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It was expected that the contrasting effects of MET1a/s were mediated by
removal of silencing of the paternal allele of endosperm growth inhibitors, thus
causing seed size increase and vice versa [11]. However, MET1a/s has a dominant
effect, which does not allow distinguishing whether seed size variations in wild
type (wt)×MET1a/s crosses originated from the loss of MET1 in the previous
parental generation (sporophyte) or in the haploid generation producing the gam-
etes (gametophyte). In addition, MET1a/s lines accumulate epimutations [6] and
abnormal methylation profiles [14], which could be partially responsible of the
phenotypes observed. A study based on a recessive loss-of-function allele, met1-6
[15] showed clearly that the loss of met1 during male gametogenesis reduces seed
size. This result was also in agreement with the demonstration of a gametophytic
effect of met1-3 on the silencing of the paternal alleles of the imprinted genes
FIS2 and FWA [12]. However the existence of a gametophytic maternal effect of
met1-6 on seed size remained unclear [15] and a potential effect on met1-6 loss of
function on the diploid parental sporophytic generation was not tested explicitly.
To address these concerns, we restricted our analysis to homozygous and heterozy-
gous mutants derived from a self-fertilized heterozygous met1-3/+ mother and
compared the effects on seed development of met1-3 loss of function during male
gametogenesis, female gametogenesis and the parental diploid generation.
Results and Discussion
A Distinctive Paternal Effect Is Associated to MET1 Loss-of-
Function During Male Gametogenesis
The null recessive allele met1-3 causes a loss of DNA methylation in first genera-
tion homozygous plants [16]. The loss of met1 function is caused by a T-DNA
insert linked to a gene conferring resistance to the herbicide BASTA. To confirm
specific parental contributions of met1-3 to seed size, we analyzed digital images
of seeds from crosses that varied MET1 genotype and parent of transmission
(Figure 1, Table 1). Seeds produced by crosses between wild-type ovules and pol-
len from met1-3/met1-3 plants were smaller than seeds produced between wild
type ovules and wild type pollen (Figure 1A). Quantitative analysis resolved these
two genotypes into two distinct populations based on seed width and length (n
= 108; P<.0001 for ANOVA, t-test and Mann Whitney) (Figure 1B, Table 1).
This verified that met1-3 has a paternal effect on seed size as observed in previous
studies [7], [9], [15]. We then conducted the same experiment with heterozygous
met1-3/+ plants. Half of the pollen from met1-3/+ plants carries the met1-3 al-
lele causing re-activation of imprinted genes [12] and other silenced loci [17].
It is thus possible to predict a gametophytic paternal effect of met1 with size
