engleski

Noncanonical interactions of plant seryl-tRNA synthetase

It is well known that aaRSs can be involved in diverse cellular functions beyond translation independently or by forming protein-protein interactions. However, the studies of plant aaRS assemblies are scarce. We used high- throughput interactome technologies to identify potential protein interactors of Arabidopsis thaliana SerRS. We generated transgenic plants expressing SerRS-TAP and subsequent TAP-MS analysis revealed several potential interactors, including glutathione S- transferase PM24 and aluminium induced protein AT4g27450. Yeast two hybrid screen indicated interaction with BEN1, protein involved in metabolism of steroidal plant hormones brassinosteroids. These plant growth- promoting hormones regulate a variety of physiological processes crucial for normal plant growth and development and are also involved in plant stress response. The SerRS:BEN1 interaction was confirmed using surface plasmon resonance and microscale thermophoresis (MST), that enable Kd determination. To pinpoint regions responsible for interaction, truncated variants of both SerRS and BEN1 proteins were prepared and analyzed using MST. Kd for complex containing BEN1 and truncated SerRS variant without basic C-terminal extension was similar to Kd of the SerRS:BEN1 complex indicating that basic C- terminus does not participate in interaction. N-terminal tRNA binding domain of SerRS did not form the complex with BEN1. Deletion of N- terminal extension of BEN1 containing six acidic amino acids disrupted the interaction. Therefore we conclude that interaction involves SerRS globular catalytic domain and acidic N- terminus of BEN1. Furthermore, using RT-qPCR we investigated possible correlation of these two proteins on the level of gene expression. We observed slightly lower expression of BEN1 gene in transgenic plants overexpressing SerRS compared to wild type. To test whether SerRS regulates BEN1 expression in nucleus we determined its localization using GFP- localization experiments and immunoblot analysis. Our data show that SerRS is exclusively localized in cytosol, despite its several putative nuclear localization signals. To investigate the role of SerRS in stress, we examined growth of transgenic seedlings overexpressing SerRS on growth media that induce ionic stress, osmotic stress and stress imposed by heavy metals. Our results show that transgenic seeds germinate earlier and that in most cases transgenic seedlings grow better on stress media compared to wild type seedlings. This indicates that enhanced expression of SerRS plays a role in the response of plant to various stress conditions. Considering that brassinosteroids are also involved in plant stress response we propose functional importance of SerRS:BEN1 assembly.

aminoacyl-tRNA synthetase ; plant ; protein interactions ; cellular localization ; stress response

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