Halle, Wittenberg, Univ., Habil.-Schr., 2004. / Computerdatei im Fernzugriff.

Thesis (Ph.D.) - University of Queensland, 2003. / Includes bibliography.

Halle, Wittenberg, Universiẗat, Habil.-Schr., 2004.

Entre os minerais, o fósforo é um dos mais limitantes ao crescimento e desenvolvimento vegetal. Como é altamente requerido para os mais diversos processos fisiológicos e celulares, as plantas desenvolveram complexos mecanismos para manejar sua deficiência. As respostas à limitação de fósforo são bem conhecidas, mas sobre a sua percepção e a transdução do sinal pouco se sabe. Os mutantes p9, p23 e p37 estudados neste trabalho são provenientes de uma seleção que busca identificar genes regulatórios envolvidos na sinalização de fósforo. Desta forma, fez-se através da caracterização morfológica, fisiológica e bioquímica destes mutantes condicionais de Arabidopsis thaliana, deficientes quando ácidos nucléicos são a única fonte de fosfato (Pi), a ampliação do conhecimento da rota de sinalização da limitação de Pi. Os fenótipos dos mutantes devem-se à mutação em um gene recessivo para cada mutante, sendo estes complementares. Análises do sistema radicular, acúmulo de amido e antocianinas, teor de Pi livre e do P total e atividade de nucleases em diferentes disponibilidades de fósforo e a avaliação da especificidade dos fenótipos à deficiência de P possibilitaram a criação de hipóteses para a ação dos genes mutados A mutação de p9 causa, provavelmente, alterações na sensibidade às concentrações de Pi, podendo estar relacionada tanto aos sensores da raiz quanto à interação da transdução dos sinais entre o sensor local e o status da planta. Devido à limitação de sementes não foi possível desenvolver hipótese sobre a ação do gene mutado em p23. Enquanto o gene mutado em p37 age primordialmente sobre o elongamento e a divisão das células radiculares, estas respostas podem estar sendo influenciadas pelos níveis de citocinina. Assim, a complexidade da transdução do sinal à limitação de Pi e a interação com outras rotas de sinalização em plantas vasculares evidencia a importância de estudar suas respostas e esclarecer como esses processos são regulados.

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With the recent development of nanotechnology, there has been increased production of engineered nanomaterials but limited containment strategies, resulting in inevitable release of a large amount of engineered nanoparticles (ENPs) in the environment. Many ENPs have potential adverse impacts on the environment, and one of the most commonly used ENPs, silver nanoparticle (AgNP), has attracted increased global concern. The current study focused on phytotoxicity of AgNPs to a model plant, Arabidopsis thaliana. Silver nanoparticles were introduced into the growth medium for the wild type A. thaliana plants and root uptake and translocation of AgNPs were examined in hydroponic growth condition. We also conducted growth stage based phenotypic analysis by growing A. thaliana throughout its life cycle in soil. The result indicated that: (1) wild type seeds germination was not affected by either AgNPs or their dissolved Ag+ ions; (2) AgNPs exposure resulted in a concentration- and size- dependent inhibition effect to the root elongation; (3) confocal and electron microscopy indicated that AgNPs could be taken up by seedling roots, yet most of AgNPs attached to the surface of seedling root cap; (4) AgNPs and their dissolved Ag+ ions at tested concentrations had little influence on the vegetative growth of A. thaliana, but they accelerated the floral development; and (5) the effect on the floral development stage reduced the quality of second-generation (F1) seeds, as indicated by their lower germination rate. In conclusion, AgNPs displayed both acute and chronic phytotoxicity to A. thaliana.

Accurate regulation of gene transcription is essential for organismal survival, and is orchestrated by myriad transcription factors and cofactors (TFs). Little is known about how the intrinsic activity of TFs is controlled. Recent work has indicated that the selective proteolysis of TFs provided by the ubiquitin-proteasome system (UPS) plays an important role in stimulating gene expression through a ‘destruction-activation’ mechanism, whereby the degradation of a ‘used’ TF is thought to stimulate further ‘fresh’ TF binding and reinitiate gene transcription. TFs are targeted to the proteasome via E3 ligases that mediate the addition of ubiquitin molecules to form a chain on the substrate TF. These polyubiquitin chains may be extended by E4 ligases, which recognize substrates with four or more ubiquitin molecules, amplifying substrate targeting to the proteasome. In plants the immune response to many pathogens is regulated by the hormone salicylic acid (SA), which operates through the transcriptional coactivator NPR1 to induce large scale changes in gene expression. Proteasome-mediated degradation of NPR1 appears to be required for the activation of its target genes. Mutation of the E3 ligase prevents ubiquitination of NPR1, leading its to stabilisation and suppression of transcription. Chapter 3 of this work identifies the first E4 ligase, UBE4, involved in NPR1 regulation. Mutation of UBE4 resulted in reduced capacity to polyubiquitinate substrates and stabilized NPR1. In contrast to E3 ligase mutants, however, mutant ube4 plants displayed increased NPR1 target gene expression. These results suggest that initial ubiquitination of NPR1 may stimulate its ability to initiate transcription and that subsequent ubiquitin chain elongation limits NPR1 activity by targeting it to the proteasome. Chapter 4 describes a ubiquitin-protein-ligase (UPL) which is both novel and crucial to the SA-mediated defence response. Mutation of this UPL leads a large reduction in total cellular polyubiquitinated proteins and was associated with strongly enhanced disease susceptibility. Gene expression profiling of upl mutants revealed an intimate connection between cellular polyubiquitination and appropriate activation of SA-responsive gene expression programmes. Destruction activation was first described in yeast and is required for the regulation of yeast amino acid synthesis TF GCN4. GCN4 requires proteasome-mediated degradation to induce genes involved in amino acid production. Chapter 5 investigates the role of two E4 ligases in GCN4 turnover. While one mutation had little effect of GCN4-mediated transcription a second increased basal transcriptional levels, suggesting that an E4 is required for the prevention of spurious GCN4-mediated transcription. In summary the work presented here describes cellular mechanisms by which global and substrate-specific polyubiqutination are vital to regulation of gene transcription.

Entre os minerais, o fósforo é um dos mais limitantes ao crescimento e desenvolvimento vegetal. Como é altamente requerido para os mais diversos processos fisiológicos e celulares, as plantas desenvolveram complexos mecanismos para manejar sua deficiência. As respostas à limitação de fósforo são bem conhecidas, mas sobre a sua percepção e a transdução do sinal pouco se sabe. Os mutantes p9, p23 e p37 estudados neste trabalho são provenientes de uma seleção que busca identificar genes regulatórios envolvidos na sinalização de fósforo. Desta forma, fez-se através da caracterização morfológica, fisiológica e bioquímica destes mutantes condicionais de Arabidopsis thaliana, deficientes quando ácidos nucléicos são a única fonte de fosfato (Pi), a ampliação do conhecimento da rota de sinalização da limitação de Pi. Os fenótipos dos mutantes devem-se à mutação em um gene recessivo para cada mutante, sendo estes complementares. Análises do sistema radicular, acúmulo de amido e antocianinas, teor de Pi livre e do P total e atividade de nucleases em diferentes disponibilidades de fósforo e a avaliação da especificidade dos fenótipos à deficiência de P possibilitaram a criação de hipóteses para a ação dos genes mutados A mutação de p9 causa, provavelmente, alterações na sensibidade às concentrações de Pi, podendo estar relacionada tanto aos sensores da raiz quanto à interação da transdução dos sinais entre o sensor local e o status da planta. Devido à limitação de sementes não foi possível desenvolver hipótese sobre a ação do gene mutado em p23. Enquanto o gene mutado em p37 age primordialmente sobre o elongamento e a divisão das células radiculares, estas respostas podem estar sendo influenciadas pelos níveis de citocinina. Assim, a complexidade da transdução do sinal à limitação de Pi e a interação com outras rotas de sinalização em plantas vasculares evidencia a importância de estudar suas respostas e esclarecer como esses processos são regulados.

Cellular redox homeostasis mediates a wide range of physiological and developmental processes. Various stresses trigger over-production of reactive oxygen/nitrogen species which leads to oxidative modifications of redox-sensitive proteins. Identification and characterization of redox-sensitive proteins are important steps toward understanding molecular mechanisms of stress responses. In the study, a high-throughput quantitative proteomic approach termed OxiTRAQ was developed for identifying proteins whose thiols undergo reversible oxidative modifications in Arabidopsis cells subjected to oxidative stress. In this approach, a biotinylated thiol-reactive reagent is used for differential labeling of reduced and oxidized thiols, and the biotin-tagged peptides are affinity-purified and labeled with iTRAQ reagents for quantitation. This approach allows identification of the specific redox-regulated cysteine residues in proteins and offers an effective tool for elucidation of redox proteomes. With this approach, we identified 195 cysteine-containing peptides from 179 proteins whose thiols underwent oxidative modifications in Arabidopsis cells following the treatment with hydrogen peroxide. A majority of those redox-sensitive proteins, including several transcription factors, were not identified by previous redox proteomics studies. Besides, this method was also used to identify proteins that underwent oxidative modifications in Arabidopsis cells subjected to 15 minute treatment of salicylate (a key signaling molecule in the plant defense pathway) or flg22 (a peptide from bacterial flagellin that induces pathogen associated molecular patterns-triggered immunity). In total, 127 peptides from 111 distinct proteins were identified as salicylate- and/or flg22-responsive redox-sensitive proteins. Among the identified redox sensitive proteins are many regulatory proteins including those involved in chromatin remodeling, transcription, nucleocytoplasmic shutting, and posttranslational regulation. Furthermore, in vivo 15N metabolic labeling method combined with a cysteine-containing peptide enrichment technique was applied to identify proteins that undergo oxidative modifications in plants in response to pathogen attack. The identification of redox-sensitive proteins provides a foundation from which further study can be conducted toward understanding the biological significance of redox signaling in plant stress response.

Plants regulate many physiological processes in response to adverse environmental stresses. This study focused on the seed germination and seedling establishment stage and investigated the molecular signaling events when abiotic stresses, such as osmotic, water and temperature, were applied. Seeds of Arabidopsis, mutants or wild type, were used to identify the signaling components. Cold-pretreatment (stratification) is widely used to break seed dormancy and improve germination rate. Stratification at 4. significantly broke the seed dormancy of Arabidopsis in wild-type, cyp707a2, sleepy1 and sleepy1/cyp707a2, but not in ga3ox1. Stratification and exogenous ABA treatment strongly enhanced the expression and the activity of a-amylase in the freshly harvested seeds among the wild-type and those mutants, which have relatively high ABA content. Similarly, the expression of RGL2 and ABI5 were also substantially suppressed by stratification. These results suggest that stratification firstly leads to GA biosynthesis and unlocks the inhibition of RGL2 on the expression of a-amylase. Stratification also relieves the inhibition of ABA on the germination process but the inhibition of ABA on seedling development is not affected. We have isolated an Arabidopsis mutant, dsptp1, which is hyposensitive to osmotic stress during seed germination and seedling establishment, indicated by exhibiting higher seed germination rate, lower inhibition in root elongation under osmotic stress, and more tolerance to drought compared with the wild type (Col0) plants. Osmotic stress and drought enhanced AtDsPTP1 expression in seed coats, the bases of rosette leaves and roots. Compared with the wild type, the dsptp1 mutant increased proline accumulation, reduced MDA content and ion leakage, and enhanced antioxidant enzyme activity under osmotic stress. AtDsPTP1 regulated the transcript levels of various dehydration responsive genes, ABA biosynthesis and metabolic enzyme gene under osmotic stress, resulting in reduced accumulation of ABA in dsptp1 mutant plants than wild type in response to osmotic stress. AtDsPTP1 also mediated the ABA signaling pathway under osmotic stress by suppressing the expression of ABI1 and enhancing the expression of the positive regulators ABI3 and ABI5 in ABA signaling. These data suggest that AtDsPTP1 positively regulates ABA accumulation and signaling during seed germination and seedling establishment in Arabidopsis under osmotic stress. To further investigate the regulation mechanism of DsPTP1 in osmotic stress and drought signaling, we analyzed the water holding capacity between wild type and dsptp1 mutant. The dsptp1 mutant exhibited enhanced water holding capacity compared to wild type under osmotic stress resulting from reduced water loss and increased relative water content, which shall contribute the osmotic and drought tolerance. To identify the signaling components, we investigated the activity of MAPKs under osmotic and drought stress and found that the DsPTP1 differentially regulates the activities of MAPK6 and a p38 MAPK, which is inferred as MAPK12 according to its molecular weight in Arabidopsis under osmotic and salt stress. However, there is no direct interaction between DsPTP1 and 20 MAPKs indicated by the results of the of specific interaction test. These results suggest that the differential regulation of MAPK6 and MAPK12 by DsPTP1 is indirect. In addition, we screened the interaction proteins of DsPTP1 under abiotic stress. Seventeen positive clones were acquired from the sequencing results. More work need to be done to confirmed the positive interactions and the signaling cascades. In summary, seed germination and seedling growth are closely regulated by environmental cues. This should be the result of evolutionary selection since successful new growth from the seed embryo depends on the sensitive perception of environmental conditions and effective regulation of many physiological processes that are involved. We have demonstrated that plant hormones, especially ABA, play central regulative roles during such regulations. Many other signaling components, such as protein kinases and phosphatases, are also involved. Identifying the detailed signaling pathways should be the focus of further research.