Investigation of the Physiological Function of Protein Phosphatase 4 (PP4) in Arabidopsis thaliana

Abstract

Protein Phosphatase 4 (PP4), a member of serine/threonine-specific phospho-protein phosphatases (PPP) family, is remarkably well conserved across eukaryotes. PP4 has been studied mainly in yeast and mammalian cells, and virtually nothing is known about PP4 in plants. In mammalian cells, PP4 plays a role in several processes not relevant to plants. The major objective of this thesis was to investigate the physiological function of PP4 in Arabidopsis thaliana as a model plant. A. thaliana has two PP4 catalytic subunits, namely PP4-1 (At4G26720) and PP4-2 (At5G55260). In addition, putative regulatory subunits for PP4 were bioinformatically detected in A. thaliana: PP4R2L (At5G17070) and PSY2L (At3G06670). Using reverse genetics approach, this thesis focuses on expression studies (semiquantitative RT-PCR) and observation of phenotype of the A. thaliana gene encoding PP4 catalytic and putative regulatory subunits in loss-of-function mutants (T-DNA insertional mutagenesis lines), artificial microRNA (amiRNA) stable lines, and gene overexpression lines. Using intensive expression analysis by the in-gel RT-PCR, we succeeded to detect knock down and/or knock out for amiRNA plants and T-DNA plants for PSY2L and PP4R2L. Moreover, we detected and isolated stable overexpression plants for PP4-1, PP4-2, and PP4R2L. Observation of phenotype showed that knock out of PSY2L gene in a T-DNA line, SALK_048064 (insertion in exon 3 of 25), show some interesting phenotypes. The homozygous mutants of this line showed dwarfism, delayed growth, and extended life span. The knock down of PSY2L through amiRNA mechanism also showed a phenotype, such as reduced size and twisted leaves. No significant phenotype was found in overexpressor plants. We also investigated the subcellular localization of these subunits in two different plant expression systems: A. thaliana mesophyll protoplasts and particle bombardment into onion epidermis cells. Main location of the catalytic subunits, PP4-1 and PP4-2, are in cytoplasm, with few in nucleus. PSY2L is strongly found in nucleus, whereas the other regulatory subunit, PP4R2L is not only found in nucleus but also tend to locate in cytoplasm. In vivo investigations of subcellular localization of PP4 subunits show resemblance to in silico analysis.