Suppression of photosynthetic gene expression by chloroplast DNA methylation: Consolidating scientific understanding

The instructions for photosynthesis, breakdown of chlorophylls, and lycopene synthesis are found in the genetic code of DNA, which consists of four different letter combinations: A, C, G, and T. Of these, the chemical structure of C is of great interest. C can have a methyl group at position 5 of its chemical structure. The following methods elucidated the function of methylated DNA in tomato fruit. (1) Some restriction enzymes that cut DNA are sensitive to methyl groups, and some are not, and DNA methylation can be detected by using these enzymes. (2) Methylated C can be detected by a technique called "reversed-phase HPLC (high-performance liquid chromatography)," which separates the letter molecules A, C, G, and T in DNA after its hydrolysis to individual bases. (3) Photosynthesis occurs in cell organelles called chloroplasts, which can be isolated, and gene expression can continue in vitro. (4) DNA extracted from chloroplasts can be used for gene expression (RNA synthesis) in vitro using an enzyme called RNA polymerase, prepared from chloroplasts or commercially available. Using these four techniques, we concluded that "photosynthetic genes in chloroplasts are methylated during the reddening process of tomato fruit, resulting in the loss of expression of these genes and the eventual cessation of photosynthetic function." We published this in 1990 in the European "EMBO Journal," which is on par with the "Proceedings of the National Academy of Sciences of the United States of America (PNAS)." The results attracted the interest of other researchers, and considerable studies were conducted on DNA methylation and gene expression in tomatoes and other plants.

I thought molecular genetic studies in a model experimental plant, Arabidopsis thaliana, might provide additional insight into the mechanisms mediating this DNA methylation. Arabidopsis does not have an organ corresponding to the tomato fruit. Therefore, we compared DNA methylation between Arabidopsis leaves, roots, and cultured cells. However, we could not find any phenomena in Arabidopsis that could be considered to be mediated by DNA methylation. For the next 30 years, I worked on other research projects. During this time, complete literature databases have been developed, and it is easy to see which of our publications have been cited in subsequent research papers. While some papers support our results, one can find a significant number of papers that dispute them. Over the next 30 years, technology has advanced where (5) C methylation can be determined at the DNA sequence level (the bisulfite sequencing method). (6) The DNA sequence's position and amount of methylation modifications can be determined using the latest technology for selective photo-crosslinking detection of methylated C in DNA (Ps-Oligo with PAA-g-Dex). (7) It is possible to determine whether or not RNA polymerase or transcription factors bind to the beginning of gene reads on DNA (ChIP: chromatin immunoprecipitation method). (8) Methylated C-specific antibodies can be used to analyze gene sequences near the methylated C (MeDIP: methylated DNA immunoprecipitation method). Furthermore, this antibody can detect highly sensitively methylated DNA and stain tissues. (9) DNA with methylated Cs in specific sequences can be chemically synthesized, and gene expression (transcription) of such methylated sequences can be examined in the in vitro systems described above. Although these experiments with tomatoes are desirable, we understand the following now. (I) Negative regulation by DNA methylation occurs only in irreversible processes, suppressing the expression of critical photosynthetic genes. This is evident in phenomena such as the reddening process in tomato fruit, cultured cells that have lost their ability to re-differentiate after years of passaging, and cell-specific gene expression in photosynthesis (C4 photosynthesis) in maize. In other words, this system does not work as DNA demethylation in situations where photosynthetic function is acquired, such as when seedlings grown in the dark are greened by light. (II) Results have been reported in which methylation of chloroplast DNA during tomato reddening was not detected by methods (1) and (2) above. The Argentine variety Platense was used in this experiment, and experiments (3) and (4) were not performed. We mainly used the Japanese variety Firstmore. Comparing these results, (1) is not comparable because the probes used to identify the DNA fragments differ. In (2), since the same methodology is used, the elution time of methylated C in the variety Platense has a shoulder instead of a peak in the variety Firstmor. There may be differences in the degree of DNA methylation among the varieties. In method (1), we must also consider that the restriction enzyme recognition site may not be located within the sequence that controls gene expression. Also, in process (2), 26% of C is methylated in our experimental system, but if it is less than 5%, it is at the detection limit. Based on our experiment (4) result, the DNA methylation rate of Platense is probably around 1/5 of Firstmore's. Even with low methylation, the genes will likely not be expressed if the methylation is within the regulatory sequences. A comprehensive analysis of methylated DNA (methylome) has recently become available. A paper on the analysis of chloroplast DNA methylation during ripening in rice strongly supports our observation of tomato fruits turning red.


June 17, 2024, released      Hirokazu Kobayashi