Enabling the Spaceflight Methylome: DNA Isolated from Plant Tissues Preserved in RNAlater^® Is Suitable for Bisulfite PCR Assay of Genome Methylation

It is likely that alteration to the methylome of plants flown aboard the International Space Station (ISS) is one of the modalities engaged to cope with this unfamiliar environment (Paul et al., unpublished observations). Methylation occurs at the 5 position of cytosine bases and coordinates chromatin modification by recruitment of chromatin remodelers, resulting in regulation of gene expression, X-chromosome inactivation, genomic imprinting, and embryonic development (Jaenisch and Bird 2003; Mohandus et al., 1981; Li et al., 1993; Finnegan et al., 1996), and has a vast amount of relevance to all kingdoms of life. DNA methylation on cytosine bases is utilized across the genome to withdraw the availability of DNA from interactions with transcriptional machinery, generating alterations in the plants’ transcript profile and resulting physiology. Cytosine methylation is involved in a variety of biological processes in plants (Stroud et al., 2013), including development, pathogen response, temperature, and salt stress. Given that the environment of spaceflight is essentially unknown within the evolutionary history of plants, it is likely that a range of adaptation mechanisms, including DNA methylation, will occur as plants adapt to this novel environment. Therefore, the goal of this study is to establish a method for genomic DNA isolation suitable for the investigation of the methylation status, and that is consistent with accepted practices for plants that have been grown and harvested aboard the ISS.

APEX04-EPEX is an upcoming Arabidopsis plant growth experiment currently planned for launch on SpaceX-10. The focus of EPEX, which is an acronym for Epigenetic Expression, is to examine the spaceflight methylome of Arabidopsis. In preparation for that experiment, a ground-based Experiment Verification Test (EVT) was performed at Kennedy Space Center (KSC). The focus of this paper is to document the approaches used in the EVT, which tests the utilization of the resources and preservation techniques that will be available for the upcoming spaceflight experiment. In the design of this EVT, methylation mutants were included to give a base of evidence from which we could make conclusions as to the quality of the methods and approach for preserving the methylome and eventually for investigating the role of the methylome remodeling process as a response to spaceflight. The Methyltransferase 1 (MET1) and Elongator complex subunit 2 (ELP2) are key genes that encode regulators playing central roles in DNA methylation of plants (Kankel et al., 2003; Saze et al., 2003; Wang et al., 2013) and the met1-7 and elp2-5 mutants have been previously described (Kanno et al., 2008;Wang et al., 2013). Compared to the wild type control, the altered DNA methylation patterns in these mutants should reveal methylome differences, provided that the method preserves cytosine methylation across the genome. This paper describes the process that generates an examination of methylation states of two genes chosen from previous spaceflight data (Paul et al., 2013; Paul et al., unpublished data) in RNAlater™-preserved Arabidopsis plants using a modified DNA extraction method followed by bisulfite conversion, polymerase chain reaction (PCR) amplification, TA cloning, and Sanger sequencing to determine methylation status.

We expected the DNA to be of lower concentration and amount in root samples, asthese tissues were of lower fresh mass and, in general, contain lower DNA concentration per gram of fresh mass. Given the small growth phenotype present in the elp2-5 genotype, we expected DNA extractions of elp2-5 roots to be of concern (Figure 1). Since the corresponding shoot DNA extractions had such a large fresh mass input, we expected the quality of shoot DNA to be qualitatively compromised. This also raised concern for elp2-5 shoot extractions due to the comparatively large number of plants deemed necessary for adequate DNA quantity for bisulfite conversion. In general, four replicates of each material exhibited comparatively consistent DNA quality and quantity. The Col-0 roots and shoots provided DNA samples with similar concentration, while met1-7 and elp2-5 roots showed obviously lower DNA concentration compared with the corresponding shoot samples (Figure 2; Table 1), indicating the difficulty in handling root tissues of small mass. In all cases, no RNA or protein contamination or DNA degradation was observed in the gel (Figure 2), which suggested DNA quality of adequate levels for bisulfite conversion. Moreover, given the total DNA solution amount of 100 μL, most samples met the requirement we had set for bisulfite conversion (200 ng with 10 ng/uL). The exceptions were one root sample of met1-7 and two root samples of elp2-5 (Table 2). However, total DNA amounts of these three samples were above 900 ng, which is much higher than the minimum suggestion for bisulfite conversion, and were suitable for bisulfite conversion after being subjected to standard concentration treatments. Three biological replicates from Col-0 root and shoot samples were analyzed by TapeStation, and generated DIN values above 6.3 on a 10-point scale (Figure 3). This extra step of qualification verified the level of quality displayed in the agarose gel electrophoresis (Figure 2) and also generated valuable information for a variety of downstream applications.

After the sequence information was produced, manual observations of the distribution of methylated cytosine bases were undertaken, generating the information displayed in Figure 5 and Figure 6. No mixed peaks were observed in the chromatogram peaks—a tribute to the uniformity of cloned amplicons in the TA colonies. For AT2G07698 and AT2G07687, the regions of +253 to +507 and +19 to +355 (the A of ATG start codon was designated +1) were analyzed, respectively. Methylation in AT2G07698 was much more bimodal and generally much lower than in AT2G07687, typically having only 0 or 1 out of 15 colonies methylated at a given position in the CG, CHG, or CHH context in wild type (Figure 5). In met1-7, no methylation was observed in most of the cytosine sites of AT2G07698 exon region (Figure 5), which is in line with the MET1 role of maintaining CG methylation (Cao and Jacobsen, 2002). It has been reported that loss-of-function mutation of ELP2 will lead to disorder in genome-wide DNA methylation (Wang et al., 2013). Indeed, in two CG sites and most of CHH/CHG sites of AT2G07698 region, elp2-5 showed a higher methylation level than Col-0 (Figure 5). For AT2G07687, the methylation levels were somewhat consistent among three genotypes in most of CG and CHG sites (Figure 6a–b). For CHH context, most of the cytosine methylation was disrupted in both met1-7 and elp2-5 (Figure 6c). Interestingly, unlike AT2G07698, elimination of methylation in CG context was observed in AT2G07687 exon region in met1-7, suggesting the stability of CG methylation in this gene. Together, the Sanger sequencing results indicated that these extracted DNA samples were suitable for PCR-based cytosine methylation analysis.

Figure 5

Figure 6

In summary, we have generated a working method for isolating genomic DNA from RNAlater^®-preserved material, and characterrizing the methylation status of DNA from RNAlater^®-preserved plants. These data illustrate that novel approaches can be used to navigate some of the necessary limitations of spaceflight experiments, such as the reliance of RNAlater^®-preserved material in many current ISS preservation scenarios. In addition, examinations of genotypes revealed genotype-specific differences in the methylation state of genes of interest. For DNA extraction, we deemed 10 of Col-0, 16 of met1-7, and the maximum available elp2-5 (total minus 3 for RNA) plants to be necessary to get DNA solutions of adequate concentration for bisulfite conversion. We also found that the PCR conditions used to amplify bisulfite-converted DNA derived from RNAlater^® samples were highly sensitive, and in our case it was necessary to use a polymerase kit designed specifically for amplifying bisulfite-converted DNA. In our attempts to analyze the PCR products generated from our genes of interest, we adopted the TA cloning approach to not only clean up the Sanger sequencing chromatograms, but to also introduce the ability to quantify percent methylation of a locus within a diverse population of cell types. Results produced from the methylation analysis revealed that the levels of methylation varied widely between genotypes and the gene analyzed. This targeted approach adopted for the EVT will have wide application in space-related genomic research, and also revealed interesting gene-specific differences in methylation among genotypes. From the data that we collected during this experiment, we demonstrated that in addition to its intended purpose of RNA preservation, RNAlater^®-preserved plant material is suitable for high-quality DNA extraction and methylation analysis among comparably preserved controls.