THE ROLE OF DNA METHYLATION ON NUCLEAR AND CELL-DIFFERENTIATION IN THE FILAMENTOUS CAULONEMA OF THE MOSS FUNARIA-HYGROMETRICA

This paper describes the effects of hypomethylation with 5-azacytidine (5azaC) and dihydroxypropyladenine (DHPA) on protonemata of the moss Funaria hygrometrica Hedw. Following treatment with 5azaC or DHPA, hyp omethylation of the EcoR II (CCA(T)GG) and Pst I (CTGCAG) sites in rib osomal DNA (rDNA) was confirmed using restriction enzyme analysis and Southern hybridization. Hypomethylation of the genome had profound eff ects on protonemal differentiation. Whilst apical cell organization an d cell dimensions and shape remained unchanged, there was a marked ret ardation in both cytoplasmic and nuclear differentiation. Developmenta l abnormalities included: late and erratic side branch formation, some loss of the distinction between chloronema and caulonema, formation o f aberrant buds, and loss of the potential to form brood and tmema cel ls after 5azaC treatment which in general had more profound effects th an DHPA. Cytologically, caulonema cells were less highly polarized or unpolarized, and tended to retain small round chloroplasts, whilst nuc lei in the hypomethylated protonema endoreduplicated to lower levels a nd tended to remain more spherical. Increased nucleolar volumes and lo ss of intranucleolar rDNA heterochromatin following hypomethylation mi ght be the result of increased transcriptional activity of ribosomal R NA (rRNA) genes and drug-induced DNA decondensation, respectively. Gro wing hypomethylated protonema at 25 degrees C induced extremely atypic al cells and development. This temperature sensitivity and aberrations in development overall can be attributed to changes in the normal pat terns of gene expression brought about by hypomethylation of gene prom oter or regulator regions.