An efficient immunodetection method for histone modifications in plants

Abstract

Background: Epigenetic mechanisms can be highly dynamic, but the cross-talk among them and with the genome is still poorly understood. Many of these mechanisms work at different places in the cell and at different times of organism development. Covalent histone modifications are one of the most complex and studied epigenetic mechanisms involved in cellular reprogramming and development in plants. Therefore, the knowledge of the spatial distribution of histone methylation in different tissues is important to understand their behavior on specific cells.

Results: Based on the importance of epigenetic marks for biology, we present a simplified, inexpensive and efficient protocol for in situ immunolocalization on different tissues such as flowers, buds, callus, somatic embryo and meristematic tissue from several plants of agronomical and biological importance. Here, we fully describe all the steps to perform the localization of histone modifications. Using this method, we were able to visualize the distribution of H3K4me3 and H3K9me2 without loss of histological integrity of tissues from several plants, including Agave tequilana, Capsicum chinense, Coffea canephora and Cedrela odorata, as well as Arabidopsis thaliana.

Conclusions: There are many protocols to study chromatin modifications; however, most of them are expensive, difficult and require sophisticated equipment. Here, we provide an efficient protocol for in situ localization of histone methylation that dispenses with the use of expensive and sensitive enzymes. The present method can be used to investigate the cellular distribution and localization of a wide array of proteins, which could help to clarify the biological role that they play at specific times and places in different tissues of various plant species.

Figure 1

Schematic representation of the principal steps performed in the protocol for immunodetection of histone methylation. Different tissues were collected and fixed with FAA solution and paraffin-embedded. The samples were sectioned into 4-5-μm slices and the slides were dewaxed. The antigenic sites were retrieved and the tissue was blocked and incubated with the primary and secondary antibodies. The samples were counterstained with DAPI and analyzed under confocal microscope.

Figure 2

Plant material collected for the immunodetection proteins.(A) Callus of Arabidopsis thaliana. (B) Shoots and buds of Cedrela odorata. (C) Plantlets of Agave tequilana. (D) Somatic embryos of Coffea canephora obtained 56 days after induction. (E) Flowers of Capsicum chinense. A, C and D were obtained through cultures in vitro. B and E were collected from the field.

Figure 3

Immunodetection of histone modifications in tissues of several plant species. Immunodetection was performed using antibodies against H3K4me3 and H3K9me2, using the histone H3 as positive control. Panels from left to right show the confocal images of transmitted light, DAPI, histone H3, H3K4me3 and H3K9me2. Dashed squares represent the close-up of the sites detected by immunoflourescence with the antibodies mentioned above in the globular embryo of Coffea canephora(A), in meristematic zones in the callus of Arabidopsis thaliana(B), in the shoot apex of Agave tequilana(C), in the endothecium (yellow arrowhead) and in the lobes cells of anthers (white arrowhead) in Capsicum chinense(D) and in the proximal cells to epidermis of Cedrela odorata (E).

Figure 4

Visualization of H3K4me3 and H3K9me2 in specific cells in different plants. Immunodetection was carried out with specific antibodies against H3K4me3 and H3K9me2. Confocal images of DAPI, H3K4me3, H3K9me2 and merged signals are shown from left to right and represent specific zones magnified from dashed squares shown in Figure  3, to verify that the signal in section tissues is free of background noise and it is inside cells. (A) Meristematic cells of globular embryo in Coffea canephora. (B) Callus cells of Arabidopsis thaliana. (C) Shoot apex cells of Agave tequilana. (D) Epidermic layer (yellow arrowhead) and endothecium cells (white arrowhead) of lobes anthers in Capsicum chinense. (E) Epidermis and collenchyme cells of Cedrela odorata. In all species, the H3K4me3 was observed to be particularly abundant in the biggest and largest cells; whereas the H3K9me2 was visualized in the nuclei of cells preferentially as flourescent spots.