Identification of novel components and links in ubiquitin dependent protein degradation pathways of Arabidopsis thaliana

Abstract

The canonical ubiquitin 26S proteosome dependent protein degradation pathway and its sub-branch N-end rule pathway are important ubiquitin dependent processes in eukaryotes. The majority of substrates are predominantly targeted for degradation by the proteosome. Expression of a ubiquitin variant with Arg instead of Lys at position 48 (ubK48R) in the Arabidopsis RV86-5 line leads to cell death. In order to understand the downstream effects of this pathway, the ubK48R expressing line RV86-5 and the suppressor line of ubiquitin variant induced cell death, sud2, were used as tools. Fine mapping with 1239 recombinants narrowed down the sud2 mutant locus to the south arm of chromosome III, between loci At3g44400 and At3g44900. Problems caused by low recombination and repeated sequences were overcome by sub-genomic PCR-based amplification of a 350 kb region and subsequent Solexa sequencing of this region of interest. The data analysis tailored for nucleotide based comparison to reference sequence identified 15 candidates, 5 of which could be verified by conventional sequencing. In an alternative approach, microarray-based transcriptional expression differences between RV86-5 and sud2 identified 10 additional candidate suppressor genes, the majority of which are of unknown function. Among mutations in 9 of the tested candidates, 8 were able to prevent the lethal phenotype of RV86-5, indicating their involvement in the cell death process. The main interest of the ubiquitin research field is to identify E3-ligases and their interacting substrates. The second part of this work involved the search for novel E3 ligases that modify a known test protein with an aliphatic hydrophobic amino-terminal residue, Leu, which is targeted by none of the known plant N-end rule E3 ligases, PRT6 and PRT1. EMS mutagenesis on a plant line expressing a test protein with L-GUS followed by live tissue GUS staining, to screen for transgene stabilization, identified the 2 complementation groups PRT8 and PRT9, representing candidates for putative E3-ligases involved in destabilization of test proteins with amino-terminal Leu. The prt8 mutant shows delayed development. With the creation of a mapping population, the basis for the identification of locus was laid in this work. Arabidopsis mutants in the functionally unknown UBR domain proteins BIG and PRT7, which share homology with the mammalian N-end rule pathway components UBR4 and UBR7, were analyzed. A mutant in PRT7, isolated by T-DNA library screening, showed premature leaf senescence. In contrast, the big mutant showed delayed senescence and in addition no enzymatic affinity to test substrates with a basic N-terminus. Mutants were 9 isolated in two putative Arabidopsis deamidases, NTAN and NTAQ that are distantly related to mammalian deamidases. These were crossed into reporter lines expressing N-GUS and Q-GUS test proteins to deduce whether these enzymes provide substrates to Arg-t-RNA protein transferase as in mammals. These created mutants have laid the basis to analyse unknown functions of N-end rule pathway components in Arabidopsis. The importance of NO in signaling in plants has been long studied, but its molecular mechanism is still not well understood. In this work, it was found that in the Arabidopsis N-end rule pathway, NO targets test substrates with N-terminal Cys for degradation in a proteosome dependent manner and that this process is dependent on O2. With these results, strong evidence was obtained that the N-end rule pathway has a role in NO signaling and sensing. This finding has brought new insights into the plant N-end rule pathway. Taken together, the research work of this Thesis has developed new methods to overcome the low recombination problem during the mapping process, identified candidates that could potentially link the cell death processes to the ubiquitin dependent degradation pathway and identified putative E3-ligases of the N-end rule pathway by a novel way of EMS mutant screening supported by live tissue GUS assay. This research work found a connection between NO and the N-end rule pathway in A. thaliana. A complete set of mutants in all known plant N-end rule pathway components has been created, opening a window of possibility to further find natural substrates of this pathway