Identification of novel components of the zinc homeostasis mechanism in Arabidopsis thaliana

Zinc is the second most widely used transition metal in living systems. It is mainly involved in catalytic processes; it has also structural role in different proteins. Besides its widespread use in living systems zinc deficiency is one of the prevalent malnutrition challenges. Current estimates suggest that 31% of the world's population is at risk of zinc deficiency. Exponential population growth and natural resources scarcity might aggravate this problem. Therefore, in an initiative to advance the current understanding of the Zn homeostasis mechanism and aid the biofortification research, a forward genetics approach was adopted to identify molecular components of the Zn homeostasis network in plants. Previous observations have indicated that irregularity in zinc homeostasis mechanism often leads to a reduction in zinc tolerance of plants. Hence, EMS mutagenized second generation seeds of Arabidopsis thaliana were screened for reduced root growth in the presence of zinc stress (i.e. zinc hypersensitive response) in order to identify new elements of the zinc homeostasis mechanism. On the second round of screening conducted in this project 28 new Increased Zinc Sensitivity (IZS) mutants were identified and five of them (i.e. IZS 377, IZS 389, IZS 390, IZS 394 and IZS 479) were further characterized. Among these five newly characterized IZS mutants, only IZS 497 showed a specific zinc hypersensitivity phenotype (i.e. not hypersensitive to other transition metals tested). In IZS 479 a substitution of the 293rd aspartic acid by asparagine was indentified in the MTP1 gene, which could be the reason behind its zinc hypersensitivity phenotype. Furthermore, characterization of IZS 288 (one of the IZS mutants identified in the first round of screening) identified pleiotropic effects of the mutation, such as altered root architecture, changed leaf morphology, early flowering and chilling hypersensitivity, in addition to the zinc hypersensitivity phenotype. Map-based cloning of the mutated gene in IZS 288 lead to the identification of a novel WD-40 gene that is presumed to form a complex with cullin 4 ubiquitin E3 ligases and take part in the selective degradation of substrate proteins via the ubiquitin proteasome pathway. Single putative orthologs of this gene are found in Homo sapiens, D. melanogaster, C. elegans, X. laevis etc. However, functional characterization of most of these genes was still missing; hence a phenotypic analysis of three RNAi lines of the putative Drosophila ortholog was carried out. Observation in this experiment indicated the potential role of this gene at different organs and developmental stages of Drosophila. In IZS 288, the substitution of the 377th threonine by isoleucine (which is in the conserved region of the protein) might have caused a disruption in the protein structure that led to malfunctioning in the cullin 4 ubiquitin E3 ligases complex. Based on microarray analysis potential substrates (i.e. JAZ8, the TTD-A subunit of the basal transcription factor complex (TFIIH) and three histone families) of this complex were indentified. However further experiments will be required to prove the effect of the point mutation on the structure of the protein and its interaction with cullin 4 ubiquitin E3 ligase as well as to verify the potential substrates. Finally, based on prior observation flavonoids were assumed to have a role in heavy metal tolerance of plants; thus, the effect of flavonoids in zinc tolerance of Arabidopsis thaliana plants were investigated using five different flavonoids deficient mutants (i.e. transparent testa (tt) mutants). The mutant line that is completely devoid of flavonoids (tt4) and the one lacking quercetin (tt7) showed strong zinc hypersensitivity. Thus, quercetin appeared to be more effective than kaempferol in shielding the effect of zinc stress in Arabidopsis.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bayreuth

Year

2013

Author dc:creator

• Beyene Chichaibelu, Blen

Contributors dc:contributor

• Clemens, Stephan

Repository record source_url

https://epub.uni-bayreuth.de/id/eprint/1677/

OAI identifier oai:identifier

oai:epub.uni-bayreuth.de:1677