Exploring the crop epigenome: a comparison of DNA methylation profiling techniques

Abstract

Epigenetic modifications play a vital role in the preservation of genome integrity and in the regulation of gene expression. DNA methylation, one of the key mechanisms of epigenetic control, impacts growth, development, stress response and adaptability of all organisms, including plants. The detection of DNA methylation marks is crucial for understanding the mechanisms underlying these processes and for developing strategies to improve productivity and stress resistance of crop plants. There are different methods for detecting plant DNA methylation, such as bisulfite sequencing, methylation-sensitive amplified polymorphism, genome-wide DNA methylation analysis, methylated DNA immunoprecipitation sequencing, reduced representation bisulfite sequencing, MS and immuno-based techniques. These profiling approaches vary in many aspects, including DNA input, resolution, genomic region coverage, and bioinformatics analysis. Selecting an appropriate methylation screening approach requires an understanding of all these techniques. This review provides an overview of DNA methylation profiling methods in crop plants, along with comparisons of the efficacy of these techniques between model and crop plants. The strengths and limitations of each methodological approach are outlined, and the importance of considering both technical and biological factors are highlighted. Additionally, methods for modulating DNA methylation in model and crop species are presented. Overall, this review will assist scientists in making informed decisions when selecting an appropriate DNA methylation profiling method.

Keywords: DNA methylation modulation; DNA methylation profiling; bisulfite sequencing; crop epigenome; immunological techniques; mass spectrometry; next-generation sequencing.

Figure 1

Mechanisms of DNA cytosine methylation in plants. (A) Maintenance and de novo DNA methylation occur in all sequence contexts (CG, CHG, and CHH; where H = A, C, or T). Methyltransferase 1 (MET1) maintains methylation in the CG context, CHROMOMETHYLASE 2 (CMT2) or CMT3 catalyzes and maintains methylation in the CHG context, DOMAINS REARRANGED METHYLASE 2 (DRM2) and CMT2 accomplish and maintain methylation in the CHH context. The RdDM pathway conducts de novo DNA methylation in all sequence contexts. (B) The attraction of histone H3 lysine 9 (H3K9)-specific suppressor of variegation 3-9 homolog proteins (SUVH4, SUVH5 and SUVH6) results in the formation of dimethylated H3K9 (H3K9me2), and the recruitment of CMT2 and CMT3, creating a self-reinforcing feedback loop. (C) Methylation of the methylation monitoring sequence (MEMS) in the promoter region of the Repressor of silencing 1 (ROS1) gene is crucial for its transcription. Cytosine methylation at MEMS is regulated by both MET1/RdDM and ROS1, enabling sensing/monitoring of methylation levels and maintaining DNA (de)methylation homeostasis. The methyl group is denoted by “M”, and methylation state is represented as “me/m” (Adapted from Kumar and Mohapatra, 2021).

Figure 2

Principle of the conversion of cytosine to uracil by bisulfite-treatment.

Figure 3

Pipeline of the Whole Genome Bisulfite Sequencing method.

Figure 4

ONT sequencing platforms allow for real-time analysis of individual DNA strands as they pass through the nanopores embedded in electro-resistant membrane. Each nanopore is connected to a channel and sensor chip, which measures the electric current that flows through the nanopore. Each base and base modification produces a specific electrolytic signal allowing for detection of 5mC.

Figure 5

A single SMRTbell per ZMW is amplified using fluorescent-labelled nucleotides while their emission spectra are collected. The kinetic data is analyzed using bioinformatics to decipher the positions of the methylated cytosines.

Figure 6

DNA methylation nuclear distribution patterns. Quercus suber proliferating embryogenic masses. Confocal images of: (A) 5mdC immunofluorescence (green), and (B) merged image of DAPI (blue) and 5mdC immunofluorescence (green). Bar: 10 µm.

Figure 7

Variants of DNA nucleobases in plants. 5-Methylcytosine is the most well-known modified nucleobase, but seven others have been documented in plants. Mass spectrometry is a powerful tool for their detection, while some sequencing techniques can also detect and map their localisation in the genome. However, this approach is restricted to species with a reference genome.