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- © 2008 American Society of Plant Biologists
During the early stages of seed development, Arabidopsis (Arabidopsis thaliana) endosperm is syncytial and proliferates rapidly through repeated rounds of mitosis without cytokinesis. This stage of endosperm development is important in determining final seed size and is a model for studying aspects of cellular and molecular biology, such as the cell cycle and genomic imprinting. However, the small size of the Arabidopsis seed makes high-throughput molecular analysis of the early endosperm technically difficult. Laser capture microdissection enabled high-resolution transcript analysis of the syncytial stage of Arabidopsis endosperm development at 4 d after pollination. Analysis of Gene Ontology representation revealed a developmental program dominated by the expression of genes associated with cell cycle, DNA processing, chromatin assembly, protein synthesis, cytoskeleton- and microtubule-related processes, and cell/organelle biogenesis and organization. Analysis of core cell cycle genes implicates particular gene family members as playing important roles in controlling syncytial cell division. Hormone marker analysis indicates predominance for cytokinin signaling during early endosperm development. Comparisons with publicly available microarray data revealed that approximately 800 putative early seed-specific genes were preferentially expressed in the endosperm. Early seed expression was confirmed for 71 genes using quantitative reverse transcription-polymerase chain reaction, with 27 transcription factors being confirmed as early seed specific. Promoter-reporter lines confirmed endosperm-preferred expression at 4 d after pollination for five transcription factors, which validates the approach and suggests important roles for these genes during early endosperm development. In summary, the data generated provide a useful resource providing novel insight into early seed development and identify new target genes for further characterization.
Most of the world's food calories come from seeds, and extensive research has been directed at improving nutritional value and traits such as seed size and number. However, seeds are complex organs, and improvement by rational design requires an understanding of the contribution of specific tissues during important stages of seed development.
Seed development in most angiosperms begins with double fertilization, where the haploid egg cell and the double haploid central cell are both fertilized by identical haploid sperm cells contributed by a single pollen grain. This generates the diploid embryo and the triploid endosperm, respectively. The embryo and the endosperm grow rapidly in a coordinated manner that is heavily influenced by the surrounding maternal integument tissues that later form the seed coat (Olsen, 2004; Garcia et al., 2005). During early stages of seed development, maternal resources are mainly used for rapid cell division and growth of new tissues. Once cell division slows, the seed enters a maturation phase during which resources are reallocated to the synthesis of storage compounds, such as starch followed by the accumulation of oils and proteins. Biosynthetic activity then slows as the seed moves through a late maturation phase and prepares to desiccate prior to dormancy.
The developing endosperm plays several important roles during seed development (Berger, 2003; Olsen, 2004). In many plant species, including Arabidopsis (Arabidopsis thaliana), the triploid primary endosperm nucleus undergoes several rounds of free nuclear division, growing rapidly as a syncytium (Olsen, 2004). During the first phase of endosperm development, nuclei divisions are synchronous, but by the fourth division, three mitotic domains are apparent that differ with regard to the rate of nuclei divisions (Boisnard-Lorig et al., 2001; Brown et al., 2003). These are termed the micropylar endosperm, which surrounds the embryo, the peripheral endosperm, which lines the wall of the developing embryo sac, and the chalazal endosperm, which develops adjacent to the vascular connection with the seed parent (Boisnard-Lorig et al., 2001). Nuclear division continues through a third phase of endosperm development, which is marked by a migration of nuclei to the chalazal region, leading to the formation of chalazal nodules and cysts. Toward the end of this phase, the endosperm consists of approximately 200 nucleocytoplasmic domains and the embryo reaches the globular stage of development. Phase 4 sees the initiation of endosperm cellularization and reduced rates of mitosis (Scott et al., 1998; Boisnard-Lorig et al., 2001; Ingouff et al., 2005).
A role for endosperm in supporting the formation and growth of the embryo during early stages of development is suggested by the positioning of the chalazal endosperm and by the fact that seeds with severely defective endosperm cannot complete development (Scott et al., 1998). During the maturation stages, endosperm cellularizes and storage reserves are produced that accumulate in the endosperm cells (Sorensen et al., 2002). In plants that have ephemeral endosperms, such as Arabidopsis and oilseed rape (Brassica napus), the embryo develops at the expense of the endosperm and absorbs these reserves, storing them in the cotyledons (Scott et al., 1998; Olsen, 2004). Seeds that generate large endosperms during the early stages of development produce large embryos at maturity. The early proliferation of the endosperm, therefore, is associated with the growth of seeds and final seed size (Scott et al., 1998; Bushell et al., 2003). The alteration of the rate and duration of cell division in the endosperm has been proposed as a biotechnological strategy for altering seed size (Tiwari et al., 2006).
Arabidopsis provides an important model system for studying the underlying mechanisms of early seed development. Extensive and rapid analysis of many aspects of seed biology can be conducted in Arabidopsis due to the established protocols for producing and analyzing mutant and transgenic lines and the availability of a genome sequence facilitating the generation of tools for high-throughput molecular analysis. However, a major drawback to studying seed biology in Arabidopsis is its very small seeds. Laser microdissection is an important method for obtaining individual tissues or cell types for biochemical analysis. Originally developed for isolating cancerous cells from normal tissue (Emmert-Buck et al., 1996), laser microdissection has been used successfully to obtain DNA, RNA, proteins, and metabolites from a range of plant species and tissue types (for review, see Day et al., 2005, 2007a; Nelson et al., 2006). Therefore, it provides an ideal tool for analyzing gene expression changes in specific cell types during the early stages of Arabidopsis seed development (Spencer et al., 2007).
In a previous study, we compared different methods of transcriptome amplification from small amounts of RNA for use with printed long-oligonucleotide microarrays (Day et al., 2007b). A two-round IVT-based amplification was selected and used to obtain array data for proliferating syncytial endosperm at 4 d after pollination (DAP). This corresponded to the end of the third phase of endosperm development, when the syncytial endosperm contains many nucleocytoplasmic domains, but is prior to cellularization at 5 to 6 DAP (Scott et al., 1998; Boisnard-Lorig et al., 2001; Ingouff et al., 2005). The microarray data that formed the basis of this study were generated by hybridizing laser capture microdissected (LCM) endosperm-derived target alongside target from similarly treated silique tissues using a two-color microarray approach (Day et al., 2007b). A total of 18,220 unique probes gave signal higher than 2-fold background, and t testing identified 12,710 probes as being significantly differentially expressed between the whole silique and endosperm samples using a P value cutoff of <0.05. Analysis of embryo, seed coat, and endosperm markers within the data indicated that a 2-fold differential expression in the endosperm direction provided a stringent cutoff for identification of endosperm-preferred expression (Day et al., 2007b). This procedure identified 2,568 individual loci as being preferentially expressed in the endosperm. Here, we present extensive validation of the LCM endosperm array data by quantitative reverse transcription (qRT)-PCR and GUS reporter lines and provide a comprehensive analysis of the endosperm transcriptome in the context of existing online resources. This analysis has enabled novel insight into early endosperm development and has identified 793 genes as having early seed-specific and endosperm-preferred expression.
Microarray Analysis from Laser-Microdissected Endosperm Reliably Identifies Differential Expression in the Endosperm
To ensure that the microarray platform was correctly measuring differential expression between the endosperm and silique samples, 16 differentially expressed genes from the array data were selected for concurrent qRT-PCR analysis. Excess amplified RNA produced during the microarray target preparation was used to provide template for the qRT-PCR. The expression ratios produced by qRT-PCR and the microarray experiments were very similar (Table I ), and all genes were confirmed as preferentially expressed in the endosperm sample by both the microarray and qRT-PCR.
Identification of Endosperm-Preferred Genes Specifically Expressed during Early Seed Development Using Online Data Sets
To help identify genes with early endosperm-specific roles, we searched three online data sets that included a wide range of different tissues and at least one early seed or silique sample. This identified many genes with apparent early silique/seed-specific expression.
Massively parallel signature sequencing (MPSS) data (available from http://mpss.udel.edu/at/GeneQuery.php; Meyers et al., 2004) include two independent silique libraries (1–2 DAP) that are consistent with the early proliferative stage of endosperm development as well as data for inflorescences, leaves, roots, and germinating seedlings. We identified 200 genes that showed enrichment in the silique libraries. Of these, 68 were preferentially expressed in the endosperm and were MPSS silique library specific. A summary of the 35 endosperm-preferred genes with the highest MPSS tag frequencies and silique-specific MPSS profiles is given in Supplemental Figure S1.
The AtGenExpress developmental series (Schmid et al., 2005) uses Affymetrix GeneChip technology to profile Arabidopsis transcripts from different organs and at different stages of development. This library contains data for seeds dissected from the silique 6 DAP onward, but the earlier stages (2–3, 3–4, and 4 DAP) use whole silique material. SUC5 has strong endosperm-preferred expression during the proliferative stages of seed development (Baud et al., 2005) and was used as an expression template to pull out 196 genes with similar expression (based on an r value cutoff of 0.75; Supplemental Table S1). This list was filtered based on endosperm-preferred expression in our data, a median expression level in AtGenExpress of <100 units in non-seed-containing tissues, and a median expression of >100 across the seed series to generate entries for Figure 1 .
A more recent Affymetrix GeneChip data set profiles a range of tissues, including ovules and seeds dissected from gynoecia and siliques, respectively (available at http://estdb.biology.ucla.edu/genechip/). This data set includes immature seeds at 1, 3 to 4, and 7 to 8 DAP and was generated in the Goldberg (University of California, Los Angeles [UCLA]) and Harada (University of California, Davis) laboratories by Brandon Le (UCLA), Anhthu Bui (UCLA), and Julie Pelletier (University of California, Davis). We refer to it here as GHL data. We identified genes with similar expression patterns to early endosperm markers (see "Materials and Methods") and cross-referenced these to genes with differential expression from our arrays. This created a subgroup of 2,608 putative early seed-specific genes (Supplemental Table S2).
Partitioning the Data for Further Analysis
To gain insight into the differential processes in operation during early silique and endosperm development at 4 DAP, we analyzed endosperm-preferred (EP; >2-fold differentially expressed in the endosperm sample compared with the silique sample) and other silique tissue-preferred (OST; >2-fold differentially expressed in the silique sample compared with the endosperm sample) gene groups. We also looked at subgroups containing genes that were thought to be early seed specific from our analysis of the GHL data that we termed ESS-EP and ESS-OST. The GHL data were found to be the most reliable source for identifying early seed-specific expression, since they displayed high sensitivity toward known endosperm markers compared with the AtGenExpress data (see "Materials and Methods"; Supplemental Fig. S2). The list of Arabidopsis Genome Initiative (AGI) numbers used for each partition is available in Supplemental Table S3.
Analysis of the Representation of Gene Ontologies and Functional Categories
The Arabidopsis genome has been extensively annotated. The Arabidopsis Information Resource (TAIR), as part of the Gene Ontology (GO) Consortium (Rhee et al., 2003), and the Munich Information Center for Protein Sequences (MIPS; Mewes et al., 2008) both provide annotation schemes that use controlled vocabularies aimed at providing descriptions of the roles of genes that are applicable to all organisms. We identified statistically significant enrichment of annotation terms using both the TAIR GO and FunCat schemes. Since both gave similar insight into our microarray data, we present the analysis based only on the TAIR GO terms. However, complete analysis using both vocabularies is provided in Supplemental Tables S4 to S7. Analysis of the EP partition indicated enrichment for GO terms associated with the rapidly proliferating nature of the endosperm at 4 DAP (i.e. molecular biosynthesis, protein formation, the cell cycle, DNA metabolism, replication, and microtubule-based movement; Supplemental Tables S4).
The OST partition represents genes that are predominantly expressed in nonendosperm tissues of the silique. The less proliferative nature of the growth and development of the majority of nonendosperm tissues manifests as an enrichment of GO terms for growth, development, cell communication, signal transduction, and hormone-mediated signaling (Supplemental Table S4). We also saw an enrichment of a large number of GO terms associated with endogenous and environmental stimuli that presumably reflect the need for the silique to provide a buffered environment for immature seeds to develop. Unlike the syncytial endosperm, the nonendosperm tissues of the silique are mostly composed of cells encased in a cell wall matrix, a difference that is corroborated in our data by enrichment for cell wall organization/biogenesis and cell wall loosening. Also enriched in this partition are terms for carbohydrate metabolism and biomolecular transport (Supplemental Table S4).
The GO analysis was refined to only include genes expressed specifically during the early stages of seed development. The ESS-EP partition was heavily enriched for genes associated with aspects of the cell cycle, DNA and chromatin biochemistry, microtubule-associated processes, and protein synthesis. The ESS-OST partition was enriched for relatively few GO terms (development, ovule development, carpel development, gynoecium development, and organ development), consistent with a less proliferative type of tissue development in nonendosperm seed tissues (ESS-EP and ESS-OST GO analysis shown in Table II ).
Representation Analysis of Selected Gene Families
Several gene families of interest have been characterized in the recent literature or collected in online resources. Representation analyses of selected gene families are summarized in Table III , and details are given below. Significance during this stage of the analysis was based on a P value cutoff of <0.005, unless otherwise stated.
Analysis of Cell Cycle Genes
Plant syncytial development requires a rapid progression through the cell cycle, suppression of phragmoplast formation, and an uncoupling of cytokinesis from mitosis (Otegui and Staehelin, 2000). To gain further insights into this process, we analyzed our data to identify endosperm-preferred genes that have been implicated in controlling cell cycle progression (Table IV ). The core cell cycle genes of Arabidopsis (Vandepoele et al., 2002) and genes shown to be regulated by the E2F members of this family were overrepresented in the EP data (Table IV; Supplemental Table S8). Motifs associated with E2F binding were also highly enriched in this partition, such that the "E2FAT motif" (TYTCCCGCC) was enriched at the P < 1 × 10−5 level in both the EP and ESS-EP partitions and the "E2F-binding site motif" (TTTCCCGC) was enriched in the EP and ESS-EP partitions at the P < 1 × 10−9 and P < 1 × 10−10 levels, respectively.
Progression through the cell cycle occurs via coordinated sequential activation of distinct phases. M phase-specific expression is associated with an M-specific activator sequence (MSA) in the promoter region of a gene. A total of 161 differentially expressed genes from our array analysis contained the MSA sequence in the 500 bp upstream of the ATG. Of these putative M phase-specific genes, 27 were in the EP partition and 16 were in the ESS-EP partition (Supplemental Table S8). Hypergeometric testing of these putative M phase-specific transcripts indicated significant enrichment in the EP and ESS-EP partitions at the P < 0.01 and P < 0.0005 levels, respectively.
Analysis of Hormone Response Pathways
The varied distributions of phytohormones and their well-documented ability to regulate growth and development of the seed make them obvious candidates for identifying important components in the control of early endosperm development (Lur and Setter, 1993; Yang et al., 2002, 2006). A recent study by Nemhauser et al. (2006) used the AtGenExpress hormone series to identify genes that were only expressed in response to particular hormones, suggesting that these genes can be used as markers for hormone action. To gain insight into the influence of plant hormones in the developing silique at 4 DAP, we looked for the presence of these markers in our gene groups. The OST partition (representing many tissues) was significantly enriched for hormone markers, whereas the EP partition (single tissue) had significant underrepresentation (Table III).
Analysis of the full lists of genes responsive to the hormones ethylene, abscisic acid, brassinosteroid, cytokinin, gibberellin, auxin, and methyl jasmonate (1-aminocyclopropane-1-carboxylic acid [ACC], ABA, brassinolide [BL], CK, GA, indole-3-acetic acid [IAA], and MJ, respectively) showed that all but one of the hormone-responsive gene groups (GA) were significantly enriched in the OST partition (Table III; Supplemental Table S9), reinforcing the observations made using the marker list. The ACC-, ABA-, and GA-responsive genes were well represented in the endosperm-preferred partition, but only the CK-responsive genes were significantly enriched (Table III). Endosperm-preferred genes involved in CK signaling are given in Table V .
The hormone-responsive gene lists include genes that are up-regulated, down-regulated, or have a complex regulatory pattern in response to exogenous hormone application. The distribution of up-regulated, down-regulated, and complex CK-regulated genes in the data partitions were compared using a χ2 test. Significant differences from the expected distribution for CK-regulated genes were seen for both the EP and OST partitions. The EP partition included a much larger than expected number of CK up-regulated genes (observed, 94%; expected, 66%) and fewer than expected CK down-regulated genes (observed, 4%; expected, 32%). Conversely, in the OST partition, we saw a much larger than expected number of CK down-regulated genes (observed, 52%; expected, 32%) and fewer than expected CK up-regulated genes (observed, 47%; expected, 66%).
Interestingly, none of the 48 ARF and AUX-IAA transcription factors represented in the differentially expressed gene list gave evidence for endosperm-preferred expression (data not shown). Conversely, 19 of these transcription factors were present in the OST partition. Interactions between these two groups of proteins mediate auxin-dependent transcriptional regulation, and when taken together as an "auxin signaling group" (ARFs plus AUX-IAAs), hypergeometric testing showed that the underrepresentation in the EP partition was significant (P = 0.0032).
Analysis of Chromatin-Related and DNA Methylation-Sensitive Genes
Transcriptional regulation is closely related to chromatin structure, and during syncytial development, endosperm has a high proportion of euchromatin, with small chromocenters and distinct heterochromatic foci (Baroux et al., 2007). Euchromatin is associated with active transcription, and alterations in chromatin structure have been associated with the onset of cell division, morphogenesis, and differentiation (Zhao et al., 2001; Berger and Gaudin, 2003; Williams et al., 2003; Baroux et al., 2007; De Veylder et al., 2007). Enrichment analysis revealed a significant overrepresentation of chromatin-related genes (obtained from ChromDB [http://www.chromdb.org/]) in the endosperm (Table III; Supplemental Table S10). Dynamic changes in chromatin structure are associated with epigenetic alterations, such as DNA methylation and histone modifications. DNA methylation tends to be associated with transcriptional repression, and a recent study has identified a number of genes that appear to have methylation-sensitive transcription (Zhang et al., 2006). Enrichment analysis of the methylation-sensitive genes in our partitioned data generated a similar distribution pattern to that observed for transcription factors (Table III; Supplemental Table S11), which implies that DNA methylation is a widespread form of transcriptional control throughout developing siliques.
Analysis of Transcription Factors
The Database of Arabidopsis Transcription Factors (DATF [http://datf.cbi.pku.edu.cn/]) includes information about 1,922 transcription factors classified into 64 families (Guo et al., 2005). Our analysis identified differential expression for 943 of these, 187 of which were endosperm preferred (Supplemental Table S12). Furthermore, 71 transcription factors were found to be endosperm preferred and early seed specific (Supplemental Table S12). Table VI highlights the transcription factors that our analysis validates (see below) to be early seed specific, with strong evidence for endosperm-preferred expression from the microarrays. To see if any transcription factor families were overrepresented in the EP and ESS-EP lists, we calculated the frequency of each family in our data (our analysis was limited to the 28 families that had 10 or more members showing differential expression in our array data). However, little evidence for enrichment of particular types of transcription factors was observed (data not shown).
Evidence for Biological Significance of Protein Interactions for MADS Box Transcription Factors Expressed during Proliferative Endosperm Development
Twelve MADS box genes were found in the EP partition and, interestingly, all but one were type I MADS box genes (Table VII ). MADS box proteins often form homodimers and heterodimers, and a comprehensive analysis using yeast two-hybrid technology has identified interactions within the members of the Arabidopsis family (de Folter et al., 2005). By combining these data with our cell type-specific experiments, we can help infer in planta biological significance during endosperm development for the following MADS box transcription factor interactions: AGL45-AGL40, AGL40-PHE2, AGL40-PHE1, PHE2-AGL62, AGL62-PHE1, AGL62-AGL36, PHE1-AGL99, AGL99-AGL78, AGL78-AGL102, AGL86-AGL23, and AGL86-AGL40 (Table VII; Fig. 2 ). We also identified seven MADS box genes that gave strong evidence for endosperm-specific expression at 4 DAP (AGL35, PHE1, PHE2, AGL33, AGL40, AGL62, and AGL91; Table VII; Fig. 2).
Validation of Early Seed and Early Seed-Specific Expression
All of the endosperm-preferred genes discussed in detail as part of our analysis had their expression levels assessed in different plant tissues by qRT-PCR (Fig. 3 ). Samples were taken from leaves, stems, roots, flower buds, whole siliques, and seeds dissected from 4-DAP siliques. All genes detected showed higher expression in the seed sample than in whole siliques, consistent with the original LCM endosperm array data. Data analysis also predicted early seed-specific expression for a number of transcription factors (Table VI). Of the 25 novel candidates in Table VI, only two (At4g23750 and At5g11510) showed significant transcript expression in a nonseed tissue sample (Fig. 3). Both were only additionally expressed in the flower buds, perhaps suggesting prior expression in the male and/or female gametophytes prior to fertilization. At4g00140 was not detected in any conventional samples by qRT-PCR.
Identification of Promoters Driving Expression in the Early Endosperm
GUS reporter constructs were made for a selection of transcription factors to assess their use as markers for early endosperm development (Fig. 4 ). The promoter for At1g65300 (AGL38/PHE2) drove expression during very early embryo and endosperm development but became restricted to the chalazal endosperm region around the late globular stage of embryo development. Expression was also seen in pollen. The At1g49190 (ARR19) promoter was expressed specifically in the chalazal endosperm during globular and early heart stages of seed development, with some evidence of expression in stomatal guard cells of the silique. The At4g21080 (DOF4.5; Yanagisawa, 2002) promoter showed very strong chalaza-specific expression at the globular stage but then became more widespread throughout the endosperm, with the intensity of staining decreasing around the heart stage. The At4g18870 (HSF14; Guo et al., 2008) promoter also had very strong expression in the chalazal endosperm plus strong expression in the peripheral endosperm, but staining was not apparent in the micropylar domain. At around the heart stage, expression became restricted to the chalazal endosperm. Expression was also apparent in the cotyledons of the embryo during the mature green stages of seed development. At5g60440 (AGL62) was expressed specifically throughout the early developing endosperm at low levels, but staining was not apparent from the heart stage (Fig. 4).
Using laser microdissection and microarray analysis, we have obtained the transcriptome of the syncytial endosperm (Day et al., 2007b). Here, we present a detailed analysis of the transcriptome, focusing on the genes differentially expressed in the endosperm compared with other silique tissues. We also identified a subset of approximately 800 of these genes that are specifically expressed in the seed and therefore probably play key roles in seed development. Analysis of our data was consistent with the idea that the syncytial endosperm at 4 DAP is locked into a proliferative state dominated by transcripts associated with the regulation of the cell cycle, DNA processes, chromatin assembly, protein synthesis, cytoskeleton/microtubule-related processes, and cell/organelle biogenesis and organization. In the discussion below, we focus on the biological significance of the endosperm-preferred expression of particular genes involved in the cell cycle, hormone biology, transcriptional regulation, and early endosperm development.
Analysis of Cell Cycle Genes Suggests Roles for Gene Family Members in the Regulation of Syncytial Division
In Arabidopsis, eight mitotic divisions occur during the syncytial phase of development, until there are approximately 200 nuclei (Boisnard-Lorig et al., 2001). Plant syncytial development requires a rapid progression through the cell cycle, suppression of phragmoplast formation, and an uncoupling of cytokinesis from mitosis (Otegui and Staehelin, 2000). To gain further insights into this process, we analyzed our data to identify endosperm-expressed genes that have been implicated in controlling cell cycle progression.
Cyclin-Dependent Kinase/Cyclin Regulation of the Cell Cycle
The components of the cell cycle are largely conserved across the eukaryotes (Mironov et al., 1999). Fluctuations of distinct combinations of cyclin-dependent kinases (CDKs) and cyclins are necessary for progression through the different phases of the cell cycle (Gutierrez, 2005; De Veylder et al., 2007). In Arabidopsis, there are 12 CDKs and at least 49 cyclins divided into nine classes (Vandepoele et al., 2002; Wang et al., 2004). Different CDK/cyclin heterodimer combinations phosphorylate different protein targets, which then coordinate entry into the next phase of the cell cycle. A- and B-type CDKs are the main drivers of the plant cell cycle, and CDKB1;1, CDKB1;2, CDKB2;2, and CDKD1;1 (CDKD3 is just below the 2-fold cutoff) are present in the endosperm-preferred partition and probably play a key role in controlling endosperm proliferation (Table IV).
A most striking aspect of our analysis of core cell cycle genes (Vandepoele et al., 2002) is the predominance of cyclin A and B genes in the endosperm-preferred partition. Five of the seven A-type cyclins (CYCA1;1, CYCA1;2, CYCA2;1, CYCA2;2, and CYCA3;1) and all six of the B-type cyclins (CYCB1;1, CYCB1;2, CYCB1;3, CYCB2;1, CYCB2;3, and CYCB3;1) with differential expression on our arrays were endosperm preferred (Table IV). In contrast to the A and B types, only one of seven D-type cyclins present on the array (CYCD3;3; Vandepoele et al., 2002) was detected in the endosperm-preferred partition. Both A- and B-type cyclins are associated with mitotic cycles, and it has been predicted that cyclin accumulation is part of the overall program responsible for syncytial development (Boisnard-Lorig et al., 2001). The maize (Zea mays) CYCA1 is concentrated in the phragmoplast during cytokinesis (John et al., 2001). The Arabidopsis CYCA1 ortholog is endosperm preferred in our analysis, yet the precise nuclear localization of CYCA1 is not known and formation of the phragmoplast is suppressed during syncytial endosperm development. CYCB1;1 has been studied extensively during early endosperm development (Boisnard-Lorig et al., 2001), and CYCB1;1 to -3 are endosperm preferred in our analysis. It has been shown that a nondegradable form of CYCB1 suppresses phragmoplast formation at the end of each nuclear division (Weingartner et al., 2004). Therefore, studies on the regulation of CYCB1 genes should provide further insights into their possible roles in syncytial endosperm development.
Regulation of CDK/cyclin complexes through the cell cycle is also mediated through inhibitors of cyclin-dependent kinases. In plants, inhibitors of cyclin-dependent kinases are more similar to Kip protein (KRP, for Kip-related protein; De Veylder et al., 2001). There are seven KRPs present in the Arabidopsis genome. The putative CDK inhibitory protein KRP4 is endosperm preferred (Table IV), which is consistent with its reported expression in mitotically active cells (Ormenese et al., 2004). This indicates that genes capable of negatively regulating the cell cycle are also present in the EP partition.
Regulation of G2-M Phase Expression by Myb3R Genes
Proliferating plant cells show periodic transcription of a large portion of their genes (Breyne et al., 2002). In tobacco (Nicotiana tabacum), M phase-specific expression is regulated by Myb3R genes, with Myb3RA1 and Myb3RA2 showing phase-dependent transcription and their proteins binding MSA sequences in the promoter region of their target genes (Ito et al., 1998, 2001). The third tobacco gene, Myb3RB, which is thought to antagonize Myb3RA to control expression of the G2-M phase-specific genes, is expressed at a constant level during the cell cycle and is incapable of activating MSA-containing promoters (Araki et al., 2004). All five Arabidopsis Myb3R genes show evidence of endosperm expression in our data, and AtMyb3R2 and AtMyb3R4 have endosperm-preferred expression, suggesting that they play an important role in the regulation of G2-M phase-specific genes in the syncytial endosperm. Furthermore, transcripts of their putative M phase-specific target genes were enriched in the endosperm. Analysis of reporter constructs during early endosperm development should help elucidate the role of individual AtMyb3R genes, such that phase-dependent expression would indicate an MSA-activating role and phase-independent expression would indicate an inhibitory role.
E2F Transcription Factors
The orderly fluctuation of particular CDK/cyclins is required for progression through the cell cycle. These fluctuations are mediated in part by E2F transcription factors (Otegui and Staehelin, 2000; Kosugi and Ohashi, 2002; Mariconti et al., 2002). Consistent with a pronounced role during early endosperm development, E2F target genes (and upstream E2F-binding motifs) were significantly enriched in the EP and ESS-EP groups, indicating that some E2F-responsive genes have endosperm-specific roles (Table III). All six Arabidopsis E2F transcription factors (E2Fa to E2Fc and DEL1 to DEL3) gave signals consistent with endosperm expression, although data for E2Fb and DEL1 were relatively variable. E2Fa to E2Fc have important roles regulating transcription during the G1-S transition, with E2Fa and E2Fb acting as positive regulators of the cell cycle (De Veylder et al., 2002; Kosugi and Ohashi, 2003; Sozzani et al., 2006), activating targets that are necessary for DNA replication during S phase (Ramirez-Parra et al., 2003; del Pozo et al., 2006), and E2Fc suppressing cell division by acting as a transcriptional repressor (del Pozo et al., 2002; Vandepoele et al., 2005). E2F-DP-retinoblastoma protein complexes bind but do not activate the expression of S phase factor targets until the retinoblastoma protein becomes phosphorylated by a G1-specific cyclin/CDK (Nakagami et al., 2002).
Unlike E2Fa to E2Fc, DEL1 to DEL3 do not interact with DPa and DPb and bind E2F-binding sites in a monomeric form (Kosugi and Ohashi, 2002; De Veylder et al., 2007). The DEL proteins have been shown to be abundant in meristematic cells and were thought to balance the activities of E2Fa/bDPa/b transcription factors by restraining cell proliferation (Ohashi-Ito et al., 2002). DEL3 and DEL2 were present in the endosperm (Table IV), indicating a role for DEL proteins during proliferative syncytial endosperm development, in which repeated rounds of mitosis occur in the absence of cytokinesis and cell wall formation. Indeed, recent data suggest that DEL genes function as important negative regulators of cell differentiation (Ramirez-Parra et al., 2004; Vlieghe et al., 2005), consistent with a role during the syncytial phase of endosperm development. Intriguingly, DEL1 inhibits the endocycle and preserves the mitotic state of proliferating cells, and DEL3 represses transcripts associated with cell wall biosynthesis and expansion (Ramirez-Parra et al., 2004; Vlieghe et al., 2005).
In summary, analysis of the core cell cycle genes and their putative downstream targets reveals genes that appear to play important roles in the proliferation of the endosperm. Further insight into the roles of particular cell cycle genes, such as DEL2 and KRP4, in the endosperm could be obtained using reporter constructs, in situ hybridization, or region-specific endosperm LCM to associate gene expression with either the proliferating or endoreduplicating domains of the endosperm (Boisnard-Lorig et al., 2001).
Analysis of Hormone Markers Indicates an Important Role for CKs in the Proliferating Endosperm
Analysis of hormone-responsive genes showed that only the CK-responsive genes were significantly enriched in the EP partition (Table III). This is consistent with studies in rice (Oryza sativa) and maize that show significant correlations between CK levels and the rate of cell division in the early endosperm (Lur and Setter, 1993; Yang et al., 2002). Moreover, the early endosperm appears to be a site of CK biosynthesis, with major components of the CK biosynthetic pathway (isopententyltransferase genes 4 and 8) being specifically expressed in the chalazal endosperm during early seed development (Miyawaki et al., 2006; Table I).
CK Signaling Genes Are Expressed in the Early Endosperm
The enrichment of CK-responsive genes and the presence of CK biosynthesis genes in the EP partition indicate that CK signaling is important during the early stages of Arabidopsis endosperm development (Table V). CK perception and signaling are similar to two-component phosphorelays in bacteria (Müller and Sheen, 2007). AHK4 shows evidence for endosperm expression in our data and is an example of a sensor His kinase (AHK) that is able to initiate a phosphorelay when bound to CKs. Arabidopsis Histidine-containing Phosphotransfer proteins (AHPs) are subsequently phosphorylated and translocated to the nucleus, where they transfer their phosphate to Arabidopsis type-B Response Regulators (ARRs; Müller and Sheen, 2007). These mediate the transcriptional response to CKs, which includes inducing the primary response genes, the type-A ARRs, which act as negative regulators of the primary signal transduction pathway, likely via interaction with the AHPs (Dortay et al., 2006). AHP genes have functional overlap, with only multiple AHP mutants displaying a reduced sensitivity to CKs. The AHP1-5 quintuple knockout plants show multiple abnormalities in growth and development, including seeds with uneven size and some seed abortion (Hutchison et al., 2006). The average size of nonabortive seeds was about 20% larger than in the wild type, although this might be a consequence of increased maternal resources (de Jong and Scott, 2007). Similar variation in seed development was seen in the AHP2,3,5 triple mutant, which indicated that these AHPs have roles during seed development (Hutchison et al., 2006). Interestingly, of the AHPs in the list of differentially expressed genes, AHP2 and AHP5 are very close to the arbitrary 2-fold cutoff (1.99 and 1.89, respectively; Table V), indicating relatively high expression in the endosperm compared with other silique tissues. This suggests that the reduced seed set and abortion in the triple mutant might be results of aberrant endosperm development.
CK-responsive genes were significantly overrepresented in both the EP partition and the OST partition (Fig. 4), but further analysis suggested a very different response to CK in the endosperm than in the other silique tissues, with CK predominantly activating genes in the EP group and down-regulating genes in the OST group. A differential response to CK in the endosperm compared with other silique tissues was also evident in our analysis of the ARR genes in the EP and OST partitions (Table V). The EP partition included three ARRs (ARR18, ARR19, and ARR21), all of which are B-type ARRs that act as transcriptional activators during CK signaling. In contrast, the majority of A-type ARRs (negative regulators) in our data were distributed in the OST partition and perhaps indicate a more inhibitory regulation of CK signaling in other silique tissues compared with endosperm at 4 DAP.
Recently, a second group of transcription factors, termed the cytokinin response factors (CRFs), were shown to act as part of the CK two-component pathway and to require the action of both the AHK and AHP genes to mediate a CK response (Rashotte et al., 2006). CRFs mediate a large fraction of the CK transcriptional response that functionally overlaps with the B-type ARR response. Five of the six CRFs are present in our data, confirming an important role for this gene family in developing siliques at 4 DAP. CRF5/6 double mutant embryos fail to develop past the early heart stage (Rashotte et al., 2006). This suggests that CRF5 and CRF6 are embryo expressed during early seed development, which is consistent with the presence CRF5 in our OST partition. The four other CRFs (CRF1 to CRF4) are included in our EP partition, indicating a role in early endosperm development (Table V). The roles of CRF1 and CRF3 are unclear, as these genes show little or no response to CK (Rashotte et al., 2006). However, like CRF5, CRF2 is unregulated by CK in a B-type ARR-dependent manner and likely plays an important role in CK-dependent transcriptional activation in the endosperm. CRF2 and the B-type response regulators ARR18, ARR19, and ARR21 are present in our ESS-EP partition, indicating a seed-specific role for these components of CK signaling.
In a study that characterized the expression of B-type ARRs, RT-PCR analysis showed that ARR19 and ARR21 are expressed specifically in silique tissues, although this expression was not detected using promoter-GUS reporter constructs (Mason et al., 2004). However, alternative promoter-GUS constructs (ARR21 in Tiwari et al. [2006] and ARR19 in Fig. 4 of this study) indicate expression in the chalazal endosperm during early endosperm development. Interestingly, overexpression experiments show that ARR21 induces expression of the key CK biosynthesis gene IPT4 (Kiba et al., 2005). As IPT4 is also expressed in the chalazal endosperm (Miyawaki et al., 2004), it seems likely that ARR21 and perhaps ARR19 act to ensure IPT gene expression and therefore CK biosynthesis during early wild-type seed development.
Although our analysis indicates that the chalazal endosperm plays an important role in directing proliferation of the endosperm via CK signaling, the chalazal endosperm does not appear to undergo mitosis and shows evidence of endoreduplication (Boisnard-Lorig et al., 2001). This is contrary to current thinking that high levels of CKs exist in mitotically dividing cells, the suggested sites of de novo CK biosynthesis (Moncaleán et al., 2001; Friml, 2003; Nordstrom et al., 2004; He et al., 2005). Another important function for CKs may be determining cell fate, as a Sinapis alba MADS box gene (Bonhomme et al., 2000) and a maize homolog of Arabidopsis KNAT1, important in the development and maintenance of the shoot apical meristem (Hewelt et al., 2000), are induced by CK treatment.
Auxin Signaling and Auxin-Responsive Genes Are Underrepresented in the Proliferating Endosperm
While our data suggest a primary role for CK signaling during syncytial endosperm development, there is likely to be significant interplay with other phytohormones. It is well documented that the ratio of auxins and CKs plays an important role in controlling tissue proliferation and differentiation. A study of maize endosperm showed CK levels at 9 DAP that corresponded to the maximal cell division rate in the endosperm and that were reduced sharply as auxin levels increased toward the mid to late stages of endosperm development. This is consistent with other studies on grain development, in which CK levels were maximal during early stages and auxin levels reached maximal levels later in seed development (Mengel et al., 1985; Lur and Setter, 1993). Furthermore, auxin has been shown to have a rapid negative control on CK levels by suppressing its biosynthesis (Nordstrom et al., 2004). Our data indicate strong CK-associated and weak auxin-associated transcriptional responses in the syncytial endosperm at 4 DAP (Table III), along with significant underrepresentation of auxin signaling genes in the EP partition. A hypothesis that is consistent with these observations is that at 4 DAP of endosperm development, auxin levels are low, enabling increased CK biosynthesis and high rates of cell division; subsequent increases in auxin might then promote endosperm cellularization.
The Roles of Other Hormones during Endosperm Development Are Indicated through the Representation of Ethylene-, ABA-, and GA-Related Genes and the Underrepresentation of Brassinosteroid-Responsive Genes
The distribution of both ACC- and ABA-responsive genes indicates that both ethylene and ABA signaling systems are active during early silique development and have roles during endosperm development (Table III; Supplemental Table S9). In a recent study, Yang et al. (2006) made direct comparisons of both ethylene and ABA levels with endosperm cell division in spikelets of rice. They concluded that cell division and grain-filling rates were positively associated with ABA and negatively associated with ethylene. However, during the earliest stages of endosperm development (0–12 DPA), their data show that ethylene evolution rates and ACC contents of the rice spikelets were at their highest. Conversely, ABA levels were low at anthesis and slowly increased to peak values at 12 DAP or later, depending on the cultivar. These observations are consistent with the idea that during the early syncytial stage of development (0–4 d in rice) ethylene levels are high, which may inhibit ABA biosynthesis (Ghassemian et al., 2000; del Pozo et al., 2005). It remains to be elucidated whether ethylene and ABA have an antagonistic effect during early endosperm development. The finding that the NCED6 gene, which cleaves cis-epoxycarotenoids in the first step of ABA biosynthesis, has early endosperm-specific expression suggests an important role for ABA synthesis during the proliferative stage of endosperm development in Arabidopsis (Table I; Lefebvre et al., 2006).
The other hormones, BL, MJ, and GA, play important roles in various aspects of plant development (del Pozo et al., 2005), and our silique/endosperm data would suggest a stronger influence of GA on transcription in the endosperm than of either BL and MJ (Table III; Supplemental Table S9). This is consistent with observations that jasmonic acid has a negative effect on G1/S and G2/M transitions (Świątek et al., 2002) and that GAs stimulate cell division (Fabian et al., 2000). However, the underrepresentation of BL-responsive genes was unexpected, since BLs have been shown to promote cell division and are able to up-regulate the expression of CYCD3;1 and CDKB1;1 (Yoshizumi et al., 1999; Hu et al., 2000). It is possible that the underrepresentation of BL-responsive genes in the endosperm is in some way related to the underrepresentation of auxin genes, since auxin and BLs can cooperate to promote organ growth via cell cycle activation (Bao et al., 2004).
Identification of Putative Early Seed-Specific Transcription Factors with Endosperm-Preferred Expression
Seventy-one transcription factors were found to be endosperm preferred and early seed specific. These genes likely play important roles within the endosperm, and representative genes were further characterized using promoter-GUS reporter lines (Fig. 4). This both implicates the genes (At1g65300, At1g49190, At4g21080, At4g18870, and At5g60440) as being important during proliferative endosperm development and also provides tools to misexpress genes that may promote endosperm proliferation as a rational approach to generate larger seeds.
Analysis Suggests Important Roles for Type-1 MADS Box Genes during Endosperm Development
A family of transcription factors that includes key regulators of proliferation of the syncytial endosperm in Arabidopsis are the MADS box genes. The best-known MADS box transcription factors have roles in flower development and flowering time and are classified as type II MADS box genes. However, although more than half of the MADS box transcription factors are type I, little is known about their roles in plant development (Parenicová et al., 2003). Of the 12 MADS box genes in the endosperm-preferred list, 11 are type I (Table VII). Recently, AGL23 was shown to play a role in female gametophyte and embryo development (Colombo, 2008). AGL23-GUS lines show that AGL23 is expressed throughout female gametophyte development and in the developing peripheral and chalazal endosperm, consistent with our expression data. Our ability to see biological significance in the interactions predicted by de Folter et al. (2005) using yeast two-hybrid analysis will guide future studies of type I MADS box genes and help elucidate their roles in endosperm development. For example, of the seven MADS box genes that are strongly endosperm preferred at 4 DAP, only four interact with other endosperm-preferred MADS box genes. Analysis of the MADS box interactome indicates that type I heterodimer formation requires a member of the Mα subclass (de Folter et al., 2005). Of the four endosperm-specific MADS box genes that can form dimers, two are Mα subclass genes (AGL40 and AGL62) and the other two are Mγ subclass genes (PHE1 and PHE2). AGL40 and AGL62 do not form heterodimers together in yeast, but both PHE1 and PHE2 can form heterodimers with both AGL40 and AGL62 (Fig. 2).
Since a prolonged syncytial growth pattern is limited to early endosperm development in Arabidopsis, it follows that key developmental switches that define syncytial competence are also early seed specific. This is the case for AGL62, which is required for normal syncytial endosperm development, since disruption of the AGL62 gene results in very early cellularization of the endosperm, approximately 24 h after fertilization (Kang et al., 2008). Both the Drews laboratory AGL62-GFP reporter line (Kang et al., 2008) and the AGL62-GUS reporter presented in this study confirm that AGL62 expression is specific to the syncytial endosperm. AGL62 interacts in vitro with many seed-expressed MADS box genes that may help mediate its ability to enable syncytial proliferation (which requires inhibition of cytokinesis and cell wall formation). As mentioned, AGL62 can form a heterodimer with PHE1, which has also been implicated as a positive regulator of endosperm proliferation and has endosperm-specific expression at 4 DAP (Köhler et al., 2003; this study). PHE2 has 72% homology to PHE1 at the amino acid level and has been reported to have a very similar expression pattern, although no data were provided to substantiate this claim (Köhler et al., 2003). Our PHE2 promoter-GUS analysis confirms that PHE2 expression is largely equivalent to PHE1 expression during wild-type seed development. Transcription of both AGL62 and PHE1 appears to be regulated by members of the fertilization-independent seed-Polycomb (FIS-PcG) complex, since levels of both AGL62 and PHE1 are increased and persist longer in FIS than during wild-type seed development (Köhler et al., 2003). Developing seeds of the FIS class mutant mea abort during heart stage, which coincides with an overproliferated endosperm. Abortion of mea seeds is partly due to abnormally high levels of PHE1, since reducing PHE1 transcript in mea using an antisense construct can rescue the abortion phenotype. Like PHE1, PHE2 expression was also reported to be increased in mea seeds, which is suggestive of similar regulation and perhaps a redundant function. Alternatively, PHE2 may be an antagonist of PHE1 that competes to form heterodimers with AGL62, thus acting to reduce the rate of syncytial proliferation. Our data also predict that the other endosperm-specific Mα MADS box gene AGL40 plays an important role in the molecular control circuit that enables syncytial proliferation of the early endosperm, perhaps by mediating the levels of PHE1 and/or PHE2 proteins available to form heterodimers with AGL62.
Genes Involved in Important Early Developmental Processes Are Expressed at 4 DAP
Pagnussat et al. (2005) identified 130 mutants in an extensive screen for female gametophytic mutants, some of which had aberrant endosperm. Sixty-one of the genes identified as having female gametophytic phenotypes are present in our differentially expressed gene list at 4 DAP. Eleven are in the EP partition, suggesting an important role during the proliferative stage of endosperm development at 4 DAP, as well as their prior roles in enabling fertilization (At5g02100), embryo sac development (At4g14790 and At2g35950), fusion of the polar nuclei (At2g20490, At1g72440, and At4g00140), and very early embryo and endosperm formation (At2g15890, At4g13380, At2g34880, and At4g01560).
Seedgenes.org (http://www.seedgenes.org/index.html), a database of Arabidopsis seed mutants, contains six genes described as having female gametophytic inheritance patterns (in which siliques produce approximately 50% mutant seeds following pollination of heterozygotes, regardless of pollen genotype; Tzafrir et al., 2003). These include the components of the FIS-PcG complex MEA, FIS2, FIE, and MSI1 and the DNA glycosylase DME. The sixth gene, EMB2220 (At5g12840), is not early seed specific but is approximately 2-fold enriched in our endosperm sample, similar to FIE (which is also not seed specific). EMB2220 mutants have not been fully characterized, but it is tempting to speculate that this gene is also involved in endosperm development.
The FIS-PcG complex is involved in repressing seed development prior to fertilization, the formation of distinct mitotic domains during syncytial endosperm development, and the timing of endosperm cellularization (Köhler and Makarevich, 2006). Histone modifications via the FIS-PcG complex and DNA methylation via the DNA methylase MET1 have been shown to be important components of allele-specific repression of imprinted genes in Arabidopsis (Feil and Berger, 2007). The DNA glycosylase DME is involved in activation of the maternal allele of the imprinted genes MEA, FWA, and FIS2 (Feil and Berger, 2007). This parent of origin-dependent differential repression and activation of alleles appears to be limited to the endosperm in plants (Feil and Berger, 2007). Concordantly, all confirmed imprinted genes in Arabidopsis (i.e. PHE1, MEA, FIS2, and FWA [Luo et al., 2000; Kinoshita et al., 2004; Köhler et al., 2005]) are present in our EP partition. Therefore, it is likely that our EP partition is enriched for imprinted genes and provides a short list for identifying new members of this gene family.
The use of laser-assisted microdissection technology has enabled the isolation and high-resolution transcript analysis of early endosperm. This, and work by Casson et al. (2005), Day et al. (2007b), Le et al. (2007), and Spencer et al. (2007), demonstrates the power of laser-assisted microdissection for studying tissue-specific expression during early seed development, even in Arabidopsis, which has relatively small seeds. The proliferative stage of endosperm development is unusual in that rapid nuclear division occurs in the absence of cell division. Since the proliferative stage of endosperm development is important for determining final seed size, understanding the processes that control endosperm proliferation should lead to novel ways of improving seed yield. The data generated provide a useful resource enabling novel insight into this process, as illustrated by the examples discussed here, and provide a starting point for further study.
LCM Endosperm Microarray Data
The LCM endosperm microarray data analyzed in this study have been described (Day et al., 2007b) and deposited at the National Center for Biotechnology Information gene expression omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession number GSE6703.
Preparation of qRT-PCR cDNA Template and PCR Cycling Conditions for Analysis of LCM-Derived Samples
Total RNA was obtained from LCM-dissected endosperm as described (Day et al., 2007b), and total RNA was obtained using the Picopure RNA isolation kit (Arcturus) with an optional on-column DNase step (Qiagen). The purified total RNA was quantified using the Ribogreen RNA quantification kit (Invitrogen) according to the manufacturer's instructions. IVT-based amplifications were carried out using the Message Amp II kit (Ambion) following the manufacturer's instructions. Comparisons between the ability of qRT-PCR and the microarrays to measure differential expression of 17 genes in the same samples used cDNA made from amplified RNA (aRNA) generated using one round of IVT. The remaining first-round aRNA was then used as the basis for a second round of IVT, which generated target for the microarray study. For the qRT-PCR, the aRNA was primed with random hexamers and first-strand cDNA was synthesized using SuperScript III (Invitrogen) according to the manufacturer's instructions. Real-time qRT-PCR was carried out using reagents from the LightCycler FastStart DNA MasterPlus SYBR Green I kit (Roche) in 20-μL volumes using a LightCycler 1.0 (Roche). The amplification conditions for qPCR were as follows: denaturation at 95°C for 10 min; cycling at 94°C for 5 s, 58°C for 17 s, and 72°C for 10 s (single acquire); melting at 95°C for 0 s, 55°C for 20 s, and 95°C for 0 s, with ramp at 0.2°C s−1 (continuous acquire); and cooling at 40°C for 20 s. Reaction products were confirmed by melting curve analysis and by running out the product on a 1.2% agarose gel. The primers used for qRT-PCR are listed in Supplemental Table S13.
Preparation of qRT-PCR cDNA Template, and PCR Cycling Conditions for Analysis of Conventional Fresh Tissue Samples
RNA was extracted from fresh Arabidopsis (Arabidopsis thaliana) tissues using the Qiagen Plant RNeasy kit as per the manufacturer's instructions with some alterations for the seed samples. Disruption of the silique, stem, leaf, root, and flower bud tissues was carried out by harvest into 1.5-mL Eppendorf tubes and flash freezing in liquid nitrogen. Tissues were then quickly ground to a powder in Eppendorf tubes using a precooled plastic pestle on dry ice. RNA extraction reagent was added before the samples thawed. For the dissected seed samples, developing siliques were removed from plants using tweezers and cut open using a dissecting microscope with a hypodermic needle, being careful not to damage the seed within. The majority of seeds were scraped onto the back of the needle and deposited into a precooled Eppendorf tube on dry ice. Frozen seeds were transferred to precooled plastic bags embedded in dry ice, and RNA extraction reagent was pipetted into the frozen bag and allowed to thaw. Individual developing seeds (visualized through the plastic using a dissecting microscope) were completely disrupted using pressure from the tip of blunt tweezers and used as input for the Plant RNeasy kit.
RNA was quantified using a Nanodrop spectrophotometer, and cDNA was generated using the VILO cDNA synthesis kit (Invitrogen) according to the manufacturer's instructions. Real-time qRT-PCR was carried out using reagents from the Express SYBR Green ER MasterMix kit (Invitrogen) in 7-μL volumes using a LightCycler 480 (Roche). The amplification conditions for qPCR were as follows: denaturation at 95°C for 10 min; cycling at 94°C for 5 s, 61°C or 58°C for 17 s, and 72°C for 10 s (single acquire); melting at 95°C for 0 s, 55°C for 20 s, and 95°C for 0 s, with ramp at 0.2°C s−1 (continuous acquire); and cooling at 40°C for 20 s. Reaction products were confirmed by melting curve analysis and by 1.2% agarose gel electrophoresis. The primers used for qRT-PCR are listed in Supplemental Table S13 and include primers designed by Han and Kim (2006).
Identification of Genes with Evidence of Early Silique/Seed-Specific Expression
We selected the top approximately 3,200 genes that reported preferred endosperm expression in our array data to manually search the Arabidopsis MPSS database. This database contains libraries of signatures generated from several tissue types, each containing approximately 2 million signature tags (Meyers et al., 2004). These libraries are normalized so that transcript abundance can be compared between libraries. At the time of query, the database could be searched using several AGI locus identifiers per search. The resulting output was used to create a summary file that was imported into The Institute for Genomic Research Multiexperiment Viewer (MeV) 3.0 software and searched using pattern matching (Pavlidis and Noble, 2001).
To identify genes with early endosperm-specific expression, we used the known marker genes FIS2, FWA, PHE1, and SUC5 (Luo et al., 2000; Kinoshita et al., 2004; Baud et al., 2005; Köhler et al., 2005) as bait to search the AtGenExpress developmental series (Arabidopsis transcript profiles from different organs and at different stages of development were determined using Affymetrix GeneChip technology) using the Expression Angler (Bio-Array Resource for Arabidopsis Functional Genomics [BAR]; http://bar.utoronto.ca/). The software calculates the similarity of expression patterns to the marker genes for the other genes in the database using a Pearson correlation coefficient (r value). FIS2, FWA, and PHE1 had very few genes correlated with their expression patterns, probably caused by ill-defined expression patterns. In contrast, SUC5, which has strong endosperm-preferred expression during the proliferative stages of development (Baud et al., 2005), gave a well-defined pattern in the AtGenExpress seed data. The SUC5 expression template was used to identify genes similarly expressed during early seed development in the AtGenExpress data. This list was then entered into the BAR DataMetaFormatter tool (http://bar.utoronto.ca/) to create a heat map of probe intensities for genes in the AtGenExpress seed development series.
The GHL Affymetrix GeneChip data sets were downloaded from the National Center for Biotechnology Information gene expression omnibus (http://www.ncbi.nlm.nih.gov/geo/), imported into The Institute for Genomic Research MeV software, and screened using the pattern-matching function to identify those genes with similar expression patterns to the early endosperm markers SUC5, PHE1, FWA, and FIS2 (Supplemental Table S2). Pattern-matched genes for all markers were combined and cross-referenced to genes with differential expression on our arrays. This ESS subgroup was used in subsequent analysis due to its enhanced sensitivity toward known endosperm markers. For example, Supplemental Figure S2 shows a heat map of normalized intensity data from both the GHL GeneChip data and the AtGenExpress seed development series. Negligible signal was apparent for the known endosperm markers PHE1, IPT8, FWA, FIS2, and MEA in the AtGenExpress data, whereas expression of all of these markers was easily apparent in the GHL data. This likely reflects the fact that the GHL data were derived from seeds removed from the surrounding silique tissues, whereas the samples representing early seed development in the AtGenExpress data included the whole silique.
Other Data Processing and Analysis
The BAR (http://bar.utoronto.ca/) duplicate remover, DataMetaFormatter, Expression Angler, and Arabidopsis Interactions Viewer (Toufighi et al., 2005) were used for list processing, heat map generation, pattern matching, and interaction plot generation, respectively. TAIR (http://www.Arabidopsis.org/) was used to gather gene annotation information and to assemble some gene lists. Hormone-responsive genes were obtained from Nemhauser et al. (2006). Arabidopsis transcription factors were obtained from DATF (http://datf.cbi.pku.edu.cn/). Core cell cycle genes were obtained from TAIR (Vandepoele et al., 2002), and E2F-regulated genes were obtained from Vandepoele et al. (2005). We assessed the representation of E2F target genes in our data using a list of genes shown (1) to be induced by ectopic expression of E2Fa and DPa transcription factors, (2) to contain a distinctive cis-acting element in their promoters (E2F box), and (3) to have rice orthologs that also have promoters containing an E2F box (Vandepoele et al., 2005). Arabidopsis genes that contain the MSA sequence in the 500 bp upstream of the ATG were identified using the Patmatch tool from the TAIR Web site (Garcia-Hernandez et al., 2002). The Athena promoter analysis database (http://www.bioinformatics2.wsu.edu/cgi-bin/Athena/cgi/home.pl; O'Connor et al., 2005) was used to assess for enrichment of E2F-binding motifs in the 500-bp upstream regions of our EP and ESS-EP partitions.
Genes with methylation-sensitive transcription were obtained from Zhang et al. (2006). Chromatin-related genes were obtained from ChromDB (http://www.chromdb.org/). In some instances, a particular locus was represented by more than one probe on our array. In a few cases, different probes for the same locus were observed in different partitions, since cutoff values were based on a specific signal ratio. For subsequent analysis, therefore, we included the locus identifier in both partitions.
TAIR Gene Ontology (TAIR-GO) and MIPS Functional Category (MIPS-FunCat) enrichment analyses were performed using the Virtual Plant 0.9 Web site (http://virtualplant-prod.bio.nyu.edu/cgi-bin/virtualplant.cgi). This enabled us to calculate the frequency of genes with a particular TAIR-GO or MIPS-FunCat as a percentage of the whole genome. If a subgroup of genes contains significantly different proportions of a GO or FunCat, biological importance can be inferred. To assess the presence of inherent bias in the GO or FunCat groups represented by the probes on our array platform, we queried the whole genome with the list of loci on the array. This analysis revealed that several GO and FunCat groups were already overrepresented on our arrays (data not shown). To get an accurate assessment of the representation of biological processes in our partitioned data, therefore, we used our array list as the background population. t tests were carried out in MeV with no correction for multiple testing.
Construction and Analysis of GUS Reporter Lines
Five promoters were selected to validate the endosperm-preferred expression of the downstream genes predicted by the LCM endosperm microarray data. The genomic regions between the 2,356, 2,089, 1,110, and 582 nucleotide positions upstream of the translational start codons were amplified by PCR for At1g65300/PHE2, At1g49190/ARR19, At4g21080, and At4g18870, respectively. These fragments were cloned upstream of the uidA gene in the binary vector pCAMBIA1391-Z (CAMBIA) and transformed into Agrobacterium tumefaciens strain GV3101. The genomic region between −1,453 and +787 of At5g60440/AGL62 (relative to the translational start codon) was amplified and cloned in frame upstream of the uidA gene in the binary vector pCAMBIA1381xc (CAMBIA) and transformed into A. tumefaciens strain LBA4404. A list of the primers used is provided in Supplemental Table S13. Arabidopsis was transformed using the standard floral dipping protocol (Clough and Bent, 1998). Developing seeds from hygromycin-resistant primary transformants were assessed for GUS activity essentially following Stangeland and Salehian (2002). Briefly, fruits at various stages of development were dissected and placed in GUS staining buffer (50 mm phosphate buffer [pH 7.2], 0.5 mm potassium ferricyanide/ferrocyanide, and 1 mg mL−1 5-bromo-4-chloro-3-indolyl-β-glucuronic acid) overnight at 37°C. Younger fruits were cleared in Hoyer's medium (100 g chloral hydrate in 30 mL of water) for 2 to 4 h. Older fruits were placed in 3:1 ethanol:acetic acid for 4 to 8 h and cleared overnight in Hoyer's medium. Developing seeds were fully dissected from the fruits and mounted in Hoyer's mounting medium with a coverslip. Prepared slides were viewed using differential interference contrast optics on an Olympus BX51 microscope equipped with an Optronics Magnafire 2.1A digital imaging system.
The microarray data discussed in this article can be found in the National Center for Biotechnology Information gene expression omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession number GSE6703.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Heat map showing endosperm-preferred genes in MPSS data.
Supplemental Figure S2. Heat maps comparing endosperm marker detection in online data sets.
Supplemental Table S1. Genes with AtGenExpress expression profiles correlating with the early endosperm-specific gene SUC5.
Supplemental Table S2. List of putative ESS genes from the GHL data.
Supplemental Table S3. AGI list for gene partitions.
Supplemental Tables S4 to S7. Frequency of genes within a particular TAIR-GO or MIPS-FunCat from the different partitions.
Supplemental Table S8. Representations of putative M phase-specific and E2F target genes within the different partitions.
Supplemental Table S9. List of hormone-responsive genes in each partition.
Supplemental Table S10. List of chromatin-related genes (obtained from ChromDB; http://www.chromdb.org/) in each partition.
Supplemental Table S11. List of genes affected by reduced methylation in each partition.
Supplemental Table S12. List of transcription factor genes in each partition.
Supplemental Table S13. Sequences of the primers used for qRT-PCR and to generate the genomic fragments for the GUS constructs.
We thank Peter Stockwell for formatting MPSS data for pattern matching and R. Kaji for making the AGL62-GUS plants.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Richard C. Macknight (richard.macknight{at}otago.ac.nz).
↵[C] Some figures in this article are displayed in color online but in black and white in the print edition.
↵[OA] Open Access articles can be viewed online without a subscription.
↵[W] The online version of this article contains Web-only data.
- Received August 19, 2008.
- Accepted October 6, 2008.
- Published October 15, 2008.


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Hale Kamaole
Hale Kamaole 268
$192 - $270 / night
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QTTX (sleeps 6)
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Hale Kamaole 268
===The Beach === This condo is less than 300 steps to Kamaole Park III Beach, one of the most beautiful beaches in the world (gentle surf, breathtaking beauty, full-time lifeguards). Yelp gives it 5/5 stars. Excellent for swimming & snorkeling or just relaxing while the sun warms your skin. === The Condo === This 1,100 sq. ft. condo features 2 bedrooms (1 Queen and 2 twins) with ample closet space and 2 bathrooms along with a large living & dining area with a lofty cathedral ceiling that provides wide spacious effects three-dimensionally and a fully equipped kitchen. There is a queen-sized pull out bed couch in the living area. This condo is strategically located at the closest end to the beach in Building 7 and the lanai has two open sides. As a result, there is no other unit blocking your view thus enabling you to enjoy an unobstructed wide ocean view when you look toward the ocean. You can enjoy a majestic mountain view as well. Building 7 is the second closest building to the beach in the complex. As a result, this unit is quiet and private unaffected by noise from the street despite the closeness to the beach unlike building 1 or 8 which are right along the road and often affected by traffic noise. The lanai, with two open sides, provides you a spectacular unobstructed view toward the ocean as well as toward Haleakala Mountain in serenity since our condo is located at the closest end to the beach in building 7 with no obstacle in front toward ocean and mountain as well, unlike the overwhelming majority of other units where the view toward the ocean is significantly blocked by other units in front or those with lanai having only one open side. The ocean view from our lanai is wide and spectacular, unobstructed and one of the best, the best in my opinion, in the complex. You can enjoy a great sunset view and sunrise view from our lanai in serenity. Furthermore, this condo is located just in front of the pool and BBQ facility and thus it is very convenient for the family. It is in the best location in the complex for all purposes. This condo is located on the south side (Wailea side) of the complex and thus the lanai is in the shade nearly all day year-round enabling you to enjoy relaxing afternoon on the lanai indulging in majestic ocean view in serenity, unlike those units on the northern side (Kihei side) of the complex (Building 1,2,3,4) where the lanais are directly exposed to strong tropical afternoon sunlight. The unit was updated with new furniture, flat-screen TVs, free WiFi, and more. The kitchen is fully equipped, cable television on each level, ceiling fan in every room and a/c in each bedroom. A new low back-friendly mattress was installed in all beds along with a new bed cover, the queen-sized one in January 2018. In June 2014 both bathrooms were entirely updated and modernized through a complete renovation. In June 2018, a stove/oven unit, a refrigerator, and a downstairs air conditioner were renewed. In February 2019 new washer & dryer were installed. Guests are allowed to use our beach equipment, boogie boards, snorkel gears, etc. kept in the lanai storage. Since our unit is in Building 7 which is on the southern side (Wailea side) of the complex, front door parking is guaranteed unlike those in Building 1, 2, 3 and 4 on the northern side ( Kihei side) of the complex where such is impossible because there are more numbers of units than the front door parking space. After all, our unit is in the best location in the complex for all purposes; closeness to the beach, unobstructed majestic views from the lanai in the shade and serenity, closeness to the pool and BBQ facility, front door parking, etc.. You will find everything working and in good condition; no surprises. You can expect a stress-free, relaxing sun-soaked vacation in this recently renovated and updated comfortable place. This is a NON SMOKING unit. While pets can be an important part of your family, unfortunately, no pets are allowed. === The Complex === Not only is this condo close to the beach, but it is also located in the beautifully landscaped Hale Kamaole Complex, an 8-acre facility just a few minutes north of Wailea on Maui's sunny South Shore. This complex is located in a very desirable part of Maui, southern end of Kihei, just across the street from the beach of Kamaole Park III. It is within 1.5 miles of world-renowned resorts such as the Grand Wailea, A Waldorf Astoria Resort and the Four Seasons Maui – Wailea. Our complex has 2 pools (with beautiful views); community grills, tennis court, laundry facilities, tour desk, and relaxing and very spacious landscapes with various tropical plants such as palm trees, banana trees, papaya trees, plumeria, and hibiscus, etc. === Nearby === Within a minute walk, you are at the beach of Kamaole Park III. A scenic 5-15 minute walk along the South Kihei Road will take you to various shops and eateries ranging from ordinary restaurants to fancy scenic beachfront restaurants. The boat ramp for tours to Molokini and many diving, snorkeling, and whale watching charters is a 3 minutes walk. Wailea golf courses (Blue, Emerald, and Gold) are 1.5 miles away. === The Town of Kihei === Kihei is just North (and next to) of Wailea, a well-known tourist destination with luxury hotels, shopping, and great golf courses. It is about a 20-minute drive from Kahului Airport. The town of Kihei has a variety of shopping and grocery stores and eateries. Kihei- Wailea area is known to have a predictably sunny and dry climate without evening shower which is typical to many other parts of the island. Here you will find a relaxed lifestyle, beautiful beaches, great diving, snorkeling, local golf, and memorable sunsets. Thank you for inquiring about our home away home in Maui for your vacation. If you have any concerns or questions, please send us an email or call us. (HI license number: 207-836-3648-01) Registration Number: TA-207-836-3648-01, GE-20
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Warmly welcome to visit Shanghai Lulu from China!
Shanghai LULU Toy Car is a manufacture specialized in the research, development and production of walking animal ride car. Animal scctoter, animal train and kiddy rides. We rae located in shanghai. With convenient transportation access. All of our products comply with international quality standards and are greatly appreciated in a variety of different markets throughout the world.
If you are intested in any of our products or would like to discuss a custom order, please feel free to contact us.


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Intergenerational family communication about mammography
Publication Details
Browne, J. L. & Chan, A. YC. (2007). Intergenerational family communication about mammography. In M. Albion & P. Collins (Eds.), Education, Employment and Everything: The Triple Layers of a Woman's Life: Refereed Proceedings of the International Women's Conference (pp. 55-59). Toowoomba, QLD: USQ Women's Network Inc.
In Australia, almost 50% of target women (aged between 50-69 years) do not adhere to the breast screening guidelines that make early detection possible. This study explores the efficacy of everyday family communication as a mode of mammography promotion to target women. Upward communication (communication from the younger generations up to the older generations) was the focus of this study. Young women (mean age = 21;05, N = 60) participated in a two-stage questionnaire study. During Stage One they reported their family history of breast cancer and any previous conversations they had with an older female family member about mammography. Intention to engage in a future conversation about mammography with a female relative was measured, and participants were encouraged to initiate such a conversation in the future. At Stage Two, participants reported whether or not they had initiated a conversation, and their perceived outcome of the conversation. Although initial intention scores were high, only 35% initiated a conversation. Intention was the only independent variable that significantly predicted behaviour. However, intentions best predicted the behaviour of those that failed to initiate a conversation. Perceived outcomes of the conversation included the female relative being more likely to have a mammogram, and the participant being more likely to seek further information about mammography. These results suggest that intergenerational family communication about mammography is a viable promotion tool, but young women may require additional support to convert intention into behaviour.


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#SummerofFun is back for 2019 and has so much on offer for all the family. With adults catered for as carefully as the kids, there will never be a dull moment. Here is an A-Z of some of the things you can expect...
- Air rifle shooting - Shoot over a distance of 10 metres from a standing position to static targets (aged 13 and over)
- Bucking Rodeo Bull – yee-ha! A ride to remember as you buck, spin and twist, and see how long you can hold on. It is guaranteed to make everybody watching laugh-out-loud, as you try and hold on to the spinning, jumping and bucking bull for as long as you can. A fall is almost guaranteed as it gets faster!
- Cliffhanger – race against the clock to see who can make it to the top of the climbing wall first
- Dance – put your best foot forward for dance classes, designed for all ages and abilities
- Entertainment every night – In the evenings take a seat in the Atlas Theatre as we travel around the world, courtesy of Potters Theatre Company, who will be here all Summer performing their dazzling NEW show, Broadway & Beyond. Plus join us in the Terrace Bar afterwards for live music and late night entertainment
- Football coaching – ex-Leeds and England football star Paul Reaney will be on hand to coach your little ones in the beautiful game
- Greatest Show workshop - learn a song and dance routine in the workshop and perform it that night live on stage
- Heights – swing to great heights and have a go on our 16 meter outdoor thrill ride, The Giant's Swing
- Inclusive Value – with pretty much EVERYTHING included in the price of your break, you and your wallet can relax
- Jam-packed schedule – there's so much to do all day long at Potters and you'll receive a programme for each day on arrival. What will you do first?
- Kids' Clubs – are you a Bouncing Boo, a Bamboozle, an Inbetweenie or part of the T Club? Up to four Kids' Clubs run on each family break
- Laugh out Loud – the comedy team, including Mark Brewer, are here to entertain you all Summer with their antics
- Midnight Dish – enjoy a delicious late night snack to keep you dancing all night long
- New Age Kurling – have a go at this fun sport which requires you to deliver stones to the target
- Open mic – there's lots of opportunities for your voice to be heard - just let Mark Brewer know if you want to get on stage and show off your talents!
- Potters Summer Fayre – have a go at classic games such as hook a duck, darts and hoopla
- Quizzes - take part in a range of quizzes, from TV themes to general knowledge
- Rally Karts – enjoy the thrill of the race as you speed around the track. Who will get the fastest lap?
- Sweeper – jump as quickly as you can as you attempt to beat the opposition as it rotates faster and faster. Fast, fun and perfect for all the family to take on, mayhem is guaranteed as you work hard to keep your balance.
- Treasure Hunt – can you solve Buzby's clues to crack the code and open the safe first?
- Unwind in our sports and leisure facilities, including a 10-seater luxury whirlpool. Pure bliss!
- Very small person - Little ones will love having a go on Pony Cycles, Monkey Bikes and making a splash in the baby pool featuring a mountain fountain and bubble pad. There's something for everyone on #SummerofFun
- Whack a mole - be a human mole on our new inflatable challenge
- Xtreme Segways – indoors or out, it's the most fun you can have on two wheels
- You – Our Summer breaks are built with all your family in mind, plus grandparents, teenagers, and aunties and uncles
- Zumba – shake your hips with this fun and energetic class
This in addition to everything you loved last Summer, including alfresco music on the Family Green, Challenge Us, where you take on Potters staff, Laser Clays, Segways, and so much more. And don't forget our special family GameZone area, featuring a soft play area, family-friendly gaming machines, an iPad bar and four Ten Pin bowling lanes.
With all this, a stunning coastline, and food and drink to cater for every taste, get ready to join us for an amazing family Summer break. We can't wait!


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After the fatal shootings at a Colorado Springs Planned Parenthood clinic on Nov. 27, the model Chrissy Teigen responded almost immediately. A growing activist who has donated food for protesters in Ferguson, Mo., and verbally sparred with Donald J. Trump, she told her million-plus followers on Twitter that every time such incidents happen, "John and I immediately make another donation to Planned Parenthood," referring to her husband, the singer John Legend, who then tweeted out his support of his wife's stance to his 7.4 million followers.
Her tweets quickly drew praise from others horrified by the shootings, with some replying "Amen" or the like. But they also created a backlash, with others disagreeing with Ms. Teigen in the social medium's special argot ("all your brains must be elsewhere in your body! #PlannedParenthood #idiots #MoronAlert #nobrains.")
Ms. Teigen, who is pregnant, followed that up with another post: "Extremely fine with any unfollows. Would prefer it over your ignorant rants but I'm prepared either way. Nothing I haven't seen before." And to someone who suggested that her money might be better spent in a donation to the Colorado Springs Police Department, she replied, "It might seem insane, but it's possible to support two things at once. I suggest you do the same: policefoundationofcoloradosprings.com/donate."
Many other celebrities, mostly female, were quick to voice support for reproductive rights, often using the hashtag #StandwithPP.
Eric Ferrero, vice president for communications at Planned Parenthood, said that the organization had tracked over 33,000 users posting #StandwithPP or #IStandwithPP on Twitter on Friday and over 50,000 since that day.
The actress Kerry Washington, whose "Scandal" character, Olivia Pope, recently had an abortion, tweeted an emoji of praying hands with the message "Prayers to #ColoradoSprings & #PlannedParenthood." At least one follower was dismayed, replying: "Prayers for Planned Parenthood? What is the matter with u. Does being a celebrity make you godless."
The comedian Jenny Slate ("This tragedy&violence wont stop the fight for affordable health care for women&my right to make my own choices for my body"), the comedian Kathy Griffin ("I went 2 PlannedParenthood 4 my yearly exam because I was too poor to afford insurance") and the actress and director Elizabeth Banks also all shared their support with followers.
Lena Dunham, who stopped using Twitter because "It truly wasn't a safe space for me," as she said on a Re/code Decode podcast, showed her support on Instagram instead, which has no character limit on posts,; as did the actress Elisabeth Moss.
Rose McGowan, the outspoken actress and director, posted a photo of herself on Nov. 30 with a shaved head and a raised fist that read "#istandwithpp #womensrights #ourchoice if you don't like it, leave, but ask yourself if you're willing to help a woman with her lifelong medical care like they are." Of the more than 200 comments, many of her followers used an epithet to emphasize their agreement, others simply responded with a flexed arm emoji.
Connie Britton, the actress who played Tami Taylor on the television show "Friday Night Lights," has a history of working with Planned Parenthood. She collaborated with it in 2013 for a "WWTD? What Would Tami Taylor Do?" T-shirt in support of Wendy Davis, a member of the Texas Senate, who became known that year for leading a filibuster of legislation restricting abortion rights.
When asked how she planned to respond to the recent shootings, Ms. Britton wrote in an email: "I'm going to do what I do every time I see a woman or a man attacked in this country, every time I see a basic freedom like health care attacked in this country ... I am going to use my voice, and encourage those who are fighting for those freedoms to use their voices, to remember who we are as a nation. To value our freedom and our rights to equality and justice first and foremost, without fear of hatred, prejudice and violence.
"Which is, I believe, the American way."
Though large brands refrained from weighing in, some independent players in the fashion world were also eager to show support for Planned Parenthood.
On Nov. 30, the online boutique Bona Drag offered to donate 10 percent of profits from the sale of certain merchandise, including a Lindsey Thornburg $1,300 cloak, to the organization. The designer Rachel Antonoff also pledged to donate 10 percent of profits from sales of her Randy's Reproductive System Sweater, a $218 wool blend knit with intarsia female reproductive system.
The fashion blogger Bryanboy paused from his usual tweets about shopping to show some outrage about Colorado. Phillip Picardi, the digital editorial director of Teen Vogue, posted a long letter of support for reproductive rights and called the shooting "an act of domestic terrorism."
And Stephanie Mark, a founder and the head of business development for the blog the Coveteur, which is based in Toronto and SoHo, used the #standwithPP hashtag and linked to a story the site had run on Cecile Richards, the Planned Parenthood president. Ms. Mark also donated $100.
"Being in fashion, there are so many different types of people — people who have lifestyles that were maybe alienated or looked down upon, that the industry is quick to embrace change and acceptance," Ms. Mark said in an interview. "That transfers to a scenario like Planned Parenthood."
"It's disappointing because there have been amazing movements happening right now like gay marriage and feminism," added Ms. Mark, who is originally from Canada and "getting used to" the health care system in the United States. "Then something like this happens and it negates all the progress we have been pushing for, it deflates the balloon a little bit."
Their followers had nothing less than positive to say, but she thought it was still important to speak out. "When things like this happen, it's done to silence people and make them afraid," Ms. Mark said. "The best thing you can do is show that's not going to happen."


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Dr. Karen Evans
- Position: Instructor
- Department: Teacher Education and Educational Leadership
- Office Location: Building 85, Room 178
- firstname.lastname@example.com
- Campus: (850) 474-2810
Dr. Karen Evans is an Instructor and Project Coordinator of the Partnership for Professional Innovation, an alliance between UWF and the Escambia County School District.
Evans, who has a Doctor of Ministry from Drew University, teaches online and blended courses in Social Studies Education and supervises interns and secondary practicum students.
Evans, an ordained deacon in the United Methodist Church, received a B.A. in Christian Education, Psychology and Sociology from Huntingdon College and a Master's degree in Religious Education from Duke University. She is certified as a Specialist in Curriculum and Instruction by the University of Florida.
She joined UWF in 2011 after 24 years as Minister of Spiritual Formation at First United Methodist Church in Pensacola. Her responsibilities there included planning, organizing and implementing all educational experiences for children, youth and adults.
Degrees & Institutions:
Evans received a bachelor's degree in in Christian Education, Psychology and Sociology from Huntingdon College and a Master's degree in Religious Education from Duke University. She went on to earn her Doctor of Ministry from Drew University.
Keywords: Partnership for Professional Innovation, Educational Psychology, Social Studies Education, spiritual education for Methodists


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Toddler Time is a sweet toddler story-time outing, perfectly suited for little ones aged 2-4 years old! Enjoy a story, sweet treat and craft based on what you've read!
Jan - Feb.: Happy Valentine's Day, Curious George
March - April: Tales of Peter Rabbit
May-July: Pete the Cat and His Four Groovy Buttons
Aug - Oct: Llama, LLama Red Pajamas
Nov-Jan 3: Rudolph the Red nosed Reindeer
Registration is required and guarantees your spot. Please register for your child(ren) only: and be sure to arrive 15 minutes prior to class time to allow adequate time for check-in.
*Toddler Times for private groups and birthday parties are available: please email email@example.com or visit our party page.
Class and Tour Policy: Please arrive 15 minutes before the class start time to check in at the first floor/retail counter. Our classes and tours promptly start at the time you've selected. Refunds for classes and tours are not available. You may reschedule your booking up to 72 hours before class start time for a different date/time by calling 1-855-SWT-PETE.
No, thanks.


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Bacon Jalapeno Popper Bread Recipe!
During the winter, I love making homemade bread! One of my favorite sweets is this Bacon Jalapeno Popper Bread recipe. My family loves this bread so much that we've made this dish three times this month. Want to make this bread as well? If so, be sure to pick up the following ingredients:
- 8-10 slices bacon, after cooked and chopped you'll need 2 cups
- 3 cups all purpose flour
- 1 tablespoon baking powder
- 1 teaspoon salt
- 2 tablespoon granulated sugar
- 8 ounces cream cheese, softened
- ¼ cup diced jalapeno, this will be about 1 large jalapeno, I diced seeds as well
- 2 cups mild cheddar cheese
- 12 ounce buttermilk
- 1 tablespoon vegetable oil
For additional directions on making this bacon jalapeno popper bread, be sure to visit the Call Me PMC Webpage! Let us know what you thought of this bread in the comment section below. We'd love to know.
Anything to Add? Leave a Comment!
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Tailor-made service Why don't you ask us to dye your own household linen?
L'Officiel Voyage Cuisine Actuelle n°40 n°258 Aug-sept. 2012 June 1, 2012
Maison Française Plaisir de la maison elle.fr n°576Feb. 2012 Feb-march 2012 Dec. 2011
do not hesitate to contact us to get press information as well as photos.
At Vaux, enjoy a different holiday cottage
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Opera 15 final for Android has also been released after the release of the desktop version.
- Based on Chromium 28
- Improved startup-time
- Download video. Possible to download video files that can be played in Opera
- Set download location (where your files are downloaded to)
- Improved tab-UI animations
- Opening files from external SD Card now possible
- New "open in private tab" context menu
- Cookie clearing mechanism improved
- Fix for bug causing Opera to be uninstalled from selected Samsung devices
- Memory leaks plugged and general stability improvements.


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Price: $30.00
Quantity: 1 available
Book Condition: Very Good
Spine sunned and with minute tears to spine ends. Boards a bit darkened and with light rubbing to extremities. ; A summary of South American history; 8vo; 366 pages
Title: SOUTH AMERICA'S STORY
Categories: South America, Available Inventory,
Publisher: New York, Robert M. McBride & Company: 1931
Binding: Hardcover
Book Condition: Very Good
Item: 1.00 Item
Order Number: 10654


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Dr Louise Sheridan
COO, Nest Startup Academy
Dr Louise Sheridan is COO of the Nest Startup Academy, which aims to level the playing field for underrepresented founders and address the lack of diversity in tech. She was previously Head of International Tech Hub network at the UK Government's Department for Digital, Culture, Media and Sport (DCMS), where she led on establishing tech hubs across Africa, India, Brazil and Indonesia, and helping to build thriving digital ecosystems that supported inclusive innovation and growth.
She has worked as International Adviser to the UK's National Technology Adviser, and represented the UK on the Digital Nations committee. She has also worked on international policies on the rights of women and girls, and holds a PHD and MRes in gender, ethnicity and diaspora studies.


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Welcome to SINTERLAKEN 2019!
Review of a great weekend
Sinterlaken 2019 began with a vision – to meet each other, exchange ideas, experience something great together. And I think I can say that we succeeded:
The atmosphere was relaxed and friendly, we talked and laughed together – German, French, English – no matter. The workshops, the lectures, the Speleo cinema were all well attended, the premiere of the new film about the Siebenhenste-Hohgant a complete success. The SpeleOlympics pure fun, the photo and plan exhibition full of surprises. In the trade hall there was everything the SpeleoHeart could desire, and by the exhibitors there was a lot to discover and discuss. We ate well, drank well and celebrated the SSS and HRH. The excursions could take place despite the rain and everyone came out well and hopefully got home safely.
The 14th National Congress is over but lives on in our memories and as part of the history of the SSS. Thanks to everyone who participated in any way!
On behalf of your Organising Committee
Marie-José Gilbert
President of the Organising Committee