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Significance Plant gene imprinting (allele-specific chromatin modification) results in parent-of-origin–dependent expression in endosperm, a nutritive tissue essential for embryo viability. Imprinted genes function in species reproductive isolation by causing embryo abortion in crosses. We analyzed the evolution of rice gene imprinting using four cultivars spanning domestication and the divergence of two subspecies 9,000 and 300,000 y ago, respectively. Most imprinted genes are imprinted across cultivars and enriched in regulatory functions. However, approximately 10% of imprinted genes have lost or gained imprinting, often associated with stably inherited epigenetic and genetic variation, suggesting a role in rice diversification. Our results highlight the role of transposable elements and epigenetic variation in shaping heritable changes in gene expression during rice evolution., Parent-of-origin–dependent gene expression in mammals and flowering plants results from differing chromatin imprints (genomic imprinting) between maternally and paternally inherited alleles. Imprinted gene expression in the endosperm of seeds is associated with localized hypomethylation of maternally but not paternally inherited DNA, with certain small RNAs also displaying parent-of-origin–specific expression. To understand the evolution of imprinting mechanisms in Oryza sativa (rice), we analyzed imprinting divergence among four cultivars that span both japonica and indica subspecies: Nipponbare, Kitaake, 93-11, and IR64. Most imprinted genes are imprinted across cultivars and enriched for functions in chromatin and transcriptional regulation, development, and signaling. However, 4 to 11% of imprinted genes display divergent imprinting. Analyses of DNA methylation and small RNAs revealed that endosperm-specific 24-nt small RNA–producing loci show weak RNA-directed DNA methylation, frequently overlap genes, and are imprinted four times more often than genes. However, imprinting divergence most often correlated with local DNA methylation epimutations (9 of 17 assessable loci), which were largely stable within subspecies. Small insertion/deletion events and transposable element insertions accompanied 4 of the 9 locally epimutated loci and associated with imprinting divergence at another 4 of the remaining 8 loci. Correlating epigenetic and genetic variation occurred at key regulatory regions—the promoter and transcription start site of maternally biased genes, and the promoter and gene body of paternally biased genes. Our results reinforce models for the role of maternal-specific DNA hypomethylation in imprinting of both maternally and paternally biased genes, and highlight the role of transposition and epimutation in rice imprinting evolution.

Significance The angiosperm life cycle has alternating diploid (sporophyte) and haploid (gametophyte) generations. The sporophyte generation begins with fertilization of haploid gametes and the gametophyte generation begins after meiosis. In Arabidopsis, the DEMETER (DME) DNA demethylase is essential for reproduction and is expressed in the central cell and vegetative cell of the female and male gametophyte, respectively. Little is known about DME function in the sporophyte. We show that DME activity is required for sporophyte development—seed germination, root hair growth, and cellular proliferation and differentiation during development—and we identify sporophytic genes whose proper expression requires DME activity. Together, our study provides important clues about the genetic circuits regulated by the DME DNA demethylase that control Arabidopsis sporophyte development., The flowering plant life cycle consists of alternating haploid (gametophyte) and diploid (sporophyte) generations, where the sporophytic generation begins with fertilization of haploid gametes. In Arabidopsis, genome-wide DNA demethylation is required for normal development, catalyzed by the DEMETER (DME) DNA demethylase in the gamete companion cells of male and female gametophytes. In the sporophyte, postembryonic growth and development are largely dependent on the activity of numerous stem cell niches, or meristems. Analyzing Arabidopsis plants homozygous for a loss-of-function dme-2 allele, we show that DME influences many aspects of sporophytic growth and development. dme-2 mutants exhibited delayed seed germination, variable root hair growth, aberrant cellular proliferation and differentiation followed by enhanced de novo shoot formation, dysregulation of root quiescence and stomatal precursor cells, and inflorescence meristem (IM) resurrection. We also show that sporophytic DME activity exerts a profound effect on the transcriptome of developing Arabidopsis plants, including discrete groups of regulatory genes that are misregulated in dme-2 mutant tissues, allowing us to potentially link phenotypes to changes in specific gene expression pathways. These results show that DME plays a key role in sporophytic development and suggest that DME-mediated active DNA demethylation may be involved in the maintenance of stem cell activities during the sporophytic life cycle in Arabidopsis.

Significance Flowering plants reproduce via a unique double-fertilization event, producing the zygote and the nutritive endosperm. The genome of the central cell, the precursor of the endosperm, undergoes extensive demethylation prior to fertilization. This epigenetic reconfiguration, directed by the DEMETER (DME) glycosylase at thousands of loci in Arabidopsis, differentiates the epigenetic landscapes of parental genomes and establishes parent of origin-specific expression of many imprinted genes in endosperm essential for seed development. However, how DME is targeted to various locations remains unknown. Here we show that the multidomain DME is organized into 2 functional regions: the C-terminal region, which guides localization and catalysis, and the N-terminal region, which likely recruits chromatin remodelers to facilitate demethylation within heterochromatin., The Arabidopsis DEMETER (DME) DNA glycosylase demethylates the maternal genome in the central cell prior to fertilization and is essential for seed viability. DME preferentially targets small transposons that flank coding genes, influencing their expression and initiating plant gene imprinting. DME also targets intergenic and heterochromatic regions, but how it is recruited to these differing chromatin landscapes is unknown. The C-terminal half of DME consists of 3 conserved regions required for catalysis in vitro. We show that this catalytic core guides active demethylation at endogenous targets, rescuing dme developmental and genomic hypermethylation phenotypes. However, without the N terminus, heterochromatin demethylation is significantly impeded, and abundant CG-methylated genic sequences are ectopically demethylated. Comparative analysis revealed that the conserved DME N-terminal domains are present only in flowering plants, whereas the domain architecture of DME-like proteins in nonvascular plants mainly resembles the catalytic core, suggesting that it might represent the ancestral form of the 5mC DNA glycosylase found in plant lineages. We propose a bipartite model for DME protein action and suggest that the DME N terminus was acquired late during land plant evolution to improve specificity and facilitate demethylation at heterochromatin targets.

The Arabidopsis DEMETER (DME) DNA glycosylase demethylates the maternal genome in the central cell prior to fertilization, and is essential for seed viability. DME preferentially targets small transposons that flank coding genes, influencing their expression and initiating plant gene imprinting. DME also targets intergenic and heterochromatic regions, and how it is recruited to these differing chromatin landscapes is unknown. The C-terminal DME catalytic core consists of three conserved regions required for catalysis in vitro. We show that the catalytic core of DME guides active demethylation at endogenous targets, rescuing the developmental and genomic hypermethylation phenotypes of DME mutants. However, without the N-terminus, heterochromatin demethylation is significantly impeded, and abundant CG-methylated genic sequences are ectopically demethylated. We used comparative analysis to reveal that the conserved DME N-terminal domains are only present in the flowering plants, whereas the domain architecture of DME-like proteins in non-vascular plants mainly resembles the catalytic core, suggesting that it might represent the ancestral form of the 5mC DNA glycosylase found in all plant lineages. We propose a bipartite model for DME protein action and suggest that the DME N-terminus was acquired late during land plant evolution to improve specificity and facilitate demethylation at heterochromatin targets.

Significance Rice seeds comprised of embryo and endosperm are generated when the vegetative cell pollen tube delivers two sperm cells that fertilize central and egg cells, respectively. We show that the ROS1a DNA glycosylase actively demethylates DNA of rice vegetative cell genomes, which is required for viable seed production. Thus, DNA glycosylase-mediated DNA demethylation, conserved for 150 million years between rice and Arabidopsis, is likely an essential feature of seed development in all flowering plants. We also reveal that global DNA methylation levels in rice sperm and egg cells are different both from each other and also compared with the embryo, suggesting that dynamic DNA methylation reprogramming occurs during plant embryogenesis., Epigenetic reprogramming is required for proper regulation of gene expression in eukaryotic organisms. In Arabidopsis, active DNA demethylation is crucial for seed viability, pollen function, and successful reproduction. The DEMETER (DME) DNA glycosylase initiates localized DNA demethylation in vegetative and central cells, so-called companion cells that are adjacent to sperm and egg gametes, respectively. In rice, the central cell genome displays local DNA hypomethylation, suggesting that active DNA demethylation also occurs in rice; however, the enzyme responsible for this process is unknown. One candidate is the rice REPRESSOR OF SILENCING 1a (ROS1a) gene, which is related to DME and is essential for rice seed viability and pollen function. Here, we report genome-wide analyses of DNA methylation in wild-type and ros1a mutant sperm and vegetative cells. We find that the rice vegetative cell genome is locally hypomethylated compared with sperm by a process that requires ROS1a activity. We show that many ROS1a target sequences in the vegetative cell are hypomethylated in the rice central cell, suggesting that ROS1a also demethylates the central cell genome. Similar to Arabidopsis, we show that sperm non-CG methylation is indirectly promoted by DNA demethylation in the vegetative cell. These results reveal that DNA glycosylase-mediated DNA demethylation processes are conserved in Arabidopsis and rice, plant species that diverged 150 million years ago. Finally, although global non-CG methylation levels of sperm and egg differ, the maternal and paternal embryo genomes show similar non-CG methylation levels, suggesting that rice gamete genomes undergo dynamic DNA methylation reprogramming after cell fusion.

The Arabidopsis female gametophyte contains seven cells with eight haploid nuclei buried within layers of sporophytic tissue. Following double fertilization, the egg and central cells of the gametophyte develop into the embryo and endosperm of the seed, respectively. The epigenetic status of the central cell has long presented an enigma due both to its inaccessibility, and the fascinating epigenome of the endosperm, thought to have been inherited from the central cell following activity of the DEMETER demethylase enzyme, prior to fertilization. Here, we present for the first time, a method to isolate pure populations of Arabidopsis central cell nuclei. Utilizing a protocol designed to isolate leaf mesophyll protoplasts, we systematically optimized each step in order to efficiently separate central cells from the female gametophyte. We use initial manual pistil dissection followed by the derivation of central cell protoplasts, during which process the central cell emerges from the micropylar pole of the embryo sac. Then, we use a modified version of the Isolation of Nuclei TAgged in specific Cell Types (INTACT) protocol to purify central cell nuclei, resulting in a purity of 75-90% and a yield sufficient to undertake downstream molecular analyses. We find that the process is highly dependent on the health of the original plant tissue used, and the efficiency of protoplasting solution infiltration into the gametophyte. By isolating pure central cell populations, we have enabled elucidation of the physiology of this rare cell type, which in the future will provide novel insights into Arabidopsis reproduction.

Significance We describe the spatial and temporal profiles of soybean and Arabidopsis seed methylomes during development. CHH methylation increases globally from fertilization through dormancy in all seed parts, decreases following germination, and targets primarily transposons. By contrast, CG- and CHG-context methylation remains constant throughout seed development. Mutant seeds lacking non-CG methylation develop normally, but have a set of up-regulated transposon RNAs suggesting that the CHH methylation increase may be a failsafe mechanism to reinforce transposon silencing. Major classes of seed genes have similar methylation profiles, whether they are active or not. Our results suggest that soybean and Arabidopsis seed methylomes are similar, and that DNA methylation does not play a significant role in regulating many genes important for seed development., We profiled soybean and Arabidopsis methylomes from the globular stage through dormancy and germination to understand the role of methylation in seed formation. CHH methylation increases significantly during development throughout the entire seed, targets primarily transposable elements (TEs), is maintained during endoreduplication, and drops precipitously within the germinating seedling. By contrast, no significant global changes in CG- and CHG-context methylation occur during the same developmental period. An Arabidopsis ddcc mutant lacking CHH and CHG methylation does not affect seed development, germination, or major patterns of gene expression, implying that CHH and CHG methylation does not play a significant role in seed development or in regulating seed gene activity. By contrast, over 100 TEs are transcriptionally de-repressed in ddcc seeds, suggesting that the increase in CHH-context methylation may be a failsafe mechanism to reinforce transposon silencing. Many genes encoding important classes of seed proteins, such as storage proteins, oil biosynthesis enzymes, and transcription factors, reside in genomic regions devoid of methylation at any stage of seed development. Many other genes in these classes have similar methylation patterns, whether the genes are active or repressed. Our results suggest that methylation does not play a significant role in regulating large numbers of genes important for programming seed development in both soybean and Arabidopsis. We conclude that understanding the mechanisms controlling seed development will require determining how cis-regulatory elements and their cognate transcription factors are organized in genetic regulatory networks.

We profiled soybean and Arabidopsis methylomes from the globular stage through dormancy and germination to understand the role of methylation in seed formation. CHH methylation increases significantly during development throughout the entire seed, targets primarily transposable elements (TEs), is maintained during endoreduplication, and drops precipitously within the germinating seedling. By contrast, no significant global changes in CG- and CHG-context methylation occur during the same developmental period. An Arabidopsis ddcc mutant lacking CHH and CHG methylation does not affect seed development, germination, or major patterns of gene expression, implying that CHH and CHG methylation does not play a significant role in seed development or in regulating seed gene activity. By contrast, over 100 TEs are transcriptionally de-repressed in ddcc seeds, suggesting that the increase in CHH-context methylation may be a failsafe mechanism to reinforce transposon silencing. Many genes encoding important classes of seed proteins, such as storage proteins, oil biosynthesis enzymes, and transcription factors, reside in genomic regions devoid of methylation at any stage of seed development. Many other genes in these classes have similar methylation patterns, whether the genes are active or repressed. Our results suggest that methylation does not play a significant role in regulating large numbers of genes important for programming seed development in both soybean and Arabidopsis. We conclude that understanding the mechanisms controlling seed development will require determining how cis-regulatory elements and their cognate transcription factors are organized in genetic regulatory networks.

Arabidopsis thaliana endosperm, a transient tissue that nourishes the embryo, exhibits extensive localized DNA demethylation on maternally inherited chromosomes. Demethylation mediates parent-of-origin–specific (imprinted) gene expression but is apparently unnecessary for the extensive accumulation of maternally biased small RNA (sRNA) molecules detected in seeds. Endosperm DNA in the distantly related monocots rice and maize is likewise locally hypomethylated, but whether this hypomethylation is generally parent-of-origin specific is unknown. Imprinted expression of sRNA also remains uninvestigated in monocot seeds. Here, we report high-coverage sequencing of the Kitaake rice cultivar that enabled us to show that localized hypomethylation in rice endosperm occurs solely on the maternal genome, preferring regions of high DNA accessibility. Maternally expressed imprinted genes are enriched for hypomethylation at putative promoter regions and transcriptional termini and paternally expressed genes at promoters and gene bodies, mirroring our recent results in A. thaliana . However, unlike in A. thaliana , rice endosperm sRNA populations are dominated by specific strong sRNA-producing loci, and imprinted 24-nt sRNAs are expressed from both parental genomes and correlate with hypomethylation. Overlaps between imprinted sRNA loci and imprinted genes expressed from opposite alleles suggest that sRNAs may regulate genomic imprinting. Whereas sRNAs in seedling tissues primarily originate from small class II (cut-and-paste) transposable elements, those in endosperm are more uniformly derived, including sequences from other transposon classes, as well as genic and intergenic regions. Our data indicate that the endosperm exhibits a unique pattern of sRNA expression and suggest that localized hypomethylation of maternal endosperm DNA is conserved in flowering plants.

Cytosine DNA methylation regulates the expression of eukaryotic genes and transposons. Methylation is copied by methyltransferases after DNA replication, which results in faithful transmission of methylation patterns during cell division and, at least in flowering plants, across generations. Transgenerational inheritance is mediated by a small group of cells that includes gametes and their progenitors. However, methylation is usually analyzed in somatic tissues that do not contribute to the next generation, and the mechanisms of transgenerational inheritance are inferred from such studies. To gain a better understanding of how DNA methylation is inherited, we analyzed purified Arabidopsis thaliana sperm and vegetative cells—the cell types that comprise pollen—with mutations in the DRM, CMT2, and CMT3 methyltransferases. We find that DNA methylation dependency on these enzymes is similar in sperm, vegetative cells, and somatic tissues, although DRM activity extends into heterochromatin in vegetative cells, likely reflecting transcription of heterochromatic transposons in this cell type. We also show that lack of histone H1, which elevates heterochromatic DNA methylation in somatic tissues, does not have this effect in pollen. Instead, levels of CG methylation in wild-type sperm and vegetative cells, as well as in wild-type microspores from which both pollen cell types originate, are substantially higher than in wild-type somatic tissues and similar to those of H1-depleted roots. Our results demonstrate that the mechanisms of methylation maintenance are similar between pollen and somatic cells, but the efficiency of CG methylation is higher in pollen, allowing methylation patterns to be accurately inherited across generations.

Cytosine methylation is a DNA modification with important regulatory functions in eukaryotes. In flowering plants, sexual reproduction is accompanied by extensive DNA demethylation, which is required for proper gene expression in the endosperm, a nutritive extraembryonic seed tissue. Endosperm arises from a fusion of a sperm cell carried in the pollen and a female central cell. Endosperm DNA demethylation is observed specifically on the chromosomes inherited from the central cell in Arabidopsis thaliana, rice, and maize, and requires the DEMETER DNA demethylase in Arabidopsis. DEMETER is expressed in the central cell before fertilization, suggesting that endosperm demethylation patterns are inherited from the central cell. Down-regulation of the MET1 DNA methyltransferase has also been proposed to contribute to central cell demethylation. However, with the exception of three maize genes, central cell DNA methylation has not been directly measured, leaving the origin and mechanism of endosperm demethylation uncertain. Here, we report genome-wide analysis of DNA methylation in the central cells of Arabidopsis and rice—species that diverged 150 million years ago—as well as in rice egg cells. We find that DNA demethylation in both species is initiated in central cells, which requires DEMETER in Arabidopsis. However, we do not observe a global reduction of CG methylation that would be indicative of lowered MET1 activity; on the contrary, CG methylation efficiency is elevated in female gametes compared with nonsexual tissues. Our results demonstrate that locus-specific, active DNA demethylation in the central cell is the origin of maternal chromosome hypomethylation in the endosperm.

The Arabidopsis female gametophyte contains seven cells with eight haploid nuclei buried within layers of sporophytic tissue. Following double fertilization, the egg and central cells of the gametophyte develop into the embryo and endosperm of the seed, respectively. The epigenetic status of the central cell has long presented an enigma due both to its inaccessibility, and the fascinating epigenome of the endosperm, thought to have been inherited from the central cell following activity of the DEMETER demethylase enzyme, prior to fertilization. Here, we present for the first time, a method to isolate pure populations of Arabidopsis central cell nuclei. Utilizing a protocol designed to isolate leaf mesophyll protoplasts, we systematically optimized each step in order to efficiently separate central cells from the female gametophyte. We use initial manual pistil dissection followed by the derivation of central cell protoplasts, during which process the central cell emerges from the micropylar pole of the embryo sac. Then, we use a modified version of the Isolation of Nuclei TAgged in specific Cell Types (INTACT) protocol to purify central cell nuclei, resulting in a purity of 75–90% and a yield sufficient to undertake downstream molecular analyses. We find that the process is highly dependent on the health of the original plant tissue used, and the efficiency of protoplasting solution infiltration into the gametophyte. By isolating pure central cell populations, we have enabled elucidation of the physiology of this rare cell type, which in the future will provide novel insights into Arabidopsis reproduction.

In double fertilization, the vegetative cell of the male gametophyte (pollen) germinates and forms a pollen tube that brings to the female gametophyte two sperm cells that fertilize the egg and central cell to form the embryo and endosperm, respectively. The 5-methylcytosine DNA glycosylase DEMETER (DME), expressed in the central cell, is required for maternal allele demethylation and gene imprinting in the endosperm. By contrast, little is known about the function of DME in the male gametophyte. Here we show that reduced transmission of the paternal mutant dme allele in certain ecotypes reflects, at least in part, defective pollen germination. DME RNA is detected in pollen, but not in isolated sperm cells, suggesting that DME is expressed in the vegetative cell. Bisulfite sequencing experiments show that imprinted genes ( MEA and FWA ) and a repetitive element ( Mu1a ) are hypomethylated in the vegetative cell genome compared with the sperm genome, which is a process that requires DME. Moreover, we show that MEA and FWA RNA are detectable in pollen, but not in isolated sperm cells, suggesting that their expression occurs primarily in the vegetative cell. These results suggest that DME is active and demethylates similar genes and transposons in the genomes of the vegetative and central cells in the male and female gametophytes, respectively. Although the genome of the vegetative cell does not participate in double fertilization, its DME-mediated demethylation is important for male fertility and may contribute to the reconfiguration of the methylation landscape that occurs in the vegetative cell genome.

Imprinting occurs in the endosperm of flowering plants. The endosperm, a product of central cell fertilization, is critical for embryo and seed development. Imprinting in the endosperm is mainly due to the inherited differences in gamete epigenetic composition. Studies have also shown that there are differences in genomic DNA methylation patterns between embryo and endosperm. Examining those differences, along with mutations in the DNA demethylase gene DEMETER, gives insight into the number of imprinted genes and how an antagonistic relationship between TE defense and gene regulation could evolutionarily affect imprinting establishment. Finally, studies demonstrate that DEMETER demethylase activity influences endosperm chromatin composition, and could possibly enhance DNA de novo methylation activity.

Imprinted genes are expressed primarily or exclusively from either the maternal or paternal allele, a phenomenon that occurs in flowering plants and mammals. Flowering plant imprinted gene expression has been described primarily in endosperm, a terminal nutritive tissue consumed by the embryo during seed development or after germination. Imprinted expression in Arabidopsis thaliana endosperm is orchestrated by differences in cytosine DNA methylation between the paternal and maternal genomes as well as by Polycomb group proteins. Currently, only 11 imprinted A. thaliana genes are known. Here, we use extensive sequencing of cDNA libraries to identify 9 paternally expressed and 34 maternally expressed imprinted genes in A. thaliana endosperm that are regulated by the DNA-demethylating glycosylase DEMETER, the DNA methyltransferase MET1, and/or the core Polycomb group protein FIE. These genes encode transcription factors, proteins involved in hormone signaling, components of the ubiquitin protein degradation pathway, regulators of histone and DNA methylation, and small RNA pathway proteins. We also identify maternally expressed genes that may be regulated by unknown mechanisms or deposited from maternal tissues. We did not detect any imprinted genes in the embryo. Our results show that imprinted gene expression is an extensive mechanistically complex phenomenon that likely affects multiple aspects of seed development.

DNA methylation (5-methylcytosine) in mammalian genomes predominantly occurs at CpG dinucleotides, is maintained by DNA methyltransferase1 (Dnmt1), and is essential for embryo viability. The plant genome also has 5-methylcytosine at CpG dinucleotides, which is maintained by METHYLTRANSFERASE1 (MET1), a homolog of Dnmt1. In addition, plants have DNA methylation at CpNpG and CpNpN sites, maintained, in part, by the CHROMOMETHYLASE3 (CMT3) DNA methyltransferase. Here, we show that Arabidopsis thaliana embryos with loss-of-function mutations in MET1 and CMT3 develop improperly, display altered planes and numbers of cell division, and have reduced viability. Genes that specify embryo cell identity are misexpressed, and auxin hormone gradients are not properly formed in abnormal met1 embryos. Thus, DNA methylation is critical for the regulation of plant embryogenesis and for seed viability.

We identified the Arabidopsis (Arabidopsis thaliana) tanmei/emb2757 (tan) mutation that causes defects in both embryo and seedling development. tan mutant embryos share many characteristics with the leafy cotyledon (lec) class of mutants in that they accumulate anthocyanin, are intolerant of desiccation, form trichomes on cotyledons, and have reduced accumulation of storage proteins and lipids. Thus, TAN functions both in the early and late phases of embryo development. Moreover, the TAN and LEC genes interact synergistically, suggesting that they do not act in series in the same genetic pathway but, rather, that they have overlapping roles during embryogenesis. tan mutants die as embryos, but immature mutant seeds can be germinated in culture. However, tan mutant seedlings are defective in shoot and root development, their hypocotyls fail to elongate in the dark, and they die as seedlings. We isolated the TAN gene and showed that the predicted polypeptide has seven WD repeat motifs, suggesting that TAN forms complexes with other proteins. Together, these results suggest that TAN interacts with other proteins to control many aspects of embryo development.

Position-dependent regulation of growth is important for shaping organs in multicellular organisms. We have characterized the role of JAGGED, a gene that encodes a protein with a single C2H2zinc-finger domain, in controlling the morphogenesis of lateral organs in Arabidopsis thaliana. Loss of JAGGED function causes organs to have serrated margins. In leaves, the blade region is most severely affected. In sepals, petals and stamens, the strongest defects are seen in the distal regions. By monitoring cell-cycle activity in developing petals with the expression of HISTONE 4, we show that JAGGED suppresses the premature differentiation of tissues, which is necessary for the formation of the distal region. The localization of defects overlaps with the expression domain of JAGGED, which is restricted to the growing regions of lateral organs. JAGGED expression is notably absent from the cryptic bract, the remnant of a leaf-like organ that subtends the flower in many species but does not normally develop in wild-type Arabidopsis. If misexpressed, JAGGED can induce the formation of bracts, suggesting that the exclusion of JAGGED from the cryptic bract is a cause of bractless flowers in Arabidopsis.

Two studies were conducted at two locations to evaluate growth performance and carcass characteristics of growing-finishing pigs fed diets containing either glyphosate-tolerant Roundup Ready (event nk603) corn, a nontransgenic genetically similar control corn (RX670), or two conventional sources of nontransgenic corn (RX740 and DK647). A randomized complete block design (three and four blocks in Studies 1 and 2, respectively) with a 2 x 4 factorial arrangement of treatments (two genders and four corn lines) was used. Study 1 used 72 barrows and 72 gilts (housed in single-gender groups of six; six pens per dietary treatment) with initial and final BW of approximately 22 and 116 kg, respectively. Study 2 used 80 barrows and 80 gilts (housed in single-gender groups of five; eight pens per dietary treatment) with initial and final BW of approximately 30 and 120 kg, respectively. Pigs were housed in a modified open-front building in Study 1 and in an environmentally controlled finishing building in Study 2. The test corns were included at a fixed proportion of the diet in both studies. Animals had ad libitum access to feed and water. Pigs were slaughtered using standard procedures and carcass measurements were taken. In Study 1, overall ADG, ADFI (as-fed basis), and gain:feed (G:F) were not affected (P0.05) by corn line. In Study 2, there was no effect of corn line on overall ADFI (as-fed basis) or G:F ratio. In addition, overall ADG of barrows fed the four corn lines did not differ (P0.05); however, overall ADG of gilts fed corn DK647 was greater (P0.05) than that of pigs fed the other corn lines. There was no effect (P0.05) of corn line on carcass yield or fatness measurements in either study. Differences between barrows and gilts for growth and carcass traits were generally similar for both studies and in line with previous research. Overall, these results indicate that Roundup Ready corn (nk603) gives equivalent animal performance to conventional corn for growing pigs.

The MEA Polycomb gene is imprinted in the Arabidopsis endosperm. DME DNA glycosylase activates maternal MEA allele expression in the central cell of the female gametophyte, the progenitor of the endosperm. Maternal mutant dme or mea alleles result in seed abortion. We identified mutations that suppress dme seed abortion and found that they reside in the MET1 methyltransferase gene, which maintains cytosine methylation. Seeds with maternal dme and met1 alleles survive, indicating that suppression occurs in the female gametophyte. Suppression requires a maternal wild-type MEA allele, suggesting that MET1 functions upstream of, or at, MEA. DME activates whereas MET1 suppresses maternal MEA::GFP allele expression in the central cell. MET1 is required for DNA methylation of three regions in the MEA promoter in seeds. Our data suggest that imprinting is controlled in the female gametophyte by antagonism between the two DNA-modifying enzymes, MET1 methyltransferase and DME DNA glycosylase.

We isolated mutations in Arabidopsis to understand how the female gametophyte controls embryo and endosperm development. For the DEMETER (DME) gene, seed viability depends only on the maternal allele. DME encodes a large protein with DNA glycosylase and nuclear localization domains. DME is expressed primarily in the central cell of the female gametophyte, the progenitor of the endosperm. DME is required for maternal allele expression of the imprinted MEDEA (MEA) Polycomb gene in the central cell and endosperm. Ectopic DME expression in endosperm activates expression of the normally silenced paternal MEA allele. In leaf, ectopic DME expression induces MEA and nicks the MEA promoter. Thus, a DNA glycosylase activates maternal expression of an imprinted gene in the central cell.

We identified a new gene that is interrupted by T-DNA in an Arabidopsis embryo mutant called raspberry3. raspberry3 has “raspberry-like” cellular protuberances with an enlarged suspensor characteristic of otherraspberry embryo mutants, and is arrested morphologically at the globular stage of embryo development. The predicted RASPBERRY3 protein has domains found in proteins present in prokaryotes and algae chloroplasts. Computer prediction analysis suggests that the RASPBERRY3protein may be localized in the chloroplast. Complementation analysis supports the possibility that the RASPBERRY3 protein may be involved in chloroplast development. Our experiments demonstrate the important role of the chloroplast, directly or indirectly, in embryo morphogenesis and development.

Angiosperm reproduction is characterized by alternate diploid sporophytic and haploid gametophytic generations. Gametogenesis shares similarities with that of animals except for the formation of the gametophyte, whereby haploid cells undergo several rounds of postmeiotic mitosis to form gametes and the accessory cells required for successful reproduction. The mechanisms regulating gametophyte development in angiosperms are incompletely understood. Here, we show that the nucleoporin Nup88-homolog MOS7 (Modifier of Snc1,7) plays a crucial role in mitosis during both male and female gametophyte formation in Arabidopsis thaliana. Using a mutagenesis screen, we identify the mos7-5 mutant allele, which causes ovule and pollen abortion in MOS7/mos7-5 heterozygous plants, and preglobular stage embryonic lethality in homozygous mos7-5 seeds. During interphase, we show that MOS7 is localized to the nuclear membrane but, like many nucleoporins, is associated with the spindle apparatus during mitosis. We detect interactions between MOS7 and several nucleoporins known to control spindle dynamics, and find that in pollen from MOS7/mos7-5 heterozygotes, abortion is accompanied by a failure of spindle formation, cell fate specification, and phragmoplast activity. Most intriguingly, we show that following gamete formation by MOS7/mos7-5 heterozygous spores, inheritance of either the MOS7 or the mos7-5 allele by a given gamete does not correlate with its respective survival or abortion. Instead, we suggest a model whereby MOS7, which is highly expressed in the Pollen- and Megaspore Mother Cells, enacts a dosage-limiting effect on the gametes to enable their progression through subsequent mitoses.

Very long chain lipids contribute to the hydrophobic cuticle on the surface of all land plants and are an essential component of the extracellular pollen coat in the Brassicaceae. Mutations in Arabidopsis CER genes eliminate very long chain lipids from the cuticle surface and, in some cases, from the pollen coat. In Arabidopsis, the loss of pollen coat lipids can disrupt interactions with the stigma, inhibiting pollen hydration and causing sterility. We have positionally cloned CER6 and demonstrate that a wild-type copy complements the cer6-2 defect. In addition, we have identified a fertile, intragenic suppressor, cer6-2R, that partially restores pollen coat lipids but does not rescue the stem wax defect, suggesting an intriguing difference in the requirements for CER6 activity on stems and the pollen coat. Importantly, analysis of this suppressor demonstrates that low amounts of very long chain lipids are sufficient for pollen hydration and germination. The predicted CER6 amino acid sequence resembles that of fatty acid–condensing enzymes, consistent with its role in the production of epicuticular and pollen coat lipids >28 carbons long. DNA sequence analysis revealed the nature of the cer6-1, cer6-2, and cer6-2R mutations, and segregation analysis showed that CER6 is identical to CUT1, a cDNA previously mapped to a different chromosome arm. Instead, we have determined that a new gene, CER60, with a high degree of nucleotide and amino acid similarity to CER6, resides at the original CUT1 locus.

The control of cell proliferation during organogenesis plays an important role in initiation, growth, and acquisition of the intrinsic size of organs in higher plants. To understand the developmental mechanism that controls intrinsic organ size by regulating the number and extent of cell division during organogenesis, we examined the function of the Arabidopsis regulatory gene AINTEGUMENATA ( ANT ). Previous observations revealed that ANT regulates cell division in integuments during ovule development and is necessary for floral organ growth. Here we show that ANT controls plant organ cell number and organ size throughout shoot development. Loss of ANT function reduces the size of all lateral shoot organs by decreasing cell number. Conversely, gain of ANT function, via ectopic expression of a 35S :: ANT transgene, enlarges embryonic and all shoot organs without altering superficial morphology by increasing cell number in both Arabidopsis and tobacco plants. This hyperplasia results from an extended period of cell proliferation and organ growth. Furthermore, cells ectopically expressing ANT in fully differentiated organs exhibit neoplastic activity by producing calli and adventitious roots and shoots. Based on these results, we propose that ANT regulates cell proliferation and organ growth by maintaining the meristematic competence of cells during organogenesis.

In flowering plants, two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus that replicates to generate the endosperm, which is a tissue that supports embryo development. MEDEA (MEA) encodes an Arabidopsis SET domain Polycomb protein. Inheritance of a maternal loss-of-function mea allele results in embryo abortion and prolonged endosperm production, irrespective of the genotype of the paternal allele. Thus, only the maternal wild-type MEA allele is required for proper embryo and endosperm development. To understand the molecular mechanism responsible for the parent-of-origin effects of mea mutations on seed development, we compared the expression of maternal and paternal MEA alleles in the progeny of crosses between two Arabidopsis ecotypes. Only the maternal MEA mRNA was detected in the endosperm from seeds at the torpedo stage and later. By contrast, expression of both maternal and paternal MEA alleles was observed in the embryo from seeds at the torpedo stage and later, in seedling, leaf, stem, and root. Thus, MEA is an imprinted gene that displays parent-of-origin-dependent monoallelic expression specifically in the endosperm. These results suggest that the embryo abortion observed in mutant mea seeds is due, at least in part, to a defect in endosperm function. Silencing of the paternal MEA allele in the endosperm and the phenotype of mutant mea seeds supports the parental conflict theory for the evolution of imprinting in plants and mammals.

Higher plant reproduction is unique because two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus that replicates to generate the endosperm, a tissue that supports embryo development. To understand mechanisms that initiate reproduction, we isolated a mutation in Arabidopsis , f644 , that allows for replication of the central cell and subsequent endosperm development without fertilization. When mutant f644 egg and central cells are fertilized by wild-type sperm, embryo development is inhibited, and endosperm is overproduced. By using a map-based strategy, we cloned and sequenced the F644 gene and showed that it encodes a SET-domain polycomb protein. Subsequently, we found that F644 is identical to MEDEA ( MEA ), a gene whose maternal-derived allele is required for embryogenesis [Grossniklaus, U., Vielle-Calzada, J.-P., Hoeppner, M. A. & Gagliano, W. B. (1998) Science 280, 446–450]. Together, these results reveal functions for plant polycomb proteins in the suppression of central cell proliferation and endosperm development. We discuss models to explain how polycomb proteins function to suppress endosperm and promote embryo development.

Summary Endo-β-1,4-glucanase (EGase) expression in ripening tomato fruit and abscising tomato flowers reflects the overlapping accumulation of structurally divergent gene transcripts, including EGase1 (Cel1) and EGase2 (Cel2). Transgenic plants in which cel1 expression was specifically inhibited were evaluated in order to assess the potential role(s) played by Cel1 during fruit ripening and floral abscission. The constitutive expression of an antisense cel1 gene driven by the 35S promoter of cauliflower mosaic virus resulted in the reduction of Cel1 mRNA to trace levels in both ripening fruit and abscising abscission zones of transgenic tomato plants. Transgenic fruit in which cel1 expression was reduced to less than 0.1% of wild-type levels exhibited normal growth and softening behaviour. Transgenic floral abscission zones expressing 5–7% of wild-type levels of Cel1 mRNA exhibited an incidence of abscission reduced by up to one third. The incomplete loss of abscission ability by transgenic flowers may indicate that the contribution of Cel1 activity is insufficient to account for all floral abscission and attests to the presence in tomato abscission zones of additional cell wall hydrolase activities, including Cel2.

A T-DNA-tagged, embryo-defective Arabidopsis thaliana mutant, fist, was identified and shown to exhibit defects in nuclear positioning and cell division orientation beginning at the four-cell stage of the embryo proper. Cell division orientation was randomised, with each embryo exhibiting a different pattern. Periclinal divisions did not occur after the eight-cell embryo proper stage and fist embryos lacked a histologically distinct protoderm layer. Terminal embryos resembled globular-stage embryos, but were a disorganised mass containing 30–100 cells. Some terminal embryos (5%) developed xylem-like elements in outer surface cells, indicating that the fist mutation affects radial pattern. A soybean β-conglycinin seed storage protein gene promoter, active in wild-type embryos from heart stage to maturity, was also active in terminal fist embryos despite their disorganised globular state. This indicated that some pathways of cellular differentiation in fist embryos proceed independently of both organised division plane orientation and normal morphogenesis. Endosperm morphogenesis in seeds containing terminal fist embryos was arrested at one of three distinct developmental stages and appeared unlinked to fist embryo morphogenesis. The β-conglycinin seed storage protein gene promoter, normally active in cellularised wild-type endosperm, was inactive in fist endosperm, indicating abnormal development of fist endosperm at the biochemical level. These data indicate that the fist mutation, either directly or indirectly, results in defects in cell division orientation during the early stages of Arabidopsis embryo development. Other aspects of the fist phenotype, such as defects in endosperm development and radial pattern formation, may be related to abnormal cell division orientation or may occur as pleiotropic effects of the fist mutation.

LEAFY COTYLEDON1 (LEC1) is a central regulator of seed development that plays a key role in controlling the maturation phase during which storage macromolecules accumulate and the embryo becomes tolerant of desiccation. We queried the genomes of seedless plants and identified a LEC1 homolog in the lycophyte, Selaginella moellendorffii , but not in the bryophyte, Physcomitrella patens . Genetic suppression experiments indicated that Selaginella LEC1 is the functional ortholog of Arabidopsis LEC1. Together, these results suggest that LEC1 originated at least 30 million years before the first seed plants appeared in the fossil record. The accumulation of Selaginella LEC1 RNA primarily in sexual and asexual reproductive structures suggests its involvement in cellular processes similar to those that occur during the maturation phase of seed development.

Ovules play a central role in plant reproduction, generating the female gametophyte within sporophytic integuments. When fertilized, the integuments differentiate into the seed coat and support the development of the embryo and endosperm. Mutations in the AINTEGUMENTA (ANT) locus of Arabidopsis have a profound effect on ovule development. Strong ant mutants have ovules that fail to form integuments or a female gametophyte. Flower development is also altered, with a random reduction of organs in the outer three whorls. In addition, organs present in the outer three floral whorls often have abnormal morphology. Ovules from a weak ant mutant contain both inner and outer integuments but generally fail to produce a functional female gametophyte. We isolated the ANT gene by using a mutation derived by T-DNA insertional mutagenesis. ANT is a member of a gene family that includes the floral homeotic gene APETALA2 (AP2). Like AP2, ANT contains two AP2 domains homologous with the DNA binding domain of ethylene response element binding proteins. ANT is expressed most highly in developing flowers but is also expressed in vegetative tissue. Taken together, these results suggest that ANT is a transcription factor that plays a critical role in regulating ovule and female gametophyte development.

DNA methylation is an epigenetic mark that silences transposable elements (TEs) and repeats. Whereas the establishment and maintenance of DNA methylation are relatively well understood, little is known about their dynamics and biological relevance in plant and animal innate immunity. Here, we show that some TEs are demethylated and transcriptionally reactivated during antibacterial defense in Arabidopsis . This effect is correlated with the down-regulation of key transcriptional gene silencing factors and is partly dependent on an active demethylation process. DNA demethylation restricts multiplication and vascular propagation of the bacterial pathogen Pseudomonas syringae in leaves and, accordingly, some immune-response genes, containing repeats in their promoter regions, are negatively regulated by DNA methylation. This study provides evidence that DNA demethylation is part of a plant-induced immune response, potentially acting to prime transcriptional activation of some defense genes linked to TEs/repeats.

The tomato fruit consists of a thick, fleshy pericarp composed predominantly of highly vacuolated parenchymatous cells, which surrounds the seeds. During ripening, the activation of gene expression results in dramatic biochemical and physiological changes in the pericarp. The polygalacturonase (PG) gene, unlike many fruit ripening-induced genes, is not activated by the increase in ethylene hormone concentration associated with the onset of ripening. To investigate ethylene concentration-independent gene transcription in ripe tomato fruit, we analyzed the expression of chimeric PG promoter-beta-glucuronidase (GUS) reporter gene fusions in transgenic tomato plants. We determined that a 1.4-kb PG promoter directs ripening-regulated transcription in outer pericarp but not in inner pericarp cells, with a sharp boundary of PG promoter activity located midway through the pericarp. Promoter deletion analysis indicated that a minimum of three promoter regions influence the spatial regulation of PG transcription. A positive regulatory region from -231 to -134 promotes gene transcription in the outer pericarp of ripe fruit. A second positive regulatory region from -806 to -443 extends gene activity to the inner pericarp. However, a negative regulatory region from -1411 to -1150 inhibits gene transcription in the inner pericarp. DNase I footprint analysis showed that nuclear proteins in unripe and ripe fruit interact with DNA sequences within each of these three regulatory regions. Thus, temporal and spatial control of PG transcription is mediated by the interaction of negative and positive regulatory promoter elements, resulting in gene activity in the outer pericarp but not the inner pericarp of ripe tomato fruit. The expression pattern of PG suggests that, although they are morphologically similar, there is a fundamental difference between the parenchymatous cells within the inner and outer pericarp.

Transcription of the E4 gene is controlled by an increase in ethylene concentration during tomato fruit ripening. To investigate the molecular basis for ethylene regulation, we have examined the E4 promoter to identify cis elements and trans-acting factors that are involved in E4 gene expression. In transgenic tomato plants a chimeric gene construct containing a 1.4-kilobase E4 promoter fused to a beta-glucuronidase reporter gene is rapidly induced by ethylene in ripening fruit. Deletion of E4 promoter sequences to 193 base pairs reduces the level of GUS activity but does not affect ethylene induction. Transient expression of E4 promoter-luciferase chimeric gene constructs containing various deletions, introduced into tomato fruit pericarp by particle bombardment, indicates that a positive ethylene-responsive region is localized between nucleotides -161 and -85 relative to the transcription start site. DNase I footprint analysis shows that a nuclear factor in unripe fruit interacts specifically with sequences in this element, from -142 to -110, which are required for the ethylene response. The DNase I footprint of this factor is reduced in ethylene-treated unripe fruit and undetectable in ripe fruit. Based on the correlation of a nuclear factor binding site with promoter sequences required for ethylene induction, we propose that this in vitro DNA-binding activity may represent a factor that is involved in ethylene-regulated E4 gene expression.

Most of the transcription factors (TFs) responsible for controlling seed development are not yet known. To identify TF genes expressed at specific stages of seed development, including those unique to seeds, we used Affymetrix GeneChips to profile Arabidopsis genes active in seeds from fertilization through maturation and at other times of the plant life cycle. Seed gene sets were compared with those expressed in prefertilization ovules, germinating seedlings, and leaves, roots, stems, and floral buds of the mature plant. Most genes active in seeds are shared by all stages of seed development, although significant quantitative changes in gene activity occur. Each stage of seed development has a small gene set that is either specific at the level of the GeneChip or up-regulated with respect to genes active at other stages, including those that encode TFs. We identified 289 seed-specific genes, including 48 that encode TFs. Seven of the seed-specific TF genes are known regulators of seed development and include the LEAFY COTYLEDON ( LEC ) genes LEC1, LEC1-LIKE, LEC2 , and FUS3 . The rest represent different classes of TFs with unknown roles in seed development. Promoter-β -glucuronidase ( GUS ) fusion experiments and seed mRNA localization GeneChip datasets showed that the seed-specific TF genes are active in different compartments and tissues of the seed at unique times of development. Collectively, these seed-specific TF genes should facilitate the identification of regulatory networks that are important for programming seed development.

The ripening of many fruits is controlled by an increase in ethylene hormone concentration. E8 is a fruit ripening protein that is related to the enzyme that catalyzes the last step in the ethylene biosynthesis pathway, 1-aminocyclopropane-1-carboxylic (ACC) oxidase. To determine the function of E8, we have transformed tomato plants with an E8 antisense gene. We show here that the antisense gene inhibits the accumulation of E8 protein during ripening. Whereas others have shown that reduction of ACC oxidase results in reduced levels of ethylene biosynthesis, we find that reduction of the related E8 protein produces the opposite effect, an increase in ethylene evolution specifically during the ripening of detached fruit. Thus, E8 has a negative effect on ethylene production in fruit.

Monellin is a protein that elicits a flavor approximately 100,000 times sweeter than sugar on a molar basis. The protein exists naturally as a heterodimer, with its sweet flavor lost upon denaturation. A single–chain monellin gene, encoding both polypeptide chains linked by a hinge sequence, was placed under the control of constitutive and fruit–ripening specific promoters and transferred to lettuce and tomato. Expression of these genes in transgenic tomato and lettuce resulted in the accumulation of monellin protein in fruit and leaf, respectively, to significant levels. Production of monellin in transgenic fruits and vegetables represents an alternative strategy to enhance their flavor and quality.

Dynamic Imprinting Gene imprinting—the silencing of either a maternally derived or paternally derived gene allele—is controlled in large part by DNA methylation. In plants, imprinting occurs in the endosperm, which nourishes the embryonic plant. Gehring et al. (p. 1447 ) and Hsieh et al. (p. 1451 ) analyzed the dynamics of DNA methylation in the endosperm and embryo of Arabidopsis and found extensive demethylation in the endosperm, suggesting that many imprinted genes are likely to exist. Gehring et al. characterized five imprinted genes in detail. Four of the 10 known imprinted genes are related homeodomain transcription factors. Furthermore, 5′ sequences demethylated in several of the genes were found to be derived from transposable elements, which supports the idea that imprinting arose as a by-product of silencing invading DNA.

CELL WALL STRUCTURE AND RIPENING-ASSOCIATED CHANGES .. " ......... 676 Cellulose 677 Hemicellulose 678 Pectin . .... ........ . 679 Other Cell Wall Components " . . . . . . " . . . . . . " . . . . . . " . . . . . . " . . . . . . . . . . . . . . . . . . . . . . . 681 CELL WALL HYDRO LASES . ....... . . ....... . . 681 Polygalacturonase . .. , . . . . . . . . . .. .. . . . ..... . . . . . . . . . . . . . . . . . . ... .. ... .. . ...... .. . . . . . 682 Pectinmethylesterase..... ......... . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 686 Cx-Cellulase . ......... 687 Other Cell Wall Hydrolases 688 CELL WALL HYDROLASE FUNCTION DURING FRUIT RIPENING 689 Physical. Genetic. and Biochemical Modification of Cell Wall Hydrolase Activity 689 Molecular Genetic Modification of Cell Wall Hydrolase Activity ". . . . . . . . . 690 FUTURE PROSPECTS . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .. ... .. . . . .. . . .. ........ .. . . ...... . .. 695

Gene imprinting, the differential expression of maternal and paternal alleles, independently evolved in mammals and in flowering plants. A unique feature of flowering plants is a double-fertilization event in which the sperm fertilize not only the egg, which forms the embryo, but also the central cell, which develops into the endosperm (an embryo-supporting tissue). The distinctive mechanisms of gene imprinting in the endosperm, which involve DNA demethylation and histone methylation, begin in the central cell and sperm prior to fertilization. Flowering plants might have coevolved double fertilization and imprinting to prevent parthenogenetic development of the endosperm.

The Arabidopsis DEMETER (DME) DNA glycosylase is required for the maternal allele expression of imprinted Polycomb group (MEDEA and FIS2) and transcription factor (FWA) genes in the endosperm. Expression of DME in the central cell, not in pollen or stamen, establishes gene imprinting by hypomethylating maternal alleles. However, little is known about other genes regulated by DME. To identify putative DME target genes, we generated CaMV:DME plants which ectopically express DME in pollen and stamens. Comparison of mRNA profiles revealed 94 genes induced by ectopic DME expression in both stamen and pollen. Gene ontology analysis identified three molecular functions enriched in the DME-inducible RNA list: DNA or RNA binding, kinase activity, and transcription factor activity. Semi-quantitative RT-PCR verified the candidate genes identified by GeneChip analysis. The putative target genes identified in this study will provide insights into the regulatory mechanism of DME DNA glycosylase and the functions of DNA demethylation.

Imprinting occurs in the endosperm of flowering plants. Endosperm, produced by fertilization of the central cell in the female gametophyte, is essential for embryo and seed development. Several imprinted genes play an important role in endosperm development. The mechanism of gene imprinting involves DNA methylation and histone modification. DNA methylation is actively removed at the imprinted alleles to be activated. Histone methylation mediated by the Polycomb group complex provides another layer of epigenetic regulation at the silenced alleles. Endosperm gene imprinting can be uncoupled from seed development when fertilization of the central cell is prevented. Imprinting may be a mechanism to ensure fertilization of the central cell thereby preventing parthenogenic development of the endosperm.

Genomic imprinting is the differential expression of paternal and maternal alleles. In plants, gene imprinting occurs in the endosperm and has not been found in the embryo or adult plants. Imprinting can affect every allele of a locus (locus-dependent imprinting) or be specific to a particular allele (allele-dependent imprinting). Allele-dependent imprinting was the first type of gene imprinting discovered and has only been documented in maize. Locus-specific imprinting is found in both maize and Arabidopsis. Recent studies have revealed the integral role of female and male gametophytes in gene imprinting and mechanisms by which the parental alleles are distinguished from one another. Herein we will focus on the mechanisms of locus-specific gene imprinting in Arabidopsis and maize.

DNA methylation is an epigenetic modification of cytosine that is important for silencing gene transcription and transposons, gene imprinting, development, and seed viability. DNA METHYLTRANSFERASE1 (MET1) is the primary maintenance DNA methyltransferase in Arabidopsis (Arabidopsis thaliana). Reciprocal crosses between antisense MET1 transgenic and wild-type plants show that DNA hypomethylation has a parent-of-origin effect on seed size. However, due to the dominant nature of the antisense MET1 transgene, the parent with a hypomethylated genome, its gametophyte, and both the maternal and paternal genomes of the F1 seed become hypomethylated. Thus, the distinct role played by hypomethylation at each generation is not known. To address this issue, we examined F1 seed from reciprocal crosses using a loss-of-function recessive null allele, met1-6. Crosses between wild-type and homozygous met1-6 parents show that hypomethylated maternal and paternal genomes result in significantly larger and smaller F1 seeds, respectively. Our analysis of crosses between wild-type and heterozygous MET1/met1-6 parents revealed that hypomethylation in the female or male gametophytic generation was sufficient to influence F1 seed size. A recessive mutation in another gene that dramatically reduces DNA methylation, DECREASE IN DNA METHYLATION1, also causes parent-of-origin effects on F1 seed size. By contrast, recessive mutations in genes that regulate a smaller subset of DNA methylation (CHROMOMETHYLASE3 and DOMAINS REARRANGED METHYLTRANSFERASES1 and 2) had little effect on seed size. Collectively, these results show that maternal and paternal genomes play distinct roles in the regulation of seed size in Arabidopsis.

Arabidopsis APETALA2(AP2) encodes a member of the AP2/EREBP (ethylene responsive element binding protein) class of transcription factors and is involved in the specification of floral organ identity, establishment of floral meristem identity, suppression of floral meristem indeterminancy, and development of the ovule and seed coat. Here, we show that loss-of-functionap2mutations cause an increase in seed mass relative to that of wild-type seeds. Analysis of an allelic series ofap2mutations showed that increases in seed mass corresponded with the severity of defects in flower structure, indicating thatAP2activity directly influences seed mass. Experiments with male-sterile plants and deflowered wild-type plants showed that reduced fertility ofap2mutant plants due to abnormal flower structure accounted for only part of the increase in seed mass caused by strongap2mutant alleles. Reciprocal cross experiments showed thatAP2acts maternally to control seed mass. The maternal effect ofAP2on seed mass involves the regulation of both embryo cell number and cell size. We show further thatap2mutations cause changes in the ratio of hexose to sucrose during seed development, opening the possibility thatAP2may control seed mass through its effects on sugar metabolism. Together, these results identify a role forAP2in controlling seed mass.

Helix-hairpin-helix DNA glycosylases are typically small proteins that initiate repair of DNA by excising damaged or mispaired bases. An invariant aspartic acid in the active site is involved in catalyzing the excision reaction. Replacement of this critical residue with an asparagine severely reduces catalytic activity but preserves enzyme stability and structure. The Arabidopsis DEMETER ( DME ) gene encodes a large 1,729-aa polypeptide with a 200-aa DNA glycosylase domain. DME is expressed primarily in the central cell of the female gametophyte. DME activates maternal allele expression of the imprinted MEDEA ( MEA ) gene in the central cell and is required for seed viability. We mutated the invariant aspartic acid at position 1304 in DME to asparagine (D1304N) to determine whether the catalytic activity of the DNA glycosylase domain is required for DME function in vivo . Transgenes expressing wild-type DME in the central cell rescue seed abortion caused by a mutation in the endogenous DME gene and activate maternal MEA:GFP transcription. However, transgenes expressing the D1304N mutant DME do not rescue seed abortion or activate maternal MEA:GFP transcription. Whereas ectopic expression of the wild-type DME polypeptide in pollen is sufficient to activate ectopic paternal MEA and MEA:GUS expression, equivalent expression of the D1304N mutant DME in pollen failed to do so. These results show that the conserved aspartic acid residue is necessary for DME to function in vivo and suggest that an active DNA glycosylase domain, normally associated with DNA repair, promotes gene transcription that is essential for gene imprinting.

Imprinted genes are expressed predominantly from one allele in a parent-of-origin–specific manner. The endosperm, a seed tissue that mediates the transfer of nutrients from the maternal parent to the embryo, is an important site of imprinting in flowering plants. Imprinted genes have been

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.

The Arabidopsis LEAFY COTYLEDON 2 ( LEC2 ) gene is a central embryonic regulator that serves critical roles both early and late during embryo development. LEC2 is required for the maintenance of suspensor morphology, specification of cotyledon identity, progression through the maturation phase, and suppression of premature germination. We cloned the LEC2 gene on the basis of its chromosomal position and showed that the predicted polypeptide contains a B3 domain, a DNA-binding motif unique to plants that is characteristic of several transcription factors. We showed that LEC2 RNA accumulates primarily during seed development, consistent with our finding that LEC2 shares greatest similarity with the B3 domain transcription factors that act primarily in developing seeds, VIVIPAROUS1/ABA INSENSITIVE3 and FUSCA3. Ectopic, postembryonic expression of LEC2 in transgenic plants induces the formation of somatic embryos and other organ-like structures and often confers embryonic characteristics to seedlings. Together, these results suggest that LEC2 is a transcriptional regulator that establishes a cellular environment sufficient to initiate embryo development.

We identified a T-DNA-generated mutation in thechaperonin-60α gene of Arabidopsis that produces a defect in embryo development. The mutation, termedschlepperless (slp), causes retardation of embryo development before the heart stage, even though embryo morphology remains normal. Beyond the heart stage, theslp mutation results in defective embryos with highly reduced cotyledons. slp embryos exhibit a normal apical-basal pattern and radial tissue organization, but they are morphologically retarded. Even though slp embryos are competent to transcribe two late-maturation gene markers, this competence is acquired more slowly as compared with wild-type embryos.slp embryos also exhibit a defect in plastid development–they remain white during maturation in planta and in culture. Hence, the overall developmental phenotype of theslp mutant reflects a lesion in the chloroplast that affects embryo development. The slp phenotype highlights the importance of the chaperonin-60α protein for chloroplast development and subsequently for the proper development of the plant embryo and seedling.