND1 as bait identified a novel RING finger protein, At3g62970 that may function as the ubiquitin ligase (E3). These results suggested that XND1 functions as a negative regulator of xylem cell differentiation, and that the regulation mediated by XND1 may be integrated with the ubiquitin/26S proteasome pathway. / Ph. D.

Proteins of the nuclear envelope play important roles in a variety of cellular processes e.g. transport of proteins between the nucleus and cytoplasm, co-ordination of nuclear and cytoplasmic events, anchoring of chromatin to the nuclear periphery and regulation of transcription. Defects in proteins of the nuclear envelope and the nuclear pore complexes have been related to a number of human diseases. To understand the cellular functions in which nuclear envelope proteins participate it is crucial to map the functions of these proteins. The present study was done in order to characterize the role of three different proteins in functions related to the nuclear envelope in the yeast Saccharomyces cerevisiae. The arginine methyltransferase Rmt2 was demonstrated to associate with proteins of the nuclear pore complexes and to influence nuclear export. In addition, Rmt2 was found to interact with the Lsm4 protein involved in RNA degradation, splicing and ribosome biosynthesis. These results provide support for a role of Rmt2 at the nuclear periphery and potentially in nuclear transport and RNA processing. The integral membrane protein Cwh43 was localized to the inner nuclear membrane and was also found at the nucleolus. A nuclear function for Cwh43 was demonstrated by its ability to bind DNA in vitro. A link to nucleolar functions was demonstrated by genetic analysis. Furthermore, Cwh43 is interacting with signalling pathways perhaps acting as a sensor for signals transmitted from the cytoplasm to the nucleus. The Myr1 protein was found to be membrane-associated and to interact with proteins involved in vesicular traffic. Overexpression of Myr1 affects nuclear morphology and nuclear pore distribution suggesting a function in membrane dynamics. In conclusion, the presented results aid in a deeper understanding of functions related to the nuclear envelope in revealing a novel link between arginine methylation and the nuclear periphery, identifying a novel inner nuclear membrane protein and a new membrane-associated protein.

<p>Intracellular membrane trafficking is regulated by a large number of protein complexes and lipids. Blocking of trafficking disrupts normal membrane dynamics and causes membrane stress. Two similar proteins from <i>Saccharomyces cerevisiae</i>, Myr1 and Smy2, each containing a polyproline-binding GYF domain, were discovered in separate screens for dosage suppressors of trafficking mutations. The functions of GYF domain proteins are poorly described despite its determined structure and a number of known polyproline peptide ligands. We predicted, using computational analysis, associations between mRNA decay factors and both Myr1 and Smy2, and further demonstrated that they localize to sites of mRNA degradation upon stress, in a GYF domain dependent manner.</p><p>Ypt6 is a small GTPase that regulates vesicle docking at the late Golgi in budding yeast. Myr1 was found as a novel suppressor during the screening of a genomic library in a null ypt6 mutant. Myr1 additionally was capable of rescuing the temperature sensitive growth of a Ric1 deficient strain. Importantly, Ric1 is an activator of Ypt6 and is synthetic lethal with Myr1. Biochemical characterization of the Myr1 protein revealed a limited solubility and an ability to bind cellular membranes, likely relevant to the rescue of trafficking mutants.</p><p>We further assayed the affinity of Myr1 domains to liposomes of distinct composition. Preference for negatively charged lipids suggested possible electrostatic interactions with polybasic clusters within C-terminal regions of Myr1. In contrast, the N-terminus with the GYF domain was found to be capable of self-association. Membrane stress caused by a lipid-bilayer perturbing drug resulted in induced formation of mRNA processing bodies. Cumulatively, these studies suggest that Myr1 functions in the regulation of mRNA stability via its GYF domain, and can sense membrane stress by binding to the lipid bilayer.</p>

Intracellular membrane trafficking is regulated by a large number of protein complexes and lipids. Blocking of trafficking disrupts normal membrane dynamics and causes membrane stress. Two similar proteins from Saccharomyces cerevisiae, Myr1 and Smy2, each containing a polyproline-binding GYF domain, were discovered in separate screens for dosage suppressors of trafficking mutations. The functions of GYF domain proteins are poorly described despite its determined structure and a number of known polyproline peptide ligands. We predicted, using computational analysis, associations between mRNA decay factors and both Myr1 and Smy2, and further demonstrated that they localize to sites of mRNA degradation upon stress, in a GYF domain dependent manner. Ypt6 is a small GTPase that regulates vesicle docking at the late Golgi in budding yeast. Myr1 was found as a novel suppressor during the screening of a genomic library in a null ypt6 mutant. Myr1 additionally was capable of rescuing the temperature sensitive growth of a Ric1 deficient strain. Importantly, Ric1 is an activator of Ypt6 and is synthetic lethal with Myr1. Biochemical characterization of the Myr1 protein revealed a limited solubility and an ability to bind cellular membranes, likely relevant to the rescue of trafficking mutants. We further assayed the affinity of Myr1 domains to liposomes of distinct composition. Preference for negatively charged lipids suggested possible electrostatic interactions with polybasic clusters within C-terminal regions of Myr1. In contrast, the N-terminus with the GYF domain was found to be capable of self-association. Membrane stress caused by a lipid-bilayer perturbing drug resulted in induced formation of mRNA processing bodies. Cumulatively, these studies suggest that Myr1 functions in the regulation of mRNA stability via its GYF domain, and can sense membrane stress by binding to the lipid bilayer.