Resultados de la aplicación del procedimiento

Reciprocal crosses carried out with NYR-v showed that maternal transmission was associated with active endosperm expression (37.1%) compared to paternal transmission (1.2%). Epifluorescent microscopy was backed up by qRT-PCR data showing the greater amount of YFP expression. To date, no entirely synthetic transgene has been shown to be imprinted in plants, although, it has been observed in mammals, and is associated with differential methylation patterns dependent on parent-of-origin inheritance (Chaillet et al. 1991; Reik et al. 1987). Imprinted expression has,

however, been observed in transgenes incorporating endogenous imprinted sequences in plants (Kinoshita et al. 2004). Currently only 11 genes have been identified showing imprinted expression in maize (Raissig et al. 2011). Parental genes are epigenetically coded prior to fertilisation in the gametes or in the seed tissues following fertilisation, usually observed as differing methylation profiles. The parental orientation of transmission of coded epialleles can then effect their expression, most often in plants observed in the endosperm. A large number of candidate imprinted genes have been identified in maize endosperm tissue by analysis of transcriptional differences and the identification of differential transmission of DNA methylation patterns contributed by parental alleles (Lu et al. 2013; Waters et al. 2011; Gehring et al. 2009; Haun and Springer 2008; Kinoshita et al. 2004).

In maize and other flowering plants genome-wide demethylation of repetitive DNA occurs in the endosperm. This decrease in methylation has been utilised to aid in the identification of imprinted genes, which can show lower levels of DNA methylation compared to embryo tissue (Gutiérrez- Marcos et al. 2006; Lauria et al. 2004; Gehring et al. 2009). This difference in methylation can be seen in NYR-v, comparing endosperm and embryo methylation profiles of all cross orientations tested. In Arabidopsis genome-wide demethylation of the central cell occurs prior to fertilisation through the action of DME (DEMETER) (Hsieh et al. 2009; Gehring et al. 2006). However, DME function is not conserved in maize, therefore, the current theory is that demethylation of maternal alleles occurs in endosperm when the paternal alleles remain methylated.

My results show that methylation patterns of maternally and paternally transmitted NYR-v do not differ significantly in the endosperm. Furthermore, there is no significant difference between NYR-v promoter methylation between endosperm classified as active and silent in the maternal transmission. Therefore, there are no differing epialleles of the NYR-v promoter region to explain the difference in expression. However, methylation analysis of Mee1, the only gene imprinted in embryo tissue, shows a changing methylation profile dependent on the stage of seed development (Jahnke and Scholten 2009). The results state that DNA methylation does not always correlate with transcription, but instead is replaced by dynamic methylation profiles. But, despite dynamic methylation changes in the embryo, differing endosperm methylation profiles were still observed (Jahnke and Scholten 2009). The uniform reduced methylation state shown by the NYR-v promoter in endosperm tissue of both active and silence expression suggests that methylation does not correlate with expression in endosperm tissue. This could be the result of (i) histone modifications or (ii) undetected patterns of DNA methylation at other locations.

and a number have also shown association of specific histone modifications. A study conducted in Arabidopsis investigates the histone modifications present at Mez1, ZmFie1 and Nrp1, all of which are imprinted in the maize endosperm and have differing methylation patterns (Haun and Springer 2008). Repressive histone modifications, such as H3K27me2 and me3 can be found in alleles silenced by paternal transmission and histone modifications promoting active transcription, such as, acetylation of H3 and H4 or H3K4me2 were observed in active maternal transmission of alleles (Haun and Springer 2008). Differing histone modifications may therefore provide the epigenetic variation alone for imprinting, although it has not been observed without different DNA methylation profiles.

(ii) Imprinting has also been found reliant on DNA methylation at unexpected sites. Work con- ducted inArabidopsisfound that the imprinted expression of PHERES1 was dependent on methy- lation at a downstream location (Makarevich et al. 2008). Methylation analysis of NYR-v was only conducted at the e35S promoter, therefore, the methylation profile of adjacent regions may play a role in controlling expression. The identification of new imprinted genes has also led to the hypothesis of an effect of proximity of TEs to imprinted genes (Mosher and Melnyk 2010; Gehring et al. 2009). The genome-wide reduction in DNA methylation at TEs in the endosperm suggests that there may be involvement with imprinted loci. As previously stated, flanking NYR-v is a

gypsy-like LTR TE which may have a role in silencing, however, the role of TEs and repetitive elements in imprinting is still unclear.

The proportion of seeds showing active expression in the maternal cross orientation was also only 37%, with 50% of seeds expected to carry the NYR-v transgene. The same stochastic process that effects expression in vegetative tissue may also effect endosperm expression. On the other hand, this result may be the result of the development stage of the seeds observed. At 8 DAP, no endosperm displayed active levels of reporter expression from either cross orientation. Seeds derived from paternal transmission of NYR-v were also left for up to 16 DAP to ensure that active endosperms were not detected to the same levels at 12 DAP (data not shown). A later time point for maternally transmitted NYR-v may also have been beneficial to observe the proportion of active expressing seeds.

Alternatively, it could be argued that high expression of NYR-v from the maternal transmission is a result of a dosage dependent effect, a classic argument when applied to gene imprinting in the triploid endosperm of plants. However, this result would suggest that all seeds present uniform expression within each cross orientation, but as previously discussed, a proportion of maternally transmitted NYR-v seeds showed no expression.

Endosperm expression of NYR-v may be the product of imprinting, however, the controlling factor does not appear to rely on DNA methylation at the promoter. Therefore, the expression profile observed may be the result of unknown histone modifications or DNA methylation at different locations/adjacent endogenous regions.