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Title: Role for the phytocalpain DEK1 in plant mechanosensing
Author: Neumann, Enrique Diego
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2013
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The effect of mechanical stimulation in plants has been studied in depth for more than a century. This type of stress has been shown to trigger alterations in development such as stunting, thickened stems and differential cell wall deposition. These responses are very likely to be initiated at a subcellular level, but the molecular mechanisms transducing mechanical signals into intracellular responses still remain unknown in plants. In this thesis I test the hypothesis that the membrane anchored protein Defective Kernel 1 (DEK1) could act as a plant-specific mechanosensor in plants. Constitutive overexpression of the cytoplasmic CALPAIN domain DEK1 causes a phenotype in Arabidopsis, that that resembles that of mechanically stressed plants. The CALPAIN domain of DEK1 shows a very high homology with animal calpains; a class of calcium-dependent Cysteine proteases which undergo a calciumstimulated CALPAIN domain-releasing autolytic cleavage event during activation. A similar autolytic cleavage event has been observed in DEK1 which, together with the fact that the CALPAIN domain alone can rescue the embryo-lethality associated with loss of DEK1 function, has led to the suggestion that this domain represents an activated form of the protein. I show that like mechanically stressed plants, CALPAIN overexpressing plants show a modified call wall composition. Consistent with this, transcriptional analysis of these plants shows a deregulation of genes encoding cell wall modifying enzymes, amongst others. Other characteristics of mechanically stimulated plants which I have characterized in CALPAIN overexpressing lines include late flowering and thickened stems. Therefore, I proposed a model in which the CALPAIN domain of DEK1 acts as an effector which is normally activated by mechanical stimulation. In this model, the transmembrane domains of DEK1 would regulate activation (cleavage) of the CALPAIN domain, potentially in response to mechanical stress. In order to test this model further, CALPAIN overexpressing lines were generated in a dek1 mutant background. If the model is correct, these plants should not only behave as if responding constitutively to mechanical stimulation, but should also lack appropriate responses to applied mechanical stimuli due to lack of the mechanosensory integral membrane domain of DEK1. My results confirm that the absence of the transmembrane domains of DEK1 is indeed translated into a lack of some, but not all responses to mechanical stimulation compared to wild-type plants. Furthermore, the lack of the transmembrane domains of DEK1 correlates with the absence of a mechanically-triggered calcium flux in the plant. Thus my work suggests that the transmembrane domains of DEK1 are involved in sensing mechanical stimulation, via the regulation activity of a mechano-sensitive calcium flux at the plasma membrane. In summary, my proposal is that Defective Kernel 1 (DEK1) acts both as a key mechanosensory cellular component, and as the first effector of the signalling cascade in response to mechanical stimulation, via an autolytic activation in response to mechanical stress.
Supervisor: Ingram, Gwyneth ; Fry, Stephen Sponsor: Biotechnology and Biological Sciences Research Council (BBSRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: DEK1 ; Arabidopsis ; mechanosensing