Efeito da superexpressão dos fatores de transcrição ZmDof1 e OsDof25 sobre a eficiência de uso de nitrogênio em Arabidopsis thaliana L

Abstract

To improve nitrogen usage efficiency in plants the rice transcriptional factor OsDof25 was identified and cloned, whose probably orthologe is the maize ZmDof1, already identified and partially characterized. The ZmDof1 was also cloned for comparative analysis with OsDof25, in order to confirm this last one as ZmDof1 orthologe in rice. The constructions for Arabidopsis superexpression of these transcriptional factors were made using the cloning system of gateway technology (Invitrogen), to obtain the expression vectors 35S:ZmDof1:HA and 35S:OsDof25:HA. Lineages with different expression levels of these genes were obtained, but with only one inserted copy. These transgenic lineages when grown in a half strength of MS medium (10mM of NH4 + and 20mM of NO3 -) showed phenotypes with chloroses and growth difficulty; although when they were cultured in soil they showed great vegetative development and delay in the inflorescence emission. When analyzed the gene expression changes induced by the superexpression of these transcriptional factors, it was observed that both genes produced an increase in the expression levels of high and low affinity ammonium transporters (AMT1.1 and AMT2.1, respectively), indicating that these phenotypes may be due to the toxic effect of an excess of ammonium uptake. We also verified an increase of expression for pyruvate kinase (PK1 and PK2), and phosphoenolpyruvate carboxylase (PEPC1 and PEPC2). Pyruvate kinase converts phophoenolpyruvate (PEP) to pyruvate, and phosphoenolpyruvate carboxylase converts PEP to oxalacetate, which is substrate for malate dehydrogenase to form malate. Both pyruvate and malate may feed the Krebs cycle. In addition, there was an increase in the expression of isocitrate dehydrogenase, which is present in the citosol and mitochondria, needed for converting isocitrate to 2- oxoglutarate. Thus, it was hypothesized that the increase of expression levels of these carbon metabolism enzymes was necessary to increase the production of 2-oxoglutarate and, consequently, to reduce the toxic effect of ammonium uptaked. Besides, it was observed an increase of expression levels and activity of glutamate dehydrogenase (GDH). This enzime may work as much in the direction of glutamate amination as in deamination, when the plants were submitted to ammonium excess or carbon limitation conditions, respectively.