Expression and characterization of a protein tyrosine phosphatase from soybean and kinomic profile analysis of roots under germination process

Author(s)

Luciana de Campos Leite Medeiros

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2008-08-29

Examining board members

Hiroshi Aoyama; Jörg Kobarg; Jose Mauro Granjeiro; Pietro Ciancaglini; Janaina Nicanuzia dos Prazeres

• Advisor: Hiroshi Aoyama; Celso Eduardo Benedetti

Abstract

Our group previously described the purification and characterization of four acid phosphatase isoforms obtained from mature soybean seeds, and determined the kinetic and physico-chemical properties of these enzymes. Structural and physical studies demand high amounts at purity levels of these enzymes, requiring efforts in laborious conventional purifications. Due to this fact and to the existence of few plant proteins that already has been cloned and expressed, we proposed to clone and express a protein tyrosine phosphatase (PTP) from soybean. The kinetic studies of the recombinant PTP revealed an enzyme with phosphatase typical features and higher specificity toward Tyr-phosphate when compared to other analysed substrates. The analysis of thermal inactivation of the enzyme was carried out by circular dichroism, which showed that GmPTP had low thermal stability. The immunolocalization of GmPTP, by western blot, identified the enzyme in different soybean tissues, which were germinated in the presence and absence of light. These results are in agreement with those obtained from the quantitative PCR results, that showed a higher expression of GmPTP in roots when compared with other evaluated tissues. The physiological importance of this tissue to the plant metabolism corroborates our results, because adequate levels of phosphorus are required for the good operation of metabolic functions, as growth and development. The phosphorus demand and the consequent soil absorption through the roots dramatically increase in periods of high growth rate, for instance, during germination. Therefore, phytases and phosphatases are important suppliers for plant metabolism and besides the contribution to provide nutrients, phosphatases could play an important role in regulation of signal transduction cascades. Kinase microarrays were employed as a strong tool to analyse the root kinome in soybean plantlets germinated in light and dark conditions. We concluded that abiotic factors as light presence and nutrients availability were responsible for the activation of different pathways and different phytormones prevalence. Relating the germination conditions of soybean plantlets to these factors and to the analysis of the most activated kinases on the chip, we propose a signaling model to explain that, when the hormone abscisic acid is the main response of stress stimuli, it triggers the activation of MAPK and calcium signaling, and the resulting modulation of kinases and phosphatases activities led to proliferation and cytoskeleton rearrangement in roots. In this work, we described the cloning and expression of a soybean PTP, its kinetic characterization, tissue distribution and investigation of a putative relationship with the signaling pathway triggered by light and dark. The investigation and characterization of PTPs from plants can add relevant information in the plant metabolism research field. Therefore, we provided a wealth biochemical data which suggest that soybean plantlets contain typical PTP, which is mainly expressed in roots and apparently plays a critical role during germination. (AU)