Results Phenotype of plants overexpressing ABF3 A construct was created containing the CaMV 35S pro moter followed by the coding sequence of ABF3 and then the nopaline synthase transcriptional terminator, all of which was flanked by two loxP sites. This construct was transformed into Arabidopsis selleck chemical Sunitinib thali ana to generate 35S ABF3 plants. Fifty nine independent transformants were recovered and three high expressing lines containing single insertions were selected for further analysis. Control plant lines were generated by excising the 35S ABF3 transgene, leaving only Inhibitors,Modulators,Libraries the selectable marker transgene at the site of insertion. This was achieved by crossing 35S ABF3 plants with plants expressing the Cre recombinase gene and then back crossing the progeny to wild type Arabidopsis to remove the Cre recombinase gene, generating three control lines.

The loss of both the ABF3 transgene and the Cre recombinase transgene was con firmed by PCR. Previously, Arabidopsis plants overexpressing ABF3 were Inhibitors,Modulators,Libraries found to be more tolerant to drought conditions, which could at least partially be attributed to a lower Inhibitors,Modulators,Libraries rate of transpiration. To confirm that the 35S ABF3 lines show a similar phenotype, the transpiration rate was determined. Excised leaves from 4 week old 35S ABF3 plants left at room temperature for 1 day lost less fresh weight than both wild type and control plants, indicating a lower rate of transpiration. In addition, 35S ABF3 plants showed a mild growth retar dation that became more evident as plants matured,which is also consistent with previous results.

Identification of position effects Inhibitors,Modulators,Libraries and impact of Cre recombinase on the transcriptome Microarray analysis of control plant lines was performed to identify position effects as well as to determine if use of the Cre lox system can cause unintended effects in plant systems. Position effects Inhibitors,Modulators,Libraries should be specific to each of the three control lines, as each line is expected to have a unique insertion site. In contrast, unintended effects resulting from excision at cryptic lox sites are more likely to affect independent plant lines similarly, since cryptic sites that may be present in the Arabidop sis genome will equally be found in each plant line. Using a P value cut off of 0. 05, only a small number of genes were differentially expressed in control lines compared to wild type plants.

In the Control 48 line, only a single gene was differentially expressed. selleck chem inhibitor In the Control 57 and 59 lines, 10 and 4 genes were differentially expressed and two of these, rps7 and rps12. 1, were common to both lines. These genes could represent either position effects or natural background variation in gene expression. The rps7 and rps12. 1 genes that were differentially expressed in both Control 57 and 59 lines may reflect unintended effects of the Cre lox system.