• Analysis Of CBL10 Gene Duplication In The Halophyte Eutrema salsugineum

      Schumaker, Karen S.; Magness, Courtney A.; Schumaker, Karen S.; Beilstein, Mark A.; Yadegari, Ramin (The University of Arizona., 2014)
      The buildup of salt in soils is a major abiotic stress that affects agricultural productivity, limiting the growth and yield of most crop species which cannot tolerate even modest levels of salinity (glycophytes). Genetic variability for salt tolerance exists as some plants (halophytes) have adapted to environments with high levels of salt. Understanding how salt tolerance has been acquired in halophytic species will be an important part of strategies to improve the ability of crops to grow in saline soils. The CALCINEURIN B-LIKE10 (AtCBL10) calcium sensor was identified as a component of salt signaling in the glycophyte Arabidopsis thaliana (A. thaliana) based on hypersensitivity of the Atcbl10 mutant to salt. When A. thaliana is grown in the presence of salt, AtCBL10 interacts with the AtSOS2 protein kinase to activate the AtSOS1 sodium/proton exchanger, resulting in the removal of sodium ions from the cytosol. Eutrema salsugineum (E. salsugineum), a halophytic relative of A. thaliana, has two CBL10 genes (EsCBL10a and EsCBL10b). In this research, the duplication of CBL10 in E. salsugineum was characterized and the functions of EsCBL10a and EsCBL10b in salt tolerance were determined. My analyses indicate that the coding sequences of EsCBL10a and EsCBL10b are highly conserved, as they share 85% nucleotide identity. An analysis of transcript structure indicates transcripts from EsCBL10a and EsCBL10b loci are alternatively spliced, but in distinct ways. My results suggest that EsCBL10a and AtCBL10 likely share the ancestral genomic position, while EsCBL10b might have moved to a different genomic region, and that the duplication took place prior to the divergence of expanded Lineage II species. The expression patterns of EsCBL10a and EsCBL10b are different; EsCBL10b transcript is high in shoots and low in roots while EsCBL10a transcript is detectable in both tissues. Preliminary analysis of E. salsugineum lines with reduced expression of EsCBL10a and EsCBL10b suggest that both genes might play a role during growth in the presence of salt, but that these roles are distinct.
    • GAPs in Plant Reproduction: Uncovering the Role of Glycosylphosphatidylinositol-Anchoring of Proteins in Arabidopsis Gametophyte Function

      Palanivelu, Ravishankar; Desnoyer, Nicholas James; Yadegari, Ramin; Beilstein, Mark (The University of Arizona., 2019)
      Glycosylphoshpatidylinositol (GPI) is a complex glycolipid molecule biosynthesized in eukaryotic cells that can be post-translationally attached to a broad range of proteins by the transamidase complex (GPI-T) to serve as a membrane anchor. Apart from tethering GPI-anchored proteins (GAPs) to the extracellular leaflet of plasma membranes, GPI confers many molecular attributes to the proteins it attaches to. While the synthesis and structure of GPI is well conserved within eukaryotes, the utility of GPI-anchoring as a mode of protein membrane attachment shows striking variation among the eukaryotic kingdoms. For example, in protozoan parasites, GAPs are the major form of cell surface proteins and can shield the cell from host defenses during infection. Fungi such as yeast produce GPI anchors that can covalently link to polysaccharides in the cell wall to regulate its architecture. Animal GAPs are essential for coordinated growth during embryonic development and can act as signaling molecules to mediate cell-cell communication. In land plants, GAPs play roles in the directional growth and expansion of sporophytic tissues and are critical for the fertility of the gametophyte generation. Despite GAPs constituting about 1% of the Arabidopsis thaliana genome, it is poorly understood how this family of proteins function in plant development. Therefore, in this thesis, I investigated the role of GPI in plant reproduction by reviewing all 19 A. thaliana GAPs described to date with mutant fertility defects and annotated the expression of all 250 predicted GAPs in the A. thaliana gametophytes. Additionally, I analyzed mutations in two components of the GPI-T complex to investigate the importance of GPI-anchoring in male and female gametophyte functions. I showed that GPI-anchoring in the female gametophyte is apparently dispensable but is essential in the male gametophyte for pollen tube germination and growth. Based on my work, I propose that key differences in the structure and development of the male and female gametophytes may account for the difference in necessity of GPI-anchoring within their compartments. Thus, any conclusion regarding the importance of GPI-anchoring of proteins in the female gametophyte must consider these unique attributes. Lastly, because at least two GAPs involved in the female gametophyte function associate with receptor-like kinases (RLKs) to initiate signal transduction, we established several tools that can be used to elucidate the binding site of a well-studied GAP-RLK pair, the LORELEI-FERONIA co-receptor complex. The conclusions and tools generated in this study will help close the gaps in our understanding of the role of GAPs in plant reproduction.