An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in peach reproductive tissues

Background: Perennial fruit trees display a growth behaviour characterized by annual cycling between growth and dormancy, with complex physiological features. Rosaceae fruit trees represent excellent models for studying not only the fruit growth/patterning but also the progression of the reproductive cycle depending upon the impact of climate conditions. Additionally, current developments in high‐throughput technologies have impacted Rosaceae tree research while investigating genome structure and function as well as (epi)genetic mechanisms involved in important developmental and environmental response processes during fruit tree growth. Among epigenetic mechanisms, chromatin remodelling mediated by histone modifications and other chromatin-related processes play a crucial role in gene modulation, controlling gene expression. Chromatin immunoprecipitation is an effective technique to investigate chromatin dynamics in plants. This technique is generally applied for studies on chromatin states and enrichment of post-transcriptional modifications (PTMs) in histone proteins. Results: Peach is considered a model organism among climacteric fruits in the Rosaceae family for studies on bud formation, dormancy, and organ differentiation. In our work, we have primarily established specific protocols for chromatin extraction and immunoprecipitation in reproductive tissues of peach (Prunus persica). Subsequently, we focused our investigations on the role of two chromatin marks, namely the trimethylation of histone H3 at lysine in position 4 (H3K4me3) and trimethylation of histone H3 at lysine 27 (H3K27me3) in modulating specific gene expression. Bud dormancy and fruit growth were investigated in a nectarine genotype called Fantasia as our model system. Conclusions: We present general strategies to optimize ChIP protocols for buds and mesocarp tissues of peach and analyze the correlation between gene expression and chromatin mark enrichment/depletion. The procedures proposed may be useful to evaluate any involvement of histone modifications in the regulation of gene expression during bud dormancy progression and core ripening in fruits

Maize RNA PolIV affects the expression of genes with nearby TE insertions and has a genome-wide repressive impact on transcription

Abstract Background RNA-directed DNA methylation (RdDM) is a plant-specific epigenetic process that relies on the RNA polymerase IV (Pol IV) for the production of 24 nucleotide small interfering RNAs (siRNA) that guide the cytosine methylation and silencing of genes and transposons. Zea mays RPD1/RMR6 gene encodes the largest subunit of Pol IV and is required for normal plant development, paramutation, transcriptional repression of certain transposable elements (TEs) and transcriptional regulation of specific alleles. Results In this study we applied a total RNA-Seq approach to compare the B73 and rpd1/rmr6 leaf transcriptomes. Although previous studies indicated that loss of siRNAs production in RdDM mutants provokes a strong loss of CHH DNA methylation but not massive gene or TEs transcriptional activation in both Arabidopsis and maize, our total RNA-Seq analysis of rpd1/rmr6 transcriptome reveals that loss of Pol IV activity causes a global increase in the transcribed fraction of the maize genome. Our results point to the genes with nearby TE insertions as being the most strongly affected by Pol IV-mediated gene silencing. TEs modulation of nearby gene expression is linked to alternative methylation profiles on gene flanking regions, and these profiles are strictly dependent on specific characteristics of the TE member inserted. Although Pol IV is essential for the biogenesis of siRNAs, the genes with associated siRNA loci are less affected by the pol IV mutation. Conclusions This deep and integrated analysis of gene expression, TEs distribution, smallRNA targeting and DNA methylation levels, reveals that loss of Pol IV activity globally affects genome regulation, pointing at TEs as modulator of nearby gene expression and indicating the existence of multiple level epigenetic silencing mechanisms. Our results also suggest a predominant role of the Pol IV-mediated RdDM pathway in genome dominance regulation, and subgenome stability and evolution in maize

Genome-Wide Analysis of the Complex Transcriptional Networks of Rice Developing Seeds

<div><h3>Background</h3><p>The development of rice (<em>Oryza sativa</em>) seed is closely associated with assimilates storage and plant yield, and is fine controlled by complex regulatory networks. Exhaustive transcriptome analysis of developing rice embryo and endosperm will help to characterize the genes possibly involved in the regulation of seed development and provide clues of yield and quality improvement.</p> <h3>Principal Findings</h3><p>Our analysis showed that genes involved in metabolism regulation, hormone response and cellular organization processes are predominantly expressed during rice development. Interestingly, 191 transcription factor (TF)-encoding genes are predominantly expressed in seed and 59 TFs are regulated during seed development, some of which are homologs of seed-specific TFs or regulators of <em>Arabidopsis</em> seed development. Gene co-expression network analysis showed these TFs associated with multiple cellular and metabolism pathways, indicating a complex regulation of rice seed development. Further, by employing a cold-resistant <em>cultivar</em> Hanfeng (HF), genome-wide analyses of seed transcriptome at normal and low temperature reveal that rice seed is sensitive to low temperature at early stage and many genes associated with seed development are down-regulated by low temperature, indicating that the delayed development of rice seed by low temperature is mainly caused by the inhibition of the development-related genes. The transcriptional response of seed and seedling to low temperature is different, and the differential expressions of genes in signaling and metabolism pathways may contribute to the chilling tolerance of HF during seed development.</p> <h3>Conclusions</h3><p>These results provide informative clues and will significantly improve the understanding of rice seed development regulation and the mechanism of cold response in rice seed.</p> </div

The Zea mays mutants opaque-2 and opaque-7 disclose extensive changes in endosperm metabolism as revealed by protein, amino acid, and transcriptome-wide analyses

<p>Abstract</p> <p>Background</p> <p>The changes in storage reserve accumulation during maize (<it>Zea mays </it>L.) grain maturation are well established. However, the key molecular determinants controlling carbon flux to the grain and the partitioning of carbon to starch and protein are more elusive. The <it>Opaque-2 </it>(<it>O2</it>) gene, one of the best-characterized plant transcription factors, is a good example of the integration of carbohydrate, amino acid and storage protein metabolisms in maize endosperm development. Evidence also indicates that the <it>Opaque-7 </it>(<it>O7</it>) gene plays a role in affecting endosperm metabolism. The focus of this study was to assess the changes induced by the <it>o2 </it>and <it>o7 </it>mutations on maize endosperm metabolism by evaluating protein and amino acid composition and by transcriptome profiling, in order to investigate the functional interplay between these two genes in single and double mutants.</p> <p>Results</p> <p>We show that the overall amino acid composition of the mutants analyzed appeared similar. Each mutant had a high Lys and reduced Glx and Leu content with respect to wild type. Gene expression profiling, based on a unigene set composed of 7,250 ESTs, allowed us to identify a series of mutant-related down (17.1%) and up-regulated (3.2%) transcripts. Several differentially expressed ESTs homologous to genes encoding enzymes involved in amino acid synthesis, carbon metabolism (TCA cycle and glycolysis), in storage protein and starch metabolism, in gene transcription and translation processes, in signal transduction, and in protein, fatty acid, and lipid synthesis were identified. Our analyses demonstrate that the mutants investigated are pleiotropic and play a critical role in several endosperm-related metabolic processes. Pleiotropic effects were less evident in the <it>o7 </it>mutant, but severe in the <it>o2 </it>and <it>o2o7 </it>backgrounds, with large changes in gene expression patterns, affecting a broad range of kernel-expressed genes.</p> <p>Conclusion</p> <p>Although, by necessity, this paper is descriptive and more work is required to define gene functions and dissect the complex regulation of gene expression, the genes isolated and characterized to date give us an intriguing insight into the mechanisms underlying endosperm metabolism.</p