Abstract:
Environmental response to stress is long known to be able to induce senescence, manifesting the close relationship between the two processes. Yet the interplay between them is poorly understood. At the molecular level, the course of both entails the inhibition of biosynthetic activities and the storage of resources, as well as the activation of catabolic pathways, mobilization of reserves, and production of reactive oxygen species. That metabolic reprogramming is in any case accompanied by a massive change in the transcriptome, revealing the pivotal role played by transcription factors in the regulation of the two processes, and hinting at their function as the potential link connecting stress response and senescence.
The basic region leucine zipper (bZIP) family of transcription factors are present in all eukaryotes and control important developmental and physiological processes, which in plants include abiotic and biotic stress responses and leaf senescence. In Arabidopsis, the so called C/S1 network, which includes four bZIPs from the C sub-class and five from the S1 sub-class, is involved in the regulation of the plant energy balance and the allocation of nutrients, with a prominent function in the response to energy deprivation conditions. The induction of natural leaf senescence, in turn, was found to be regulated by another bZIP, namely GBF1, belonging to the G sub-class. An important feature of the bZIPs is that they act as dimers and different monomers can combine, raising the alluring possibility that GBF1 and bZIPs from the C/S1 network could heterodimerize, providing a physical link between the stress response and the senescence processes.
Dimerization is the core of the bZIP function, since different bZIP monomers exhibit distinctive qualities with regard to their transactivation potential and DNA binding specificity. Currently, bZIP heterodimerization is seen as a combinatorial mechanism generating a large variety of dimers with unique properties from a limited set of monomers. However, that assumption has not been systematically addressed and available data usually focus on the study of few dimers, not allowing to verify that hypothesis.
This thesis is committed to the study of the bZIP dimerization and function in Arabidopsis, although it consists of two differentiated parts. The first one investigates about an eventual regulation of the senescence specific activity of GBF1 through heterodimerization, in the context of evaluating the role of that bZIP in the crosstalk between stress response and senescence. Dimerization of GBF1 with members of the C/S1 network was assessed by Bimolecular Fluorescent Complementation (BiFC) assay, identifying bZIP63 as a potential GBF1 interacting partner. The function of the GBF1/bZIP63 heterodimer was subsequently examined in regard to its transactivation capacity and its DNA binding specificity by GUS-based transactivation assay and DNA Protein Interaction-ELISA, respectively. In addition, Arabidopsis lines with altered levels of bZIP63 expression were characterized for senescence-specific phenotypes. Evidence gathered could not support the hypothesis that GBF1 acted as a link connecting the stress response to senescence. On the other hand, data obtained confirmed that bZIP63 is involved in the general development of Arabidopsis plants, although not specifically in the senescence process.
The second part of this work challenged the accepted combinatorial model of bZIP function in Arabidopsis by systematically determining the dimerization and transactivation properties of 16 bZIPs. An interaction matrix analyzing all possible dimers among the 16 bZIPs was generated by BiFC, and the transactivation effect of 47 different bZIP dimer combinations on four promoters known to be targeted by some of those bZIPs was investigated by GUS reporter gene transactivation assays. Results indicated that the 16 bZIPs are organized in three independent interacting networks and that members of the same network exhibited partially redundant transactivation properties, but distinct for each promoter. The extent of the assays and the statistical data treatment performed provided enough perspective to reasonably infer in the organization and function of the Arabidopsis bZIP network: these findings severely limit the possibility that the currently assumed combinatorial model for bZIP function actually happens in Arabidopsis, since bZIP dimers are formed between monomers which already share similar functions. Accordingly, an alternative model is proposed in which each bZIP network operates as a functional unit, while heterodimerization serves as a mechanism integrating multiple inputs. In addition, in silico analyses of the bZIP dimerization motifs suggested the existence of a novel mechanism defining bZIP dimerization specificity based on the differential positioning of certain heptads which play prominent roles in the dimerization process.
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