Tetsu Kinoshita

Maintenance of DNA Methylation during the Arabidopsis Life Cycle Is Essential for Parental Imprinting

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.

Polycomb repression of flowering during early plant development

All plants flower late in their life cycle. For example, in Arabidopsis, the shoot undergoes a transition and produces reproductive flowers after the adult phase of vegetative growth. Much is known about genetic and environmental processes that control flowering time in mature plants. However, little is understood about the mechanisms that prevent plants from flowering much earlier during embryo and seedling development. Arabidopsis embryonic flower (emf1 and emf2) mutants flower soon after germination, suggesting that a floral repression mechanism is established in wild-type plants that prevents flowering until maturity. Here, we show that polycomb group proteins play a central role in repressing flowering early in the plant life cycle. We found that mutations in the Fertilization Independent Endosperm (FIE) polycomb gene caused the seedling shoot to produce flower-like structures and organs. Flower-like structures were also generated from the hypocotyl and root, organs not associated with reproduction. Expression of floral induction and homeotic genes was derepressed in mutant embryos and seedlings. These results suggest that FIE-mediated polycomb complexes are an essential component of a floral repression mechanism established early during plant development.

MATERNALLY EXPRESSED PAB C-TERMINAL, a Novel Imprinted Gene in Arabidopsis, Encodes the Conserved C-Terminal Domain of Polyadenylate Binding Proteins

Parental imprinting is important for seed development, but few imprinted genes have been identified in plants. The four known imprinted genes in Arabidopsis thaliana encode transcriptional regulators. Here, we describe a novel imprinted gene, MATERNALLY EXPRESSED PAB C-TERMINAL (MPC), which encodes the C-terminal domain of poly(A) binding proteins (PABPs). PABPs play roles in mRNA stability and translation. MPC interacts with proteins that also interact with the C-terminal domain of typical PABPs, suggesting that MPC may regulate translation by modulating PABP activity. In the endosperm, MPC is expressed only from the maternal allele. Reduction of MPC expression affects seed development. In dna methyltransferase1 (met1) mutants, MPC is ectopically expressed, and the paternal allele is active in the endosperm. CGs in the 5′ flanking region and gene body of MPC lose methylation in a met1 background. Both regions are required to confer imprinted reporter expression, suggesting that the gene body contains imprinting control region elements. In Arabidopsis, DEMETER (DME) activates expression of maternal alleles. MPC expression is reduced in flowers and seeds in a dme-4 mutant but only after fertilization in dme-1. We conclude that other factors along with DME promote MPC expression and that DME has indirect effects on imprinted gene expression in endosperm.

Epigenetic Memory for Stress Response and Adaptation in Plants

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.

Evolution and Control of Imprinted FWA Genes in the Genus Arabidopsis

A central question in genomic imprinting is how a specific sequence is recognized as the target for epigenetic marking. In both mammals and plants, imprinted genes are often associated with tandem repeats and transposon-related sequences, but the role of these elements in epigenetic gene silencing remains elusive. FWA is an imprinted gene in Arabidopsis thaliana expressed specifically in the female gametophyte and endosperm. Tissue-specific and imprinted expression of FWA depends on DNA methylation in the FWA promoter, which is comprised of two direct repeats containing a sequence related to a SINE retroelement. Methylation of this element causes epigenetic silencing, but it is not known whether the methylation is targeted to the SINE-related sequence itself or the direct repeat structure is also necessary. Here we show that the repeat structure in the FWA promoter is highly diverse in species within the genus Arabidopsis. Four independent tandem repeat formation events were found in three closely related species. Another related species, A. halleri, did not have a tandem repeat in the FWA promoter. Unexpectedly, even in this species, FWA expression was imprinted and the FWA promoter was methylated. In addition, our expression analysis of FWA gene in vegetative tissues revealed high frequency of intra-specific variation in the expression level. In conclusion, we show that the tandem repeat structure is dispensable for the epigenetic silencing of the FWA gene. Rather, SINE-related sequence is sufficient for imprinting, vegetative silencing, and targeting of DNA methylation. Frequent independent tandem repeat formation events in the FWA promoter led us to propose that they may be a consequence, rather than cause, of the epigenetic control. The possible significance of epigenetic variation in reproductive strategies during evolution is also discussed.

HMG Domain Containing SSRP1 Is Required for DNA Demethylation and Genomic Imprinting in Arabidopsis

In Arabidopsis, DEMETER (DME) DNA demethylase contributes to reprogramming of the epigenetic state of the genome in the central cell. However, other aspects of the active DNA demethylation processes remain elusive. Here we show that Arabidopsis SSRP1, known as an HMG domain-containing component of FACT histone chaperone, is required for DNA demethylation and for activation and repression of many parentally imprinted genes in the central cell. Although loss of DNA methylation releases silencing of the imprinted FWA-GFP, double ssrp1-3;met1-3 mutants surprisingly showed limited activation of maternal FWA-GFP in the central cell, and only became fully active after several nuclear divisions in the endosperm. This behavior was in contrast to the dme-1;met1 double mutant in which hypomethylation of FWA-GFP by met1 suppressed the DNA demethylation defect of dme-1. We propose that active DNA demethylation by DME requires SSRP1 function through a distinctly different process from direct DNA methylation control.

OsATG7 is required for autophagy-dependent lipid metabolism in rice postmeiotic anther development

In flowering plants, the tapetum, the innermost layer of the anther, provides both nutrient and lipid components to developing microspores, pollen grains, and the pollen coat. Though the programmed cell death of the tapetum is one of the most critical and sensitive steps for fertility and is affected by various environmental stresses, its regulatory mechanisms remain mostly unknown. Here we show that autophagy is required for the metabolic regulation and nutrient supply in anthers and that autophagic degradation within tapetum cells is essential for postmeiotic anther development in rice. Autophagosome-like structures and several vacuole-enclosed lipid bodies were observed in postmeiotic tapetum cells specifically at the uninucleate stage during pollen development, which were completely abolished in a retrotransposon-insertional OsATG7 (autophagy-related 7)-knockout mutant defective in autophagy, suggesting that autophagy is induced in tapetum cells. Surprisingly, the mutant showed complete sporophytic male sterility, failed to accumulate lipidic and starch components in pollen grains at the flowering stage, showed reduced pollen germination activity, and had limited anther dehiscence. Lipidomic analyses suggested impairment of editing of phosphatidylcholines and lipid desaturation in the mutant during pollen maturation. These results indicate a critical involvement of autophagy in a reproductive developmental process of rice, and shed light on the novel autophagy-mediated regulation of lipid metabolism in eukaryotic cells.

RNA-directed DNA methylation regulates parental genomic imprinting at several loci in Arabidopsis

In mammals and plants, parental genomic imprinting restricts the expression of specific loci to one parental allele. Imprinting in mammals relies on sex-dependent de novo deposition of DNA methylation during gametogenesis but a comparable mechanism was not shown in plants. Rather, paternal silencing by the maintenance DNA methyltransferase 1 (MET1) and maternal activation by the DNA demethylase DEMETER (DME) cause maternal expression. However, genome-wide studies suggested other DNA methylation-dependent imprinting mechanisms. Here, we show that de novo RNA-directed DNA methylation (RdDM) regulates imprinting at specific loci expressed in endosperm. RdDM in somatic tissues is required to silence expression of the paternal allele. By contrast, the repression of RdDM in female gametes participates with or without DME requirement in the activation of the maternal allele. The contrasted activity of DNA methylation between male and female gametes appears sufficient to prime imprinted maternal expression. After fertilization, MET1 maintains differential expression between the parental alleles. RdDM depends on small interfering RNAs (siRNAs). The involvement of RdDM in imprinting supports the idea that sources of siRNAs such as transposons and de novo DNA methylation were recruited in a convergent manner in plants and mammals in the evolutionary process leading to selection of imprinted loci.

Epigenetic programming: the challenge to species hybridization.

In many organisms, the genomes of individual species are isolated by a range of reproductive barriers that act before or after fertilization. Successful mating between species results in the presence of different genomes within a cell (hybridization), which can lead to incompatibility in cellular events due to adverse genetic interactions. In addition to such genetic interactions, recent studies have shown that the epigenetic control of the genome, silencing of transposons, control of non-additive gene expression and genomic imprinting might also contribute to reproductive barriers in plant and animal species. These genetic and epigenetic mechanisms play a significant role in the prevention of gene flow between species. In this review, we focus on aspects of epigenetic control related to hybrid incompatibility during species hybridization, and also consider key mechanism(s) in the interaction between different genomes.

DNA demethylation: a lesson from the garden.

Gene silencing by DNA methylation is well documented and known to be essential for various biological phenomena in many organisms. In contrast, the processes that convert the silent state of a gene whose DNA is methylated and predicted to form facultative heterochromatin to the actively transcribed state remain elusive. In Arabidopsis, recent studies have shown that the DNA glycosylases DEMETER (DME) and REPRESSOR OF SILENCING1 (ROS1) participate in DNA demethylation. DME is necessary for genomic imprinting in the endosperm, while ROS1 is involved in pruning DNA methylation patterns in transposons and genic regions of vegetative tissues. These findings provide us with molecular clues for understanding the underlying mechanisms of DNA demethylation and gene activation. In this review, we will consider and discuss the processes of controlling gene activation through DNA demethylation, which are predicted to include the recognition of target sequences, DNA demethylation, the transformation of the chromatin to the active state, and transcription. Many of these processes remain poorly understood at this stage.