Browsing by Subject "Arabidopsis--Genetics"
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Item Distribution and expression of apyrases in pea and Arabidopsis(2003) Sun, Yu, doctor of computer sciences; Roux, Stanley J.This dissertation describes the results of experiments aimed at defining the tissue distribution, regulated expression and nuclear function of apyases in both Pisum sativum and Arabidopsis thaliana. As an approach to tissue-specific localizations in Arabidopsis, promoter regions including 5’UTR of two Arabidopsis apyrases, Atapy1 and Atapy2, were linked with GUS and transformed into Arabidopsis thaliana. As evaluated by GUS staining, the expression patterns of the two apyrases were almost identical. The highest expression level for both apyrases was found in mature pollen, the stigma surface, root cap and columella cells, the abscission zone and the top part of stipules. Both apyrases were also expressed highly in selected cell types of roots other than root tip. However, expression in hypocotyls and in flowering stems was not detectable for either apyrase in light grown seedlings. In etiolated seedlings, both apyrases are expressed in the upper region of the hypocotyl. Semi-quantitative RT-PCR measurements showed that both apyrases are upregulated by blue light and red light. Quantitative GUS assay and RT-PCR also shows that Atapy1 and Atapy2 are differently regulated by physical injury. Possible functions of AtAPYs in secretion, wound responses and in phytochromemediated light signal transduction are proposed. Transgenic plants that express AtAPY1-GFP and the antisense of Atapy1 were also made. The AtAPY1-GFP hybrid protein was found to exist as dot-like structures primarily in the cytosol. These plants showed no visible phenotype differences to wild-type plants, in conformity to the observations that plants knocked out in Atapy1 had no apparent phenotypic differences from wild-type plants. In the nuclei of etiolated pea seedlings, expression of the apyrase protein is regulated by red light but not white light. The nuclear apyrase was found to exist in a large (MW ~ 400Kd-800Kd) protein complex bound to the nuclear matrix. Specific molecular mass proteins were found to associate with apyrase when it was separated on molecular sieve chromatography and immunoprecipitated with highly specific polyclonal antibodies. In Arabidopsis, attempts to use AtAPY1 as bait in the yeast two-hybrid system to search for its binding protein were not successful.Item Molecular genetic analysis of TTG1-dependent cell fate pathways identifies a combinatorial Myb/bHLH transcription factor network in Arabidopsis(2008-08) Gonzalez, Antonio, 1973-; Lloyd, Alan M.The discovery of the Arabidopsis ttg1 mutant almost three decades ago provided a unique opportunity for the study of how several cell fates and organ identity pathways are co-regulated. Besides showing a lack of flavonoid based pigments, the pleiotropic ttg1 mutant is also deficient for the development of several epidermal characters including plant hair cells (trichomes), the non-hair cells of the root and the mucilage-secreting cells of the seed coat epidermis. Ectopic expression of the maize R bHLH transcriptional regulator of the flavonoid pigment pathway could completely suppress all the ttg1 mutant phenotypes, providing the first clue to the nature of the control mechanisms governing TTG1-dependent traits. Because it was established that a bHLH and a Myb protein are required for the regulation of anthocyanin pigment production in several plant species and an Arabidopsis Myb gene was necessary for trichome initiation, the existence of bHLH and Myb proteins that would regulate all the TTG1-dependent developmental pathways was hypothesized. This study works towards the elucidation of the transcriptional control mechanisms that regulate the TTG1-dependent developmental pathways. The identification and characterization of a key regulator, EGL3, uncovered the redundant nature of bHLH proteins operating under the TTG1 regulatory umbrella. As a result, bHLH regulators were assigned to all TTG1-dependent epidermal cell fate pathways and new roles for previously identified bHLH proteins were revealed. Roles suggested in the literature for Arabidopsis Myb factors suspected of regulating the flavonoid pigment pathway were at odds with findings from other plant models. Analysis of Myb loss-of-function RNAi lines and TTG1:GR and GL3:GR fusion lines presented here provides a clarified understanding of the regulation of anthocyanin biosynthesis by the Myb/bHLH/WDrepeat complex in Arabidopsis. Missing from the combinatorial complex model is the Myb component controlling the differentiation of the seed coat epidermis. Work presented here characterizes Myb5 as the primary Myb regulator of this differentiation pathway and defines a new role for TT2 as partially redundant with Myb5 for testa epidermis development. Myb5 also plays a minor role in trichome development and PA biosynthesis. Thus pleiotropy among the TTG1-dependent Myb regulators previously unobserved is first noted here. A more complete Myb/bHLH combinatorial transcription factor network model for the regulation of the TTG1-dependent pathways is proposed based on the results of work presented in this dissertation.Item Regulation of arabidopsis trichome patterning and anthocyanin biosynthesis by the TTG1-bHLH-MYB complex(2007) Zhao, Mingzhe, 1973-; Lloyd, Alan M.A network of three classes of proteins consisting of bHLH and MYB transcription factors and a WD40 repeat protein - TRANSPARENT TESTA GLABRA1 (TTG1) act in concert to activate trichome initiation and patterning in Arabidopsis. These proteins also regulate the flavonoid-based pigment biosynthetic pathway in almost all higher plants including Arabidopsis. Using TTG1-YFP translational fusions, I show that TTG1 is expressed ubiquitously in Arabidopsis leaves and is preferentially localized in the nuclei of trichomes at all developmental stages. Using conditional transgenic alleles I demonstrate that TTG1 directly regulates the same genes as GL3. In vivo binding of GL3, GL1 and TTG1 to the promoters of GL2, TTG2, CPC and ETC1 establishes that these genes are major transcriptional targets for the TTG1-bHLH-MYB regulatory complex. By co-precipitation, I confirm that TTG1 interacts with the GL3 (bHLH) and GL1 (Myb) proteins in vivo, forming a complex. The loss of members of the TTG1 complex through mutation, affects the subcellular distribution of other complex members. Using particle bombardment, I show that TTG1, GL3, GL1 and GL2 do not move between adjacent epidermal cells while CPC does move to neighboring cells. These data support a model for the TTG1 complex directly regulating activators and repressors and the movement of repressors to affect trichome patterning on the Arabidopsis leaf. In addition, I also show that GL3 is recruited to its own promoter in a GL1-independent manner, which results in decreased GL3 expression, suggesting the presence of a GL3 negative auto-regulatory loop. Expression studies using GL3-GR (GL3-glucocorticoid receptor) and TTG1-GR fusions reveal direct regulation of the late anthocyanin biosynthetic genes, but not of early biosynthetic genes. Taken together, our results provide insights on the molecular mechanisms by which the combinatorial TTG1-bHLH-MYB regulatory complexes activate and repress both developmental and biosynthetic pathways in Arabidopsis.