6mA DNA methylation dynamics during chloroplast development in Agave angustifolia Haw

N6-methyladenine (6mA) DNA accumulates during chloroplast maturation in Agave angustifolia Haw. and covaries with METTL4 protein abundance, identifying 6mA as a candidate epigenetic mark associated with plastid biogenesis. Chloroplast development requires precise coordination of nuclear and organellar genomes, yet the epigenetic mechanisms regulating this process remain poorly understood. Here, we provide the first description of N6-methyladenine (6mA), a dynamic DNA modification, during Agave angustifolia Haw. chloroplast biogenesis. Using naturally occurring somaclonal variants-an albino phenotype with arrested proplastid development and a green phenotype with mature chloroplasts-we show that global 6mA levels are significantly higher in tissues with mature chloroplasts compared to those with immature proplastids. Immunofluorescence analysis revealed that while nuclear 6mA distribution remains similar between phenotypes, cytoplasmic 6mA signal area is approximately 23-fold greater in the green phenotype, a pattern most consistent with 6mA accumulation in chloroplast nucleoids during plastid maturation. Through bioinformatic analysis of the A. angustifolia transcriptome, we identified two homologs of the Arabidopsis 6mA demethylase ALKBH1A and three of ALKBH1D, along with six putative METTL4 methyltransferases. These findings identify 6mA as a candidate dynamic epigenetic mark during chloroplast development and are consistent with the hypothesis that METTL4 functions as a 6mA methyltransferase in A. angustifolia, in line with recent findings in Arabidopsis. Our work provides the first correlative evidence associating 6mA dynamics with organelle biogenesis in plants and opens new avenues for future mechanistic studies of epigenetic regulation of photosynthetic capacity.