Browsing by Subject "Arabidopsis"
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Item AtSUC1 ROOT EXPRESSION AND SUCROSE RESPONSE LEADING TO ANTHOCYANIN ACCUMULATION(2019-12) Lasin, PraphapanPrevious research indicated that AtSUC1 root expression is controlled by intragenic sequences. The 5’ upstream region (promoter) of AtSUC1 directs pollen and trichome expression, but not root expression. However, the whole AtSUC1 gene can drive root expression and sucrose-induced root expression. Here I show that root expression of AtSUC1 is controlled by the interaction between the promoter and its two short introns. Deletion of either intron from whole-gene-GUS constructs resulted in no root expression, showing that both introns are required. The two introns in tandem, fused to GUS, produce high constitutive expression throughout the vegetative parts of the plant. When combined with the promoter, the expression driven by the introns is reduced and localized to the roots. AtSUC1 expression is also induced by exogenous sucrose, and AtSUC1 is also required for sucrose-induced anthocyanins (Sivitz et al., 2008). Anthocyanin accumulation due to high sucrose was lesser in the AtSUC1 mutant compared to Col-0 wild type. A whole-gene-GUS construct expressing a non-functional AtSUC1 (D152N) mutant, that is transport inactive, was defective in sucrose-induced AtSUC1 expression and anthocyanins accumulation when expressed in an atsuc1-null background. The results indicated that sucrose uptake via AtSUC1 is required for sucrose-induced AtSUC1 expression and anthocyanin accumulation, and that the site for sucrose detection is intracellular.Item Characterization of CRISPR-Cas9 Induced SAUR19 Family Mutants in Arabidopsis.(2017-04) Hovland, Austin, S.The plant hormone auxin (IAA) regulates many aspects of plant growth and development. Small auxin up RNA (SAUR) genes are highly transcribed in response to auxin, whose differential transport and accumulation creates gradients that regulate various aspects of plant development such as: tropisms, root initiation, and cellular division and differentiation. Previous studies using overexpression of GFP-tagged SAUR proteins have shown that SAURs may function as positive regulators of cellular expansion. To provide additional evidence for this hypothesis, I investigated the effect of creating knockouts of multiple members of the SAUR19 subfamily using CRISPR/Cas9. Additionally, SAUR-promoter-GUS staining was performed to identify the location of SAUR 13, 22, 27, 28, and 29 expression. These studies revealed strong SAUR expression in adult leaf vasculature, expanding stamen filaments, hypocotyls, and petioles. Through analysis of heat-induced growth, we show that knockout of SAURs 19, 20, 21, 22, 24, and 29 conferred a modest decrease in both hypocotyl and petiole length. While not severe, this difference provides supportive evidence that SAURs function as positive regulators of cell expansion, and it also reinforces the hypothesis that SAURs have extensive genetic redundancy. Higher numbers of SAUR knockouts should produce stronger phenotypes and provide definitive evidence of SAUR function.Item The eta7/csn3-3 auxin response mutant of Arabidopsis defines a novel function for the CSN3 subunit of the COP9 signalosome.(2012-07) Huang, HeThe COP9 signalosome (CSN) is an eight subunit protein complex conserved in all higher eukaryotes. In Arabidopsis thaliana, the CSN regulates plant auxin response by removing the ubiquitin-like protein NEDD8/RUB1 from the CUL1 subunit of the SCFTIR1/AFB ubiquitin-ligase (deneddylation). Previously described null mutations in any CSN subunit resulted in the pleiotropic cop/det/fus phenotype and caused seedling lethality, hampering the study of CSN functions in plant development. In a genetic screen to identify enhancers of the auxin response defects conferred by the tir1-1 mutation, we identified a viable csn mutant of subunit 3 (CSN3), designated eta7/csn3-3. In comparison with eta6/csn1-10, which was identified in the same enhancer screen (Zhang et al., 2008), both csn3-3 and csn1-10 enhanced the auxin response defects of tir1-1. Similar to csn1-10, csn3-3 also confers several phenotypes associated with impaired auxin signaling, including auxin resistant root growth and diminished auxin responsive gene expression. Surprisingly however, unlike csn1-10 as well as other previously characterized csn mutants, csn3-3 plants are not defective in either the CSN-mediated deneddylation of CUL1 or in SCFTIR1/AFB mediated degradation of Aux/IAA proteins. These findings suggest that csn3-3 is an atypical csn mutant that defines a novel CSN or CSN3-specific function. Consistent with this possibility, I observed dramatic differences in double mutant interactions between csn3-3 and other auxin signaling mutants compared to csn1-10. Lastly, unlike other csn mutants, assembly of the CSN holocomplex was unaffected in csn3-3 plants. However, I detected a small CSN3-containing protein complex (sCSN3c) that was altered in csn3-3 plants. I hypothesize that in addition to its role in the CSN as a cullin deneddylase, CSN3 functions in a smaller protein complex that is required for proper auxin signaling. Analyses on the purification of sCSN3c suggested that it is not likely a dimer of CSN3, or a CSN subcomplex. My data resulting from sCSN3c purification using various chromatographic steps provide useful information necessary for identifying the components of the complex.Item An exposition on trichome development and cell shape with a focus on the function of MIXTA-like R2R3-MYBs.(2009-06) Gilding, Edward KalaniPlant development requires cell differentiation throughout the plant life cycle because plants rely upon the initiation and growth of new organs to reach reproductive maturity. Developmental programs specifying cell pigmentation, cell shape, and specification of cell type have been explored in Arabidopsis. Transcription factors are key components of these developmental programs and work in Arabidopsis and other plant systems have been essential in defining the roles that these factors play during development. A prime example of this in Arabidopsis is the trichome patterning program. The function and structural diversity of trichomes are intimately related, a relationship that this is explored in this thesis. What types of regulatory networks are involved in defining the form of a trichome is visited as well, setting the stage for deeper studies into Arabidopsis trichome development. Use of the glabra 3 shapeshifter (gl3-sst) allele as a proxy for early stages of trichome development in transcriptional profiling reveals the developmental activities of early stage trichomes. Candidate genes from these experiments were then used in a reverse genetics screen to find other genes with trichome phenotypes. Through this method, an R2R3-MYB transcription factor was discovered to play a role in determining cell shape. R2R3-MYB domain transcription factors constitute a major class of transcriptional regulators in plants. The Arabidopsis genome encodes an estimated 125 functional R2R3-MYB proteins. Additionally, R3-MYBs, R1R2R3-MYBs, R-R MYBs, and a single four-repeat MYB protein are encoded by the Arabidopsis genome. Animal genomes only contain a handful of MYB genes. Clearly plants have expanded and utilized this lineage in their evolutionary history, and not surprisingly, many of the regulatory programs these plant genes function in are prominent or specific to plants. As a group, the R2R3-MYB family has been studied previously and authorities have defined various subgroups to which members of this gene class are assigned. This thesis focuses upon members of subgroup 9, defined by the presence of the AQWESA amino acid motif. Seminal work describing the function of this group began with the Antirrhinum majus gene MIXTA. This gene is required for the proper differentiation of conical cells in the floral epidermis. AmMYB MIXTA-like 1, AmMYB MIXTA-like 2, AmMYB MIXTA-like 3, have since been described in Antirrhinum and all have been shown to be functionally similar to MIXTA by heterologus expression in tobacco in the control of cell shape, albeit to varying degrees. Collectively, the available Antirrhinum gene data supports the notion that subgroup 9 R2R3-MYBs are determinants of cell shape be it floral trichomes or conical cells. Here the technical capabilities we possess in Arabidopsis are used to define the function of the subgroup 9 R2R3-MYB NOECK, (NOK, AT3G01140). NOK functions as a negative regulator of trichome branching, leading to trichome cells with increased volume in the mutant line. This phenotype is opposite that of the reduction in cell volume that might occur in mixta Antirrhinum floral epidermal cells that do not become conical by growing out of the epidermal plane. Expression profiling of trichome cells of various mutants including nok revealed coordinately regulated genes that are extracellular matrix components. These findings coupled with the published data indicates that NOK, and perhaps all other subgroup 9 R2R3-MYBs, control cell shape by altering properties of the extracellular matrix. Preliminary data testing the functional equivalence of selected MIXTA-like genes from Antirrhinum majus, Arabidopsis, Dendrobium crumenatum, and Medicago truncatula are given. These data support the portability of the NOK functional characterization data to other plant species. Furthermore, these findings illustrate that subgroup 9 R2R3-MYBs alter cell shape regardless of phylogenic origin.Item Identification and characterization of EMS mutant trm5a-1 and its interactions with SPY(2020-01) Grandt, KristinSPINDLY (SPY) is an O-fucosyltransferase involved in several processes in Arabidopsis thaliana, including gibberellin signaling, cytokinin signaling, and plant development. spy plants display a number of easily scored phenotypes such as increased stem elongation, decreased leaf serration, and early flowering. Despite its apparent importance, SPY’s function has not been fully characterized, as very few SPY substrates have been identified. In order to address this gap in knowledge, a suppressor screen was conducted to identify potential SPY interactors. An M2 population of ethyl methanesulfonate mutagenized spy-4 plants was screened for suppression of spy phenotypes. Three alleles that showed suppression of spy early flowering and spindly floral shoot phenotypes were identified. The strongest of those alleles was selected for analysis. However, spy-4 is partially male sterile, so the suppressor allele was crossed into spy-3 to facilitate genotypic and phenotypic analysis. Bulk segregant analysis using next generation sequencing was employed to identify the mutation responsible for the partial spy suppression observed. This analysis identified a point mutation that introduces a premature stop codon into the coding region of the TRM5a gene as the most likely candidate. This EMS allele is called trm5a-1 here-in. Analysis of another trm5a allele (T-DNA insertion line trm5a-2) and transgene rescue experiments confirmed that this mutation was responsible for suppressing spy. trm5a-1 and trm5a-2 mutants grow slowly and flower late, phenotypes that have been previously reported in other trm5a alleles. The trm5a-1/spy-3 double mutant phenotypes for these traits suggest a complex interaction between the two genes. Other phenotypes which have not been previously characterized in trm5a mutants are also observed. Exogenous application of cytokinin results in outgrowths at the valve margin in trm5a-1, trm5a-2, spy-3, and trm5a-1/spy-3, but not, at the concentrations used, in Col-0. In all trm5a-1, trm5a-2, and spy-3 single mutants, the outgrowths are small hair- or fan-like protrusions . However, in trm5a-1/spy-3, the outgrowths are larger and may even appear to have stigmatic papillae on their surface. There is also a phenotype of floral clustering in trm5a mutants, which is occasionally observed in some spy mutants but not spy-3. In trm5a-1 and trm5a-2 mutants, the phenotype manifests mainly as flower doublets with occasional small clusters of 4-5 flowers, while in trm5a-1/spy-3 the clusters are larger often comprised of ten or more flowers. The trend of trm5a-1/spy-3 plants to display more severe valve margin outgrowth and clustering phenotypes than the single mutants suggests additive or synergistic interactions between TRM5a and SPY. Broadly, the phenotypes of the trm5a-1, trm5a-2, and spy-3 demonstrate the importance of the genes in proper plant growth and development, and the phenotypes observed in the trm5a-1/spy-3 double mutant suggest a degree of genetic interaction between the two genes and areas of interest for future research into both.Item Identification and characterization of three Arabidopsis sugar insensitive genes(2008-10) Huang, YadongCarbohydrates have signaling functions in regulating gene expression, metabolic pathways and developmental processes. Eukaryotic organisms have evolved conserved and novel mechanisms for sensing and responding to sugars. Plant sugar response pathways are complex and exhibit cross-talk with other response pathways. Sugar responses and signaling pathways have been studied via physiological, biochemical and genetic approaches. Genetic screens have identified sugar response mutants with altered seedling growth phenotypes. The Gibson lab has isolated an array of sugar insensitive (sis) mutants by screening mutagenized Arabidopsis seeds on high concentrations of sugars. The identification and characterization of three of the SIS genes, SIS7, SIS3 and SIS8, are presented here. SIS7 is allelic to NCED3/STO1, an abscisic acid (ABA) biosynthetic gene, which is involved in drought and salt stress responses. Lateral root (LR) development of sis7 mutants is resistant to the inhibitory effects of osmotica. Transcriptomic analysis revealed that a set of auxin-related genes are expressed at lower levels in sis7 seeds than in wildtype seeds when incubated with glucose, suggesting that these genes may be involved in controlling LR development by both ABA and auxin. SIS3 encodes a RING finger protein that functions as an E3 ligase in in vitro ubiquitination assays. The sis3 seeds display wild-type germination responses to ABA and GA. However, the root growth of sis3 mutants has slightly reduced sensitivity to ABA. The sis8 mutants have decreased sensitivity to high sugars and hyperosmolarity. Positional cloning of sis8 revealed that the mutation is in a putative mitogen-activated protein kinase kinase kinase gene. Seed germination assays indicate that sis8 mutants have wild-type sensitivity to ABA and GA, whereas overexpression of SIS8 causes slight hypersensitive responses. Potential interaction partners of SIS8 have been identified via yeast two-hybrid screening. A T-DNA insertion in the gene encoding one potential SIS8- interacting protein, UGT72E1, causes a sis phenotype. Further studies of the SIS3 and SIS8 genes will provide more insight into the mechanisms of sugar signaling in plants.Item Investigating Interaction of Tissue-Specific Circadian Rhythms in Arabidopsis(2024-08-31) Crenshaw, Eiley C.; Zulfiqar, Alveena; Menon, AnandaItem Positive And Negative Regulation Of Defense Responses Against Pseudomonas Syringae In Arabidopsis(2014-03) Sreekanta, SumaImmune signaling in plants involves both positive and negative regulators. Maintaining a balance between growth and defense responses is important because there is a fitness cost to the plants if immune responses are left unchecked. Suppression of immune responses in the absence of pathogens as well as after the threat has passed is critical in maintaining such a balance between growth and defense responses. Upon pathogen perception, the positive regulators counter the immune repression to induce defense responses. We investigated the roles of two genes, CBP60a and PCRK1 in the regulation of defense responses against Pseudomonas syringae pathogen in the model system Arabidopsis thaliana . CBP60a is a negative regulator of immune responses. We showed that CBP60a is a CaM binding protein and that CaM binding is important for its function in transducing defense signals. Mutants of CBP60a were more resistant to Pseudomonas syringae infection suggesting that CBP60a was a negative regulator of defense responses. We found that CBP60a functions in repressing immune signaling under conditions where the plants are not challenged by a pathogen. We also investigated the role of a putative kinase, PCRK1, in immune signaling. We showed that pcrk1 mutants are more susceptible to Pseudomonas syringae than wild type plants suggesting that PCRK1 has a positive role in immune responses. We also showed that PCRK1 is important for immunity triggered by some of the conserved Microbe Associated Molecular Patterns (MAMP) as well endogenous signals generated as a result of pathogen activity called Damage Associated Molecular Patterns (DAMP).Item Raw data for: Biphasic Control of Cell Expansion by Auxin Coordinates Etiolated Seedling Development(2021-07-27) Du, Minmin; Bou Daher, Firas; Liu, Yuanyuan; Steward, Andrew; Tillman, Molly; Zhang, Xiaoyue; Wong, Jeh Haur; Ren, Hong; Cohen, Jerry D; Li, Chuanyou; Gray, William M; grayx051@umn.edu; Gray, William M; William Gray LabSeedling emergence is critical for food security. It requires rapid hypocotyl elongation and apical hook formation, both of which are mediated by regulated cell expansion. How these events are coordinated in etiolated seedlings is unclear. Here, we show that biphasic control of cell expansion by the phytohormone auxin underlies this process. Shortly after germination, high auxin levels restrain elongation. This provides a temporal window for apical hook formation, involving a gravity-induced auxin maximum on the eventual concave side of the hook, triggering PP2C.D1-controlled asymmetrical H+-ATPase activity, resulting in differential cell elongation. Subsequently, auxin concentrations decline acropetally and switch from restraining to promoting elongation, driving hypocotyl elongation. Our findings elucidate how differential auxin concentrations throughout the hypocotyl coordinate etiolated development, leading to successful soil emergence.Item A role for UV-B -induced DNA damage in photomorphogenic responses in etiolated Arabidopsis seedlings(2014-01) Biever, Jessica JoUltraviolet (UV) radiation is a constituent of sunlight that influences plant morphology and growth. It induces photomorphogenic responses but also causes damage to DNA. Plant responses to DNA damage caused by UV-B light are often categorized as general mechanisms that get activated by other environmental stresses. Photodimers are formed through the direct absorption of UV-B light by DNA and are removed, in part, by nucleotide excision repair (NER). UV-B irradiation resulted in the accumulation of the two most common photodimers, cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6,4)-pyrimidinone dimers (6,4PPs), in etiolated wild type (wt) Arabidopsis seedlings. Arabidopsis mutants of the endonucleases that function in NER, xpf-3 and uvr1-1, show hypersensitivity to UV-B (280-320 nm) in terms of hypocotyl growth inhibition. I hypothesized that the accumulation of UV-B-induced photodimers was responsible for the hypocotyl growth phenotype of these NER mutants after UV-B irradiation. It was also predicted that the accumulation of photodimers could ultimately trigger signaling pathways that result in cell-cycle arrest through stalled replication sites or double-strand breaks. This was tested using the suppressor of gamma 1 (sog1-1) mutant, which lacks a transcription factor responsible for gene induction and cell-cycle arrest after gamma irradiation, and a Col-0 line containing a CYCB1;1-GUS reporter construct. CYCB1;1 encodes a cyclin that accumulates in response to cell-cycle arrest at the G2/M transition. The main conclusion from this work is that hypocotyl growth inhibition induced by UV-B light in etiolated Arabidopsis seedlings, which is a classic photomorphogenic response, is influenced by signals originating from UV-B light absorption by DNA that lead to cell-cycle arrest. Furthermore, this process is shown to occur independently of UVR8 and its signaling pathway responsible for CHS induction. This work also demonstrates that UV-B-induced DNA damage can be responsible for specific photomorphogenic responses, at least in etiolated Arabidopsis seedlings, and does not simply induce general stress responses.