Plant Biology Graduate Program Masters Theses Collection

Development of a Cytosolic pH Reporter for Tobacco BY2 Cells

Urbanowski, Michael E. — Fri, 23 Nov 2012 07:53:32 PST

The regulation of pH is a critical homeostatic function of plant cells. In addition to acting as the primary cationic species responsible for energizing the plasma membrane, protons likely act as an important regulator and messenger. Despite this importance, few studies have thoroughly described cytosolic pH patterns as the plant cell progresses through the cell cycle. To investigate pH in plant cells, I chose Nicotiana tabacum (tobacco) Bright Yellow-2 (BY-2) cells as a model system. My research has two aims. First, I will measure and report the interphase cytosolic pH of BY-2 cells. Next, I will assay the cytosolic pH as BY-2 cells progress through mitosis and cytokinesis. I hypothesize that pH patterns are be temporally or spatially associated with structures such as the mitotic spindle or the phragmoplast. To investigate cytosolic pH in BY-2 cells, I will develop a cytosolic pH reporter based on a pH sensitive ratiometric fluorescent dye. This dye will be able to resolve both temporal and spatial changes in pH throughout the cytosol while imposing a minimal amount of stress on BY-2 cells. I found that pH-GFP, a variant of eGFP, had qualities of a robust pH reporter. To introduce the dye, explored biolistic bombardment, Agrobacterium mediated transient transformation, and polyethylene glycol mediated transformation as methods for introducing the pH-GFP gene into BY-2 cells. I observed very few transformation events using these methods and my observations did not support these approaches as suitable for introducing pH-GFP into BY-2 cells.

Investigation of Maize Yellow-stripe1 and Iron Signaling in Arabidopsis thaliana

Vasques, Kenneth A. — Thu, 23 Aug 2012 05:54:19 PDT

Iron is an essential micronutrient that plays a role in essential processes in all living organisms. Because iron deficiency anemia is the number one human nutritional deficiency worldwide, research has been focused on studying biofortification, a method of plant breeding focused on increasing the nutrient content of the grain. In order to effectively implement this strategy, research to understand the molecular mechanisms surrounding iron uptake and maintenance within plants is necessary. A major goal of this work lies in exploring iron signaling in Arabidopsis thaliana and further characterization of the yellowstripe1-like1yellowstripe1-like3 (ysl1ysl3) double mutant plant. As shown here, iron signaling in arabidopsis appears to be regulated by a long distance signal derived in the shoots. The ysl1ysl3 double mutant is a plant shown to have severe mis-regulation of iron regulated genes. Here, I show ysl1ysl3 mutant plants are unable to respond to iron deficiency properly and lose signaling ability. It has been proposed that these defects are due to iron accumulation in the shoots but that appears untrue suggesting alternative functions for YSL1 and YSL3 in iron signaling. Another goal of the work included here seeks to elucidate alternative roles for ZmYS1 in maize outside of primary acquisition of iron from the soil. Here, ZmYS1 expression is shown via promoter::GUS analysis expressing throughout the shoot in many cell-types consistent with a role in the movement of iron between leaf tissues. Interestingly, expression in germinating seeds suggests ZmYS1 may also play a role in remobilization of stored iron in developing embryos.

Functional Analysis of Receptor-like Kinases in Pollen-Pistil Interactions in Arabidopsis thaliana

Aggarwal, Mini — Wed, 02 Dec 2009 11:25:31 PST

Rac/Rop GTPases are molecular switches in plants that control the growth of polarized cells such as pollen tubes and root hairs, differentiation, development, actin dynamics, production of reactive oxygen species (ROS) and disease resistance. These small GTPases are activated by guanine nucleotide exchange factors (GEFs) that replace GDP for GTP and are referred to as RopGEFs in plants. To identify upstream components of the RopGEF regulated signaling pathways, GEF1 from Arabidopsis thaliana was used as a bait to screen a seedling cDNA library in a yeast two-hybrid system. This yielded members of a small family of the Catharanthus roseus Receptor-like kinase (CrRLK), referred to as the Feronia-like Receptor-like kinase (FlRLK) family as the potential GEF interactors. A synergid cell-expressed member of this family, FERONIA/SIRENE (RLK-10), regulates pollen tube reception by the female gametophyte and along with the other two plasma-membrane bound receptors of this family, THESEUS (RLK-3) and HERCULES (RLK-4) promote cell elongation in Arabidopsis.

I have chosen two of these FlRLKs for my studies: RLK-5, the most abundant pollen expressed member of the family and RLK10. My studies with RLK-5 suggest that it plays a significant role in pollination or fertilization since homozygous rlk-5 was never recovered from selfed heterozygous rlk-5 mutant. In addition to this, the reciprocal crosses with RLK-5/rlk-5 and wild type resulted in severe male transmission defect indicating that the rlk-5 mutation induces male sterility. This observation was found consistent with the pollen-specific expression pattern of RLK-5 suggested by microarray data and confirmed by histochemical GUS staining analysis of the RLK5p-GUS transgenic Arabidopsis. The RLK-5/rlk-5 pollen displayed no pollen-viability defects and the pollen tube growth in-vivo appears normal. Despite the unaffected pollen viability and apparent normal in-vivo pollen tube growth, the RLK-5/rlk-5 plants formed low seed set suggesting compromised fertilization. Additional analysis will be required to determine the basis of male deficiency and reduced seed sets in RLK5/rlk5 mutants.

Loss of function mutations of RLK-10 (FERONIA/SIRENE) gene show failure in pollen tube growth arrest upon penetration of the female gametophyte, supernumerary pollen tube penetration of the ovule and reduced female fertility. Our studies with RLK-10 suggest that it is important for mediating a proper oxido-reductive condition within the ovule necessary for pollen tube rupture and fertilization of the female gametophyte. The multiple pollen tube entry phenotype of the Arabidopsis knockout mutant, rlk-10 correlated with decreased ROS level and de-esterified pectin in the filiform apparatus lining the synergid cell of its female gametophyte. These results establish an important signaling link between RLK-10 and ROS and pectin in a GEF-Rac/Rop regulated pathway in pollen tube-ovule interaction.

Investigating the Role of YSLs in Oryza sativa and Arabidopsis thaliana through RT-PCR and Yeast Functional Complementation

Chiecko, Jeffrey — Wed, 02 Dec 2009 11:03:15 PST

Analysis of Myosin VIII Function in the Moss Physcomitrella Patens

Ritchie, Julie — Fri, 03 Apr 2009 11:52:41 PDT

Site Characteristics and Plant Invasion: Light Limitation of Invasive Establishment and Impacts of Elaeagnus Umbellata on Soil Nitrogen Availability and Co-occurring Species

Mostoller, Erin L. — Mon, 15 Dec 2008 07:51:33 PST

Invasive species have become an increasing threat to many habitats worldwide. This research addressed the questions of whether several invasive woody plants can germinate, survive and grow in light levels typical of an undisturbed forest understory, and also whether alteration of the nitrogen cycle by one invader is likely to increase the rate of invasion by others.

Self-Incompatibility in African Lycium (Solanaceae)

Feliciano, Natalie M. — Mon, 18 Aug 2008 07:08:34 PDT

Chapter one of my thesis has been published in the May 2008 issue of the journal Evolution under the title “A TALE OF TWO CONTINENTS: BAKER’S RULE AND THE MAINTENANCE OF SELF-INCOMPATIBILITY IN LYCIUM (SOLANACEAE).” This chapter was co-authored by Dr. Jill S. Miller and Dr. Rachel Levin.

A Molecular Phylogenetic Assessment of Pseudendoclonium

Mullins, Richard F. — Wed, 09 Jan 2008 11:07:11 PST

ABSTRACT A MOLECULAR PHYLOGENETIC ASSESSMENT OF PSEUDENDOCLONIUM SEPTEMBER 2007 RICHARD F. MULLINS JR., B.S., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Professor Daniel R. Cooley Pseudendoclonium was established in 1900 by N. Wille to include a crust-forming green microalga occurring near the high water line on jetties in Drobak, Norway. Ordinal and familial affiliation of the genus have remained uncertain due to a lack of distinguishing morphological characteristics and because molecular phylogenetic data have not been generated for the type species. Ribosomal SSU rDNA sequence data for Pseudendoclonium submarinum, the type species, are presented. Phylogenetic analysis of these data place Pseudendoclonium within the Ulvales. SSU rDNA sequence data from three additional species, Pseudendoclonium basiliense, Pseudendoclonium akinetum and Pseudendoclonium fucicola are included in the analyses and clearly support the hypothesis that Pseudendoclonium is polyphyletic. Based on the sequence data, P. submarinum and P. fucicola share ulvalean lineage, but these algae are not congeneric and P. fucicola must be removed from Pseudendoclonium. Sequence data support the classification of P. basiliense and P. akinetum as distinct species of a single genus. The close affiliation of these two species with Ulothrix and other Ulotrichalean genera, however, reveals their ordinal separation from P. submarinum. P. basiliense and P. akinetum must also be removed from Pseudendoclonium and require generic reassignment within the Ulotrichales.