Characterizing changes in genome-wide methylation in response to elevated carbon dioxide in Arabidopsis thaliana: a methylation sensitive amplified fragment length polymorphism (MS-AFLP) approach

Abstract

In this study, Arabidopsis thaliana (A. thaliana) were grown in either ambient carbon dioxide (CO2) concentrations or elevated CO2 concentrations where half of the plants from each group were treated with 5-azacytidine during plant development; these treated groups were used as a positive control. At the rosette state, A. thaliana leaves were collected for DNA extraction, then were concentrated. The extracted DNA was used for the technique, methylation-sensitive amplified fragment length polymorphism (MS-AFLP) where restriction enzymes, HpaII and MspI were applied. These restriction enzymes cleave CCGG sites differently and are responsible for identifying differential methylation states throughout the entire A. thaliana genome. After the MS-AFLP protocol, the samples were visualized on 4% agarose gels where it was determined that the majority of the CCGG sites were unmethylated, non-polymorphic fragments. Regarding 5-azaC as a positive control, it was successful since more unmethylated sites were visualized in the 5-azaC treated groups, compared to the control groups. As for the differential methylation states, there was evidence that A. thaliana growth in different CO2 concentrations cause differential methylation states since a total of 28 polymorphisms were identified where 10 polymorphisms were identified between control groups grown in either low or high CO2 while 18 polymorphisms were identified between 5-azaC treated groups grown in either low or high CO2. Although these polymorphisms were produced, differential methylation seemed to be randomized where patterns among the polymorphisms could not be identified.