The yeast regulatory protein kinase, general control non-derepressible-2 (GCN2) plays a key role in general amino acid control. have also been identified in (Santoyo (Sattlegger (Lbackground. Arabidopsis plants were either grown in sterilized nutrient medium plates for herbicide treatments or in non-sterilized soil for virus infection. For herbicide treatment experiments, Arabidopsis seeds were surface-sterilized and sown on a piece of sterile filter paper (3MM) laid on the surface of B5 medium (Sigma G5893; Gamborg (TYMV) or (2005) were examined for their expression stability and gene At1G13320 encoding a protein Rabbit Polyclonal to ADRB1 Hh-Ag1.5 supplier phosphatase 2A subunit (PP2A) was chosen for use in all subsequent experiments (data not shown). Table 1. Nucleotide sequences (5 to 3) of primers used for real-time quantitative PCR analysis Two biological replicates were used for each of the 18 treatment combinations: two lines, wild-type and GT8359, by nine treatments (Control, Glyphosate, Glyphosate+FWY, IRL 1803, IRL 1803+H, Acifluorfen, Diuron, Chlorsulfuron, and Hh-Ag1.5 supplier Chlorsulfuron+ILV). For each biological replicate there were three technical replicates. Technical replicates were kept together on 96-well plates, but treatment combinations were spread across five plates, for each of the biological replicates separately. This was done in order to have as many genes under investigation as possible on any given plate complete with the control (reference) gene. Data were processed using the window of linearity method (Ramakers and arbitrary target gene on plate (((and are the mean efficiencies for plates whilst and are the tests on the corresponding degrees of freedom (df). These analyses were performed using the GenStat? (2007) statistical system with reference to Payne (2007). Electron microscopy Electron microscopy was performed by Jean Devonshire of the Rothamsted Centre for Bioimaging, UK. Plant tissue was prepared for examination under the electron microscope by leaf dip in 2% phosphotungstic acid (PTA) negative stain and transfer to pre-prepared formvar/carbon coated Cu grids (200 mesh). The electron microscope used was a JEM2010 FasTEM (JEOL UK) with an Ultrascan 1000 camera (GATAN UK) for imaging. Results Immunodetection of AtGCN2 in Arabidopsis seedlings and production of a homozygous null line of mutant GT8359 Antiserum was raised to a peptide with the amino acid sequence KLRPYSKDMGYEDTD, which is present in the N-terminal region of Arabidopsis GCN2 (AtGCN2) (Zhang (Lelement in the first intron … Mutant GT8359, a Genetrap line containing a element insertion in the gene, was obtained from Cold Spring Harbor Laboratory (http://genetrap.cshl.org). The presence of the element in the gene was confirmed by amplification of two fragments by polymerase chain reaction (PCR). The first was amplified using primers specific for the which is disrupted by insertion of the element in GT8359, and therefore was present in the wild type and heterozygote, but not the homozygous null mutant. Subsequently, all seeds Hh-Ag1.5 supplier were grown on plates containing kanamycin. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that an mRNA molecule was still being transcribed from the gene in the homozygous line (not shown). However, western analysis showed very clearly that the AtGCN2 protein was not present in extracts from homozygous null plants (Fig. 1A). DNA fragments containing both the element was inserted in the first intron with an eight base-pair duplication at the insertion site (Fig. 1B). Lack of AtGCN2 makes Arabidopsis more sensitive to herbicides that affect amino acid biosynthesis Mutant GT8359 grew normally in soil (Fig. 2A) and.