The roles of Polycomb-Group proteins in the development of Arabidopsis thaliana

The roles of Polycomb-Group proteins in the development of Arabidopsis thaliana The Polycomb-group (Pc-G) complex determines animal cell fate by regulating the expression of the homeotic genes that specify the body pattern. Several Pc-G proteins form a complex, termed Polycomb Repressive Complex 2 (PRC2), which can methylate histone tails. In plants, cell fates are less rigidly determined, suggesting the Pc-G play minor roles in development, or that its effects are more readily reversible. In Arabidopsis, there are three homologues of the catalytic unit of the PRC2, encoded by the MEDEA (MEA), CURLY LEAF (CLF) and SWINGER (SWN) genes. CLF and SWN are expressed throughout development, whereas MEA is confined to seed development. The swn- mutants appear normal, clf- mutants are early flowering, but swn- clf- double mutants are only viable in tissue culture, and develop into immortal callus-like material. This suggests CLF and SWN function is masked by redundancy. SWN is widely conserved in flowering plants, suggesting it may have functions independent of CLF. The severity of the swn- clf- phenotype indicated the Pc-G might play broad roles in plant development, but few targets are known. The aims of this thesis were to determine whether SWN had discrete functions in development, and uncover target genes and developmental pathways controlled redundantly by CLF and SWN. Phenotypic analysis suggested that SWN is required to promote the juvenile to adult phase transition and repress leaf initiation rate. Microarray analysis was performed and combined with published “ChIP on chip” data of genome wide loci possessing histone 3 lysine 27 trimethylation (H3K27me3), a modification specifically catalyzed by the PRC2. My results suggest that there are over 1000 direct targets of Pc-G in Arabidopsis, and these tended to be the most highly over-expressed genes in Pc-G mutants. Validation of the microarray data, and phenotypic analysis revealed Pc-G complex is a global regulator of development, and SWN and CLF play novel roles in stem cell maintenance, promoting and repressing flowering, and in confining embryogenic traits to seed development

Managing understory light conditions in boreal mixedwoods through variation in the intensity and spatial pattern of harvest: A modelling approach

In the context of partial harvesting, adequately managing post-harvest light conditions are essential to obtain a desired composition of tree species regeneration. The objective of this study was to determine how varying the intensity and spatial pattern of harvest would affect understory light conditions in boreal mixedwood stands of northwestern Quebec using the spatially explicit SORTIE-ND light model. The model was evaluated based on comparisons of observed and predicted light levels in both mapped and un-mapped plots. In mapped plots, reasonably accurate predictions of the overall variation in light levels were obtained, but predictions tended to lack spatial precision. In un-mapped plots, SORTIE-ND accurately predicted stand-level mean GLI (Gap Light Index) under a range of harvest intensities. The model was then used to simulate nine silvicultural treatments based on combinations of three intensities of overstory removal (30%, 45% and 60% of basal area) and three harvest patterns (uniform, narrow strips, large gaps). Simulations showed that increasing overstory removal had less impact on light conditions with uniform harvests, and a more marked effect with more aggregated harvest patterns. Whatever the harvest intensity, uniform cuts almost never created high light conditions (GLI > 50%). Gap cuts, on the other hand, resulted in up to 40% of microsites receiving GLI > 50%. Our results suggest that either a 30% strip or gap cut or a 45–60% uniform partial harvest could be used to accelerate the transition from an aspen dominated composition to a mixedwood stand because both types of cut generate the greatest proportion of moderately low light levels (e.g., 15–40% GLI). These light levels tend to favour an accelerated growth response among shade-tolerant conifers, while preventing excessive recruitment of shade-intolerant species. A better understanding of how spatial patterns of harvest interact with tree removal intensity to affect understory light conditions can provide opportunities for designing silvicultural prescriptions that are tailored to species’ traits and better suited to meet a variety of management objectives

Antagonistic Roles of SEPALLATA3, FT and FLC Genes as Targets of the Polycomb Group Gene CURLY LEAF

In Arabidopsis, mutations in the Pc-G gene CURLY LEAF (CLF) give early flowering plants with curled leaves. This phenotype is caused by mis-expression of the floral homeotic gene AGAMOUS (AG) in leaves, so that ag mutations largely suppress the clf phenotype. Here, we identify three mutations that suppress clf despite maintaining high AG expression. We show that the suppressors correspond to mutations in FPA and FT, two genes promoting flowering, and in SEPALLATA3 (SEP3) which encodes a co-factor for AG protein. The suppression of the clf phenotype is correlated with low SEP3 expression in all case and reveals that SEP3 has a role in promoting flowering in addition to its role in controlling floral organ identity. Genetic analysis of clf ft mutants indicates that CLF promotes flowering by reducing expression of FLC, a repressor of flowering. We conclude that SEP3 is the key target mediating the clf phenotype, and that the antagonistic effects of CLF target genes masks a role for CLF in promoting flowering

Dynamic Regulation of H3K27 Trimethylation during Arabidopsis Differentiation

During growth of multicellular organisms, identities of stem cells and differentiated cells need to be maintained. Cell fate is epigenetically controlled by the conserved Polycomb-group (Pc-G) proteins that repress their target genes by catalyzing histone H3 lysine 27 trimethylation (H3K27me3). Although H3K27me3 is associated with mitotically stable gene repression, a large fraction of H3K27me3 target genes are tissue-specifically activated during differentiation processes. However, in plants it is currently unclear whether H3K27me3 is already present in undifferentiated cells and dynamically regulated to permit tissue-specific gene repression or activation. We used whole-genome tiling arrays to identify the H3K27me3 target genes in undifferentiated cells of the shoot apical meristem and in differentiated leaf cells. Hundreds of genes gain or lose H3K27me3 upon differentiation, demonstrating dynamic regulation of an epigenetic modification in plants. H3K27me3 is correlated with gene repression, and its release preferentially results in tissue-specific gene activation, both during differentiation and in Pc-G mutants. We further reveal meristem- and leaf-specific targeting of individual gene families including known but also likely novel regulators of differentiation and stem cell regulation. Interestingly, H3K27me3 directly represses only specific transcription factor families, but indirectly activates others through H3K27me3-mediated silencing of microRNA genes. Furthermore, H3K27me3 targeting of genes involved in biosynthesis, transport, perception, and signal transduction of the phytohormone auxin demonstrates control of an entire signaling pathway. Based on these and previous analyses, we propose that H3K27me3 is one of the major determinants of tissue-specific expression patterns in plants, which restricts expression of its direct targets and promotes gene expression indirectly by repressing miRNA genes

Conceptualizing and measuring strategy implementation – a multi-dimensional view

Through quantitative methodological approaches for studying the strategic management and planning process, analysis of data from 208 senior managers involved in strategy processes within ten UK industrial sectors provides evidence on the measurement properties of a multi-dimensional instrument that assesses ten dimensions of strategy implementation. Using exploratory factor analysis, results indicate the sub-constructs (the ten dimensions) are uni-dimensional factors with acceptable reliability and validity; whilst using three additional measures, and correlation and hierarchical regression analysis, the nomological validity for the multi-dimensional strategy implementation construct was established. Relative importance of ten strategy implementation dimensions (activities) for practicing managers is highlighted, with the mutually and combinative effects drawing conclusion that senior management involvement leads the way among the ten key identified activities vital for successful strategy implementation

Tissue-Specific Expression of FLOWERING LOCUS T in Arabidopsis Is Maintained Independently of Polycomb Group Protein Repression[W][OA]

This study shows that the role of Polycomb Group (PcG) proteins in the regulatory network determining tissue-specific expression is not identical for all PcG-target genes. The data suggest that a positive regulatory factor produced in differentiated phloem companion cells sets a prerequisite for FT expression

Effect of the <i>clf</i> mutation on histone methylation.

<p>ChIP analysis using 12 day old seedlings. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030715#s3" target="_blank">Results</a> show H3K27me3 levels at different genes, other than the rightmost bars which show H3K4me3 levels at the <i>AG</i> gene. The SEP3-2 primers amplify a region of the <i>SEP3</i> promoter previously implicated in regulation of <i>SEP3</i> by Pc-G proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030715#pone.0030715-Liu4" target="_blank">[63]</a>, SEP3-M amplify part of the large first intron of <i>SEP3</i>. Error bars are standard error of mean of three technical replicates. The experiment was repeated on independent samples with very similar results as shown in supplementary data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030715#pone.0030715.s003" target="_blank">Figure S3</a>.</p

Gene expression in suppressor mutants.

<p>(<b>A</b>) Western blot analysis of FPA protein levels. Three independent <i>clf-50 fpa-10</i> samples were processed. Note that no protein is detected in the null <i>fpa-7</i> control, whereas in extracts from a <i>35S::FPA-YPA</i> transgenic line a larger product corresponding to the <i>FPA-YFP</i> fusion protein is detected, confirming the specificity of the antibody for FPA. No FPA protein is detectable in <i>fpa-10</i> extracts, indicating that <i>fpa-10</i> is likely a null allele. (<b>B</b>) Real time PCR analysis of <i>FLC</i> expression. (<b>C</b>) Real time PCR analysis of <i>AG</i> expression, showing high <i>AG</i> expression in suppressor mutants. (<b>D</b>) Western blot analysis of AG protein expression. The AG antibody detects two proteins of about 29 kDa that are specific for AG, the smaller band possibly representing a truncated product or spurious translation initiation event (Riechmann et al., 1999). AG protein is strongly detected in wild type flowers but not in leaves. Weak expression is found in <i>clf-50</i> and <i>clf-50 fpa-10</i> leaves. (<b>E</b>) Real time PCR analysis of <i>SEP3</i> expression. (<b>F</b>) Histochemical staining of GUS reporter gene activity. <i>SEP3::GUS</i> is not expressed in wild type leaves but shows weak expression in vasculature of <i>clf-81</i> leaves (enlarged in inset). (<b>G</b>) Real time PCR analysis of <i>SEP2</i> expression. (<b>H</b>) Real time PCR analysis of <i>FT</i> expression (<b>I</b>) Real time PCR analysis of <i>SEP3</i> expression. Error bars in real time PCR experiments represent standard error of mean of three independent samples (biological replicates). Expression was normalised relative to the <i>EiF4A</i> gene, and is expressed relative to expression in wild type. In <b>B</b>, <b>C</b>, <b>E</b>, <b>G</b>, <b>H</b> whole seedlings less roots of 20 day old short day grown seedlings were used. In <b>I</b> rosette leaves of long day plants at 21 days were used.</p