Functional Studies of the Arabidopsis thaliana Ubc13-Uev Complex

Abstract

Ubiquitination is an important biochemical reaction found in all eukaryotic organisms and is involved in a wide range of cellular processes. Conventional ubiquitination requires the formation of polyubiquitin chains linked through Lys48 of the ubiquitin, which targets proteins for degradation, while the noncanonical Lys63-linked polyubiquitination of the proliferating cell nuclear antigen is required for error-free DNA damage tolerance (DDT or postreplication repair) in yeast. The ubiquitin-conjugating enzyme Ubc13 and a cognate Ubc enzyme variant (Uev or Mms2) are involved in this process. Because there is less information available on either Lys63-linked ubiquitination or error-free DDT in plants, the goal of my research was to study the functions of Ubc13 and Uev in plants using Arabidopsis thaliana as the model organism.

Four UEV1 genes from Arabidopsis thaliana were isolated and characterized. All four Uev1 proteins can form a stable complex with AtUbc13 and can promote Ubc13 mediated Lys63 polyubiquitination. All four UEV1 genes can replace yeast MMS2 in DDT function in vivo. Although these genes are ubiquitously expressed in most tissues, UEV1D appears to be expressed at a much higher level in germinating seeds and pollen. We obtained and characterized two uev1d null mutant T-DNA insertion lines. Compared with wild-type plants, seeds from uev1d null plants germinated poorly when treated with a DNA-damaging agent. Seeds that germinated grew slow and the majority ceased growth within 2 weeks. Pollen from uev1d plants also displayed a moderate but significant decrease in germination in the presence of DNA damage agent. These results indicate that Ubc13-Uev complex functions in DNA damage response in Arabidopsis thaliana.

Arabidopsis thaliana contains two UBC13 genes, AtUBC13A and AtUBC13B, that are highly conserved with respect to DNA sequence, protein sequence and genomic organization, suggesting that they are derived from a recent gene duplication event. Both AtUbc13 proteins are able to physically interact with human and yeast Mms2, implying that plants also employ a Lys63-linked polyubiquitination reaction. Furthermore, Both AtUBC13 genes were able to functionally complement the yeast ubc13 null mutants, suggesting the existence of an error-free DNA damage tolerance pathway in plants. The AtUBC13 genes appear to be expressed ubiquitously and were not induced by various conditions tested.

The ubc13a/b double mutant lines were created and displayed strong phenotypic changes. The double mutant plants were delayed in seed germination as well as cotyledon and true leaf development. When seedlings were grown vertically on plates, the roots of the double mutant were shorter and grew in a zig-zag manner, compared to the straight growth of wild type roots. Root length and number of lateral roots on wild type and ubc13a and ubc13b single mutant plants were about 3 times longer than those of double mutant plants after 9 and 12 days of growth. When double mutant seeds were sown directly into soil, many did not germinate and those that germinated grew much slower than wild type. At 35 days, double mutant plants were smaller with thinner, flatter, and lighter coloured rosette leaves compared to wild type plants. These phenotypes indicate that AtUbc13 not only plays a role in DDT to protect genome integrity but also is involved in plant development. Hence, this study set a cornerstone for future investigations into the roles of Ubc13 and Uev1 in plant development.