The Arabidopsis FWA gene was initially identified from late-flowering epigenetic mutants that show ectopic FWA expression associated with heritable hypomethylation of repeats around transcription starting sites. Here, we show that wild-type FWA displays imprinted (maternal origin-specific) expression in endosperm. The FWA imprint depends on the maintenance DNA methyltransferase MET1, as is the case in mammals. Unlike mammals, however, the FWA imprint is not established by allele-specific de novo methylation. It is established by maternal gametophyte-specific gene activation, which depends on a DNA glycosylase gene, DEMETER. Because endosperm does not contribute to the next generation, the activated FWA gene need not be silenced again. Double fertilization enables plants to use such "one-way" control of imprinting and DNA methylation in endosperm.

Imprinted genes are expressed predominantly from either their paternal or their maternal allele. To date, all imprinted genes identified in plants are expressed in the endosperm. In Arabidopsis thaliana, maternal imprinting has been clearly demonstrated for the Polycomb group gene MEDEA (MEA) and for FWA. Direct repeats upstream of FWA are subject to DNA methylation. However, it is still not clear to what extent similar cis-acting elements may be part of a conserved molecular mechanism controlling maternally imprinted genes. In this work, we show that the Polycomb group gene FERTILIZATION-INDEPENDENT SEED2 (FIS2) is imprinted. Maintenance of FIS2 imprinting depends on DNA methylation, whereas loss of DNA methylation does not affect MEA imprinting. DNA methylation targets a small region upstream of FIS2 distinct from the target of DNA methylation associated with FWA. We show that FWA and FIS2 imprinting requires the maintenance of DNA methylation throughout the plant life cycle, including male gametogenesis and endosperm development. Our data thus demonstrate that parental genomic imprinting in plants depends on diverse cis-elements and mechanisms dependent or independent of DNA methylation. We propose that imprinting has evolved under constraints linked to the evolution of plant reproduction and not by the selection of a specific molecular mechanism.

SummaryVacuolar processing enzyme (VPE) has been shown to be responsible for maturation of various seed proteins in protein-storage vacuoles. Arabidopsis has three VPE homologues; bVPE is speci®c to seeds and aVPE and gVPE are speci®c to vegetative organs. To investigate the activity of the vegetative VPE, we expressed the gVPE in a pep4 strain of the yeast Saccharomyces cerevisiae and found that gVPE has the ability to cleave the peptide bond at the carbonyl side of asparagine residues. An immunocytochemical analysis revealed the speci®c localization of the gVPE in the lytic vacuoles of Arabidopsis leaves that had been treated with wounding. These ®ndings indicate that gVPE functions in the lytic vacuoles as the bVPE does in the proteinstorage vacuoles. The bVPE promoter was found to direct the expression of the b-glucuronidase reporter gene in seeds and the root tip of transgenic Arabidopsis plants. On the other hand, both the aVPE and gVPE promoters directed the expression in senescent tissues, but not in young intact tissues. The mRNA levels of both aVPE and gVPE were increased in the primary leaves during senescence in parallel with the increase of the mRNA level of a senescence-associated gene (SAG2). Treatment with wounding, ethylene and salicylic acid up-regulated the expression of aVPE and gVPE, while jasmonate slightly up-regulated the expression of gVPE. These gene expression patterns of the VPEs were associated with the accumulation of vacuolar proteins that are known to respond to these treatments. Taken together, the results suggest that vegetative VPE might regulate the activation of some functional proteins in the lytic vacuoles.

In contrast to the majority of animal species, plants are sessile organisms and are, therefore, constantly challenged by environmental perturbations. Over the past few decades, our knowledge of how plants perceive environmental stimuli has increased considerably, e.g. the mechanisms for transducing environmental stress stimuli into cellular signaling cascades and gene transcription networks. In addition, it has recently been shown that plants can remember past environmental events and can use these memories to aid responses when these events recur. In this mini review, we focus on recent progress in determination of the epigenetic mechanisms used by plants under various environmental stresses. Epigenetic mechanisms are now known to play a vital role in the control of gene expression through small RNAs, histone modifications and DNA methylation. These are inherited through mitotic cell divisions and, in some cases, can be transmitted to the next generation. They therefore offer a possible mechanism for stress memories in plants. Recent studies have yielded evidence indicating that epigenetic mechanisms are indeed essential for stress memories and adaptation in plants.