Studying the extracellular loop domain of DEK1 in evolutionary context Utilizing a dominant negative effect and cross-species domain swap chimeras in Arabidopsis thaliana
Year: 2011
Type: dissertation
Abstract: DEFECTIVE KERNEL 1 (DEK1) is a highly conserved phytocalpain, embedded in the\nmembrane of all plant cells examined. Null alleles of dek1 are lethal and dek1 mutants have\nshown that functional DEK1 protein is essential for proper development of both epidermal\ncell layer in plant embryos, and for positional dependant aleurone cell formation during seed\ndevelopment in maize, Arabidopsis and in tobacco plant. Transgenic Arabidopsis lines\noverexpressing the membrane-anchored domain of DEK1 have shown a dominant negative\nphenotype, suggesting that the AtDEK1-MEM plays role in the transmission of proposed\npositional cues. In this research we used the dominant negative effect and cross-species\ndomain swap analysis in order to study conserved features of the extracellular DEK1-LOOP\ndomain in Arabidopsis thaliana in an evolutionary context. Two constructs, designed to\noverexpress the AtMEM part of DEK1, where the LOOP domain was replaced by the DEK1-\nLOOP sequence from other organisms, were successfully cloned and transformed into wild\ntype Arabidopsis thaliana background. Transgenic plants overexpressing the AtDEK1-MEM\nconstruct, where the Arabidopsis loop sequence was replaced with loop from the unicellular\nalga Mesostigma viride, showed phenotype frequencies significantly similar to those\nobserved in plants overexpressing the membrane-anchored domain of DEK1 that results in\ndominant negative phenotype. These results suggest that the DEK1-LOOP of Mesostigma\nviride most probably interacts with the same positional cues or the same family of proteins\nas the DEK1-LOOP in Arabidopsis thaliana. Transgenic lines overexpressing the AtDEK1-\nMEM construct, where the AtDEK1-LOOP sequence was replaced with DEK1-LOOP from\nmaize (Zea mays), only alleviated the dominant negative effect by exerting significantly\nlower frequency of the respective phenotype, also compatible to phenotypic frequencies\nobserved in plants overexpressing the AtDEK1-MEM-DEL construct, where the loop\ndomain is deleted. These results suggest that the DEK1-LOOP domain could have\nfunctionally diverged in species-specific manner between Arabidopsis and maize. However,\nall these speculations are true only if the observed phenotypes are result of dominant\nnegative effect caused by the DEK1-LOOP domain. Q-PCR analyses of the transgenic\ntranscripts in two and five weeks old plants could not be correlated to the observed\nphenotypes.
Author: Kostadinka Mikkelsen
Language: English
Subfield: Plant Science
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2490532194