At 4-6 dpi, a much more uniform pattern of methylation change was observed. For 13/47 genes that had heterochromatic sensing/marking domains in our previous time series (Table 1), methylation was increased in the leaf, and in some cases reduced in the sheath (Table 2). For the other 24 genes, methylation status was uniformly increased (Table 2, Supplementary Table S2). For example, WOX16 and ERF6 had increased methylation at 6 dpi at heart-specific, hypomethylated, early Ems domains (Baena-López et al., 2012; Oppermann et al., 2012). Notably, in this latter group of genes, methylation increased systematically from 1 dpi, reaching a new high of 45% at 6 dpi (Supplementary Table S3). Level of methylation at 4 wpi (start of the time series) ranged from 0.4% to 54% with an average of 10.2%, showing that this is a strong progressive increase (Supplementary Table S3).
Methylation levels were used to test the effect of heat shock (HS) treatment on the level of CHG methylation in the sample. HS causes overall genome-wide demethylation, and therefore causes hyper-methylation of many, but not all, of the CHG methylated promoters in plants (Moore-Lande and Fridborg, 2009; Franckie and Klose, 2011). In the presence of HS, 48 and 73% of the genes tested showed an increase of two methylation states and more above four methylation states, respectively. HS had a detectable effect, although not large, on the 56% of methylated promoters (Supplementary Table S4).
Developmental gene expression was measured during differentiation of XY tobacco embryos. Analysis of gene expression showed that the majority of genes that showed the earliest expression in development, 1 and 2 dpi, remained highly expressed until 6 dpi. This highlights the role of these genes in early patterning (Gebhardt and Grossniklaus, 2006; Barres and Chheda, 2008; Grossniklaus and Benfey, 2009). d2c66b5586