The maize endosperm consists of three major compartmentalized cell types: the starchy endosperm (SE), the basal endosperm transfer cell layer (BETL), and the aleurone cell layer (AL). Differential genetic programs are activated in each cell type to construct functionally and structurally distinct cells. To compare gene expression patterns involved in maize endosperm cell differentiation, we isolated transcripts from cryo-dissected endosperm specimens enriched with BETL, AL, or SE at 8, 12, and 16 days after pollination (DAP). We performed transcriptome profiling of coding and long noncoding transcripts in the three cell types during differentiation and identified clusters of the transcripts exhibiting spatio-temporal specificities. Our analysis uncovered that the BETL at 12 DAP undergoes the most dynamic transcriptional regulation for both coding and long noncoding transcripts. In addition, our transcriptome analysis revealed spatio-temporal regulatory networks of transcription factors, imprinted genes, and loci marked with histone H3 trimethylated at lysine 27. Our study suggests that various regulatory mechanisms contribute to the genetic networks specific to the functions and structures of the cell types of the endosperm.

Long noncoding RNAs (lncRNAs) affect gene regulation through structural and regulatory interactions with associated proteins. The Polycomb complex often binds to lncRNAs in eukaryotes, and an lncRNA, COLDAIR, associates with Polycomb to mediate silencing of the floral repressor FLOWERING LOCUS C (FLC) during the process of vernalization in Arabidopsis. Here, we identified an additional Polycomb-binding lncRNA, COLDWRAP. COLDWRAP is derived from the repressed promoter of FLC and is necessary for the establishment of the stable repressed state of FLC by vernalization. Both COLDAIR and COLDWRAP are required to form a repressive intragenic chromatin loop at the FLC locus by vernalization. Our results indicate that vernalization-mediated Polycomb silencing is coordinated by lncRNAs in a cooperative manner to form a stable repressive chromatin structure.

Flowering in plants is a dynamic and synchronized process where various cues including age, day length, temperature and endogenous hormones fine-tune the timing of flowering for reproductive success. Arabidopsis thaliana is a facultative long day (LD) plant where LD photoperiod promotes flowering. Arabidopsis still flowers under short-day (SD) conditions, albeit much later than in LD conditions. Although factors regulating the inductive LD pathway have been extensively investigated, the non-inductive SD pathway is much less understood. Here, we identified a key basic helix-loop-helix transcription factor called NFL (NO FLOWERING IN SHORT DAY) that is essential to induce flowering specifically under SD conditions in Arabidopsis. nfl mutants do not flower under SD conditions, but flower similar to the wild type under LD conditions. The no-flowering phenotype in SD is rescued either by exogenous application of gibberellin (GA) or by introducing della quadruple mutants in the nfl background, suggesting that NFL acts upstream of GA to promote flowering. NFL is expressed at the meristematic regions and NFL is localized to the nucleus. Quantitative RT-PCR assays using apical tissues showed that GA biosynthetic genes are downregulated and the GA catabolic and receptor genes are upregulated in the nfl mutant compared with the wild type, consistent with the perturbation of the endogenous GA biosynthetic and catabolic intermediates in the mutant. Taken together, these data suggest that NFL is a key transcription factor necessary for promotion of flowering under non-inductive SD conditions through the GA signaling pathway.

Ethylene is one of the most important hormones for plant developmental processes and stress responses. However, the phosphorylation regulation in the ethylene signaling pathway is largely unknown. Here we report the phosphorylation of cap binding protein 20 (CBP20) at Ser245 is regulated by ethylene, and the phosphorylation is involved in root growth. The constitutive phosphorylation mimic form of CBP20 (CBP20S245E or CBP20S245D), while not the constitutive de-phosphorylation form of CBP20 (CBP20S245A) is able to rescue the root ethylene responsive phenotype of cbp20. By genome wide study with ethylene regulated gene expression and microRNA (miRNA) expression in the roots and shoots of both Col-0 and cbp20, we found miR319b is up regulated in roots while not in shoots, and its target MYB33 is specifically down regulated in roots with ethylene treatment. We described both the phenotypic and molecular consequences of transgenic over-expression of miR319b. Increased levels of miR319b (miR319bOE) leads to enhanced ethylene responsive root phenotype and reduction of MYB33 transcription level in roots; over expression of MYB33, which carrying mutated miR319b target site (mMYB33) in miR319bOE is able to recover both the root phenotype and the expression level of MYB33. Taken together, we proposed that ethylene regulated phosphorylation of CBP20 is involved in the root growth and one pathway is through the regulation of miR319b and its target MYB33 in roots.

BACKGROUND: The Maternally expressed gene (Meg) family is a locally-duplicated gene family of maize which encodes cysteine-rich proteins (CRPs). The founding member of the family, Meg1, is required for normal development of the basal endosperm transfer cell layer (BETL) and is involved in the allocation of maternal nutrients to growing seeds. Despite the important roles of Meg1 in maize seed development, the evolutionary history of the Meg cluster and the activities of the duplicate genes are not understood. RESULTS: In maize, the Meg gene cluster resides in a 2.3 Mb-long genomic region that exhibits many features of non-centromeric heterochromatin. Using phylogenetic reconstruction and syntenic alignments, we identified the pedigree of the Meg family, in which 11 of its 13 members arose in maize after allotetraploidization ~4.8 mya. Phylogenetic and population-genetic analyses identified possible signatures suggesting recent positive selection in Meg homologs. Structural analyses of the Meg proteins indicated potentially adaptive changes in secondary structure from α-helix to β-strand during the expansion. Transcriptomic analysis of the maize endosperm indicated that 6 Meg genes are selectively activated in the BETL, and younger Meg genes are more active than older ones. In endosperms from B73 by Mo17 reciprocal crosses, most Meg genes did not display parent-specific expression patterns. CONCLUSIONS: Recently-duplicated Meg genes have different protein secondary structures, and their expressions in the BETL dominate over those of older members. Together with the signs of positive selections in the young Meg genes, these results suggest that the expansion of the Meg family involves potentially adaptive transitions in which new members with novel functions prevailed over older members.

Plants have evolved a number of monitoring systems to sense their surroundings and to coordinate their growth and development accordingly. Vernalization is one example, in which flowering is promoted after plants have been exposed to a long-term cold temperature (i.e. winter). Vernalization results in the repression of floral repressor genes that inhibit the floral transition in many plant species. Here, we describe recent advances in our understanding of the vernalization-mediated promotion of flowering in Arabidopsis and other flowering plants. In Arabidopsis, the vernalization response includes the recruitment of chromatin-modifying complexes to floral repressors and thus results in the enrichment of repressive histone marks that ensure the stable repression of floral repressor genes. Changes in histone modifications at floral repressor loci are stably maintained after cold exposure, establishing the competence to flower the following spring. We also discuss similarities and differences in regulatory circuits in vernalization responses among Arabidopsis and other plants.

In plants, epigenetic regulation mediates both the proper development of the plant and responses to environmental cues. Changes in epigenetic states employ DNA methylation, histone modification, and regulatory RNAs. In Arabidopsis thaliana, DNA methylation as a repressive mark is often associated with constitutively silenced loci, such as repetitive sequences, transposons, and heterochromatin. These sequences regularly give rise to small interfering RNAs, which direct DNA methylation through the RNA-directed DNA methylation (RdDM) pathway. For example, FWA locus is silenced in sporophytes and enriched with DNA methylation. Its methylated state is stable and passes to the next generation. This is an example of meiotically inherited epigenetic states. There are also epigenetic changes that can be inherited mitotically and are subsequently erased in the next generation. In this review, we use the vernalization-mediated epigenetic silencing of FLOWERING LOCUS C (FLC) as an example for this type of mitotically stable epigenetic state. Here, we discuss mechanisms of epigenetic changes that can result in meiotically or mitotically stable states with an emphasis on FWA and FLC as two examples.

Polycomb group (PcG) proteins are conserved chromatin regulators involved in the control of key developmental programs in eukaryotes. They collectively provide the transcriptional memory unique to each cell identity by maintaining transcriptional states of developmental genes. PcG proteins form multi-protein complexes, known as Polycomb repressive complex 1 (PRC1) and Polycomb repressive complex 2 (PRC2). PRC1 and PRC2 contribute to the stable gene silencing in part through catalyzing covalent histone modifications. Components of PRC1 and PRC2 are well conserved from plants to animals. PcG-mediated gene silencing has been extensively investigated in efforts to understand molecular mechanisms underlying developmental programs in eukaryotes. Here, we describe our current knowledge on PcG-mediated gene repression which dictates developmental programs by dynamic layers of regulatory activities, with an emphasis given to the model plant Arabidopsis thaliana.

Mitotic inheritance of identical cellular memory is crucial for development in multicellular organisms. The cell type-specific epigenetic state should be correctly duplicated upon DNA replication to maintain cellular memory during tissue and organ development. Although a role of DNA replication machinery in maintenance of epigenetic memory has been proposed, technical limitations have prevented characterization of the process in detail. Here, we show that INCURVATA2 (ICU2), the catalytic subunit of DNA polymerase α in Arabidopsis, ensures the stable maintenance of repressive histone modifications. The missense mutant allele icu2-1 caused a defect in the mitotic maintenance of vernalization memory. Although neither the recruitment of CURLY LEAF (CLF), a SET-domain component of Polycomb Repressive Complex 2 (PRC2), nor the resultant deposition of the histone mark H3K27me3 required for vernalization-induced FLOWERING LOCUS C (FLC) repression were affected, icu2-1 mutants exhibited unstable maintenance of the H3K27me3 level at the FLC region, which resulted in mosaic FLC de-repression after vernalization. ICU2 maintains the repressive chromatin state at additional PRC2 targets as well as at heterochromatic retroelements. In icu2-1 mutants, the subsequent binding of LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1), a functional homolog of PRC1, at PRC2 targets was also reduced. We demonstrated that ICU2 facilitates histone assembly in dividing cells, suggesting a possible mechanism for ICU2-mediated epigenetic maintenance.

Vernalization is an environmentally induced epigenetic switch in which winter cold triggers epigenetic silencing of floral repressors and thus provides competence to flower in spring. Vernalization triggers the recruitment of chromatin-modifying complexes to a clade of flowering repressors that are epigenetically silenced via chromatin modifications. In Arabidopsis thaliana, VERNALIZATION INSENSITIVE3 (VIN3) and its related plant homeodomain finger proteins act together with Polycomb Repressive Complex 2 to increase repressive histone marks at floral repressor loci, including FLOWERING LOCUS C (FLC) and its related genes, by vernalization. Here, we show that VIN3 family of proteins nonredundantly functions to repress different subsets of the FLC gene family during the course of vernalization. Each VIN3 family protein binds to modified histone peptides in vitro and directly associates with specific sets of FLC gene family chromatins in vivo to mediate epigenetic silencing. In addition, members of the FLC gene family are also differentially regulated during the course of vernalization to mediate proper vernalization response. Our results show that these two gene families cooperated during the course of evolution to ensure proper vernalization response through epigenetic changes.

Many eukaryotes, including plants, produce a large number of long noncoding RNAs (lncRNAs).Growing number of lncRNAs are being reported to have regulatory roles in various developmental processes.Emerging mechanisms underlying the function of lncRNAs indicate that lncRNAs are versatile regulatory molecules. They function as potent cis- and trans-regulators of gene expression, including the formation of modular scaffolds that recruit chromatin-modifying complexes to target chromatin. LncRNAs have also been reported in plants. Here, we describe our current understanding on potential roles of lncRNA in plants.

Heterotrimeric G proteins, consisting of Gα, Gβ, and Gγ subunits, play important roles in plant development and cell signaling. In Arabidopsis, in addition to one prototypical G protein α subunit, GPA1, there are three extra-large G proteins, XLG1, XLG2, and XLG3, of largely unknown function. Each extra-large G (XLG) protein has a C-terminal Gα-like region and a ∼400 amino acid N-terminal extension. Here we show that the three XLG proteins specifically bind and hydrolyze GTP, despite the fact that these plant-specific proteins lack key conserved amino acid residues important for GTP binding and hydrolysis of GTP in mammalian Gα proteins. Moreover, unlike other known Gα proteins, these activities require Ca(2+) instead of Mg(2+) as a cofactor. Yeast two-hybrid library screening and in vitro protein pull-down assays revealed that XLG2 interacts with the nuclear protein RTV1 (related to vernalization 1). Electrophoretic mobility shift assays show that RTV1 binds to DNA in vitro in a non-sequence-specific manner and that GTP-bound XLG2 promotes the DNA binding activity of RTV1. Overexpression of RTV1 results in early flowering. Combined overexpression of XLG2 and RTV1 enhances this early flowering phenotype and elevates expression of the floral pathway integrator genes, FT and SOC1, but does not repress expression of the floral repressor, FLC. Chromatin immunoprecipitation assays show that XLG2 increases RTV1 binding to FT and SOC1 promoters. Thus, a Ca(2+)-dependent G protein, XLG2, promotes RTV1 DNA binding activity for a subset of floral integrator genes and contributes to floral transition.

In Arabidopsis, the role of the vernalization pathway is to repress expression of a potent floral repressor, FLOWERING LOCUS C (FLC), after a sufficient period of winter cold has been perceived. Following winter, the lack of FLC expression allows unimpeded operation of the photoperiod pathway and hence rapid flowering of vernalized plants in spring via the activation of floral integrator genes. Molecular studies revealed that regulation of the key floral repressor, FLC, is under the control of the interplay between Trithorax group (TrxG)-mediated activation and Polycomb group (PcG)-mediated repression. On-off switch of genes by TrxG and PcG is an evolutionarily conserved mechanism to coordinate cellular identity in eukaryotes. Regulation of FLC by external cues provides an excellent model system to study mechanisms in which cell identity is influenced by environment. In this review, we discuss coordinated contributions by protein and long noncoding RNA components to this environmentally induced epigenetic switch of a developmental program in plants.

A report on the 10th International Congress of Plant Molecular Biology, Jeju, South Korea, October 21-26, 2012.

Long noncoding RNAs (lncRNAs) are increasingly recognized as functional regulatory components in eukaryotic gene regulation. Distinct classes of lncRNAs have been identified in eukaryotes and they play roles in various regulatory networks. Previously characterized lncRNAs include primary transcripts for small regulatory RNAs. In the era of deep sequencing, new classes of lncRNAs have emerged as potent regulatory components in gene regulation. Recent studies showed that many lncRNAs are potent cis- and trans-regulators of gene activity and they can function as scaffolds for chromatin-modifying complexes. Furthermore, differential expressions of lncRNAs suggest that transcription of lncRNAs can modulate gene activity during development and in response to external stimuli. Here, we summarize our current understanding on potential roles of lncRNAs in plants.

Proper flowering time is vital for reproductive fitness in flowering plants. In Arabidopsis, vernalization is mediated primarily through the repression of a MADS box transcription factor, FLOWERING LOCUS C (FLC). The induction of a plant homeodomain-containing protein, VERNALIZATION INSENSITIVE 3 (VIN3), by vernalizing cold is required for proper repression of FLC. One of a myriad of changes that occurs after VIN3 is induced is the establishment of FLC chromatin at a mitotically repressed state due to the enrichment of repressive histone modifications. VIN3 induction by cold is the earliest known event during the vernalization response and includes changes in histone modifications at its chromatin. Here, the current understanding of the vernalization-mediated chromatin changes in Arabidopsis is discussed, with a focus on the roles of shared chromatin-modifying machineries in regulating VIN3 and FLC gene family expression during the course of vernalization.

In some plant species, prolonged exposure to low temperature during the winter season is necessary to acquire the competence to flower in the following spring. This process, known as vernalization, is an epigenetic change in that a mitotically stable change of the developmental potential of the meristem (competence to flower) is maintained even in the absence of the inducing signal (prolonged cold exposure). In Arabidopsis, vernalization results in stable epigenetic repression of a potent floral repressor, FLOWERING LOCUS C (FLC). Increased enrichment of Polycomb Repressive Complex 2 (PRC2) and trimethylated Histone H3 Lys 27 (H3K27me3) at FLC chromatin is necessary for the stable maintenance of FLC repression by vernalization. Recent recognition of long noncoding RNAs (ncRNAs) in vernalization response indicates that long ncRNAs are evolutionarily conserved components for PRC2-mediated repression in eukaryotes.

Vernalization is an environmentally-induced epigenetic switch in which winter cold triggers epigenetic silencing of floral repressors and thus provides competence to flower in spring. In Arabidopsis, winter cold triggers enrichment of tri-methylated histone H3 Lys(27) at chromatin of the floral repressor, FLOWERING LOCUS C (FLC), and results in epigenetically stable repression of FLC. This epigenetic change is mediated by an evolutionarily conserved repressive complex, polycomb repressive complex 2 (PRC2). Here, we show that a long intronic noncoding RNA [termed COLD ASSISTED INTRONIC NONCODING RNA (COLDAIR)] is required for the vernalization-mediated epigenetic repression of FLC. COLDAIR physically associates with a component of PRC2 and targets PRC2 to FLC. Our results show that COLDAIR is required for establishing stable repressive chromatin at FLC through its interaction with PRC2.

DORMANCY ASSOCIATED MADS-BOX (DAM) genes are related to AGAMOUS-LIKE 24 and SHORT VEGETATIVE PHASE genes of arabidopsis and are differentially regulated coordinately with endodormancy induction and release in buds of several perennial plant species. DAM genes were first shown to directly impact endodormancy in peach where a deletion of a series of DAM resulted in loss of endodormancy induction. We have cloned and characterized several MADS box genes from the model perennial weed leafy spurge. Leafy spurge DAM genes are preferentially expressed in shoot tips and buds in response to cold temperatures and day length in a manner that is relative to the level of endodormancy induced by various environmental conditions. Over-expression of one DAM gene in arabidopsis delays flowering. Additionally, we show that at least one DAM gene is differentially regulated by chromatin remodeling. Comparisons of the DAM gene promoters between poplar and leafy spurge have identified several conserved sequences that may be important for their expression patterns in response to dormancy-inducing stimuli.

VERNALIZATION INSENSITIVE 3 (VIN3) is required for vernalization-mediated repression of FLOWERING LOCUS C (FLC) in Arabidopsis. The induction of VIN3 by long-term exposure to cold is one of earliest events in vernalization response. However, molecular mechanisms underlying for the VIN3 induction are poorly understood. Recently, we reported that the constitutive repression of VIN3 in the absence of the cold exposure is due to multiple repressive chromatin modifying components, including a transposable element (TE)-derived sequence, LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1) and POLYCOMB REPRESSION COMPLEX 2 (PRC2). In addition, the maximum level of VIN3 induction requires EARLY FLOWERING 7 (ELF7) and EARLY FLOWERING IN SHORDAYS (EFS), which are components of activating chromatin modifying complexes. Furthermore, dynamic changes in histone modifications at VIN3 chromatin are observed during the course of vernalization. Thus, mechanisms underlying the induction of VIN3 include changes at the level of chromatin.