Milos Tanurdzic

Epigenetic Reprogramming and Small RNA Silencing of Transposable Elements in Pollen

The mutagenic activity of transposable elements (TEs) is suppressed by epigenetic silencing and small interfering RNAs (siRNAs), especially in gametes that could transmit transposed elements to the next generation. In pollen from the model plant Arabidopsis, we show that TEs are unexpectedly reactivated and transpose, but only in the pollen vegetative nucleus, which accompanies the sperm cells but does not provide DNA to the fertilized zygote. TE expression coincides with downregulation of the heterochromatin remodeler decrease in DNA methylation 1 and of many TE siRNAs. However, 21 nucleotide siRNAs from Athila retrotransposons are generated and accumulate in pollen and sperm, suggesting that siRNA from TEs activated in the vegetative nucleus can target silencing in gametes. We propose a conserved role for reprogramming in germline companion cells, such as nurse cells in insects and vegetative nuclei in plants, to reveal intact TEs in the genome and regulate their activity in gametes.

Systems approach identifies an organic nitrogen-responsive gene network that is regulated by the master clock control gene CCA1

Understanding how nutrients affect gene expression will help us to understand the mechanisms controlling plant growth and development as a function of nutrient availability. Nitrate has been shown to serve as a signal for the control of gene expression in Arabidopsis. There is also evidence, on a gene-by-gene basis, that downstream products of nitrogen (N) assimilation such as glutamate (Glu) or glutamine (Gln) might serve as signals of organic N status that in turn regulate gene expression. To identify genome-wide responses to such organic N signals, Arabidopsis seedlings were transiently treated with ammonium nitrate in the presence or absence of MSX, an inhibitor of glutamine synthetase, resulting in a block of Glu/Gln synthesis. Genes that responded to organic N were identified as those whose response to ammonium nitrate treatment was blocked in the presence of MSX. We showed that some genes previously identified to be regulated by nitrate are under the control of an organic N-metabolite. Using an integrated network model of molecular interactions, we uncovered a subnetwork regulated by organic N that included CCA1 and target genes involved in N-assimilation. We validated some of the predicted interactions and showed that regulation of the master clock control gene CCA1 by Glu or a Glu-derived metabolite in turn regulates the expression of key N-assimilatory genes. Phase response curve analysis shows that distinct N-metabolites can advance or delay the CCA1 phase. Regulation of CCA1 by organic N signals may represent a novel input mechanism for N-nutrients to affect plant circadian clock function.

Sex-Determining Mechanisms in Land Plants

Sex determination is a process that leads to the physical separation of male and female gamete-producing structures to different individuals of a species. Even though sexually reproducing species have only three possible options—to relegate the two sexes to separate individuals, to keep them

Epigenomic consequences of immortalized plant cell suspension culture.

Plant cells grown in culture exhibit genetic and epigenetic instability. Using a combination of chromatin immunoprecipitation and DNA methylation profiling on tiling microarrays, we have mapped the location and abundance of histone and DNA modifications in a continuously proliferating, dedifferentiated cell suspension culture of Arabidopsis. We have found that euchromatin becomes hypermethylated in culture and that a small percentage of the hypermethylated genes become associated with heterochromatic marks. In contrast, the heterochromatin undergoes dramatic and very precise DNA hypomethylation with transcriptional activation of specific transposable elements (TEs) in culture. High throughput sequencing of small interfering RNA (siRNA) revealed that TEs activated in culture have increased levels of 21-nucleotide (nt) siRNA, sometimes at the expense of the 24-nt siRNA class. In contrast, TEs that remain silent, which match the predominant 24-nt siRNA class, do not change significantly in their siRNA profiles. These results implicate RNA interference and chromatin modification in epigenetic restructuring of the genome following the activation of TEs in immortalized cell culture.

Arabidopsis thaliana chromosome 4 replicates in two phases that correlate with chromatin state.

DNA replication programs have been studied extensively in yeast and animal systems, where they have been shown to correlate with gene expression and certain epigenetic modifications. Despite the conservation of core DNA replication proteins, little is known about replication programs in plants. We used flow cytometry and tiling microarrays to profile DNA replication of Arabidopsis thaliana chromosome 4 (chr4) during early, mid, and late S phase. Replication profiles for early and mid S phase were similar and encompassed the majority of the euchromatin. Late S phase exhibited a distinctly different profile that includes the remaining euchromatin and essentially all of the heterochromatin. Termination zones were consistent between experiments, allowing us to define 163 putative replicons on chr4 that clustered into larger domains of predominately early or late replication. Early-replicating sequences, especially the initiation zones of early replicons, displayed a pattern of epigenetic modifications specifying an open chromatin conformation. Late replicons, and the termination zones of early replicons, showed an opposite pattern. Histone H3 acetylated on lysine 56 (H3K56ac) was enriched in early replicons, as well as the initiation zones of both early and late replicons. H3K56ac was also associated with expressed genes, but this effect was local whereas replication time correlated with H3K56ac over broad regions. The similarity of the replication profiles for early and mid S phase cells indicates that replication origin activation in euchromatin is stochastic. Replicon organization in Arabidopsis is strongly influenced by epigenetic modifications to histones and DNA. The domain organization of Arabidopsis is more similar to that in Drosophila than that in mammals, which may reflect genome size and complexity. The distinct patterns of association of H3K56ac with gene expression and early replication provide evidence that H3K56ac may be associated with initiation zones and replication origins.

Construction of a bacterial artificial chromosome library from the spikemoss Selaginella moellendorffii: a new resource for plant comparative genomics

BackgroundThe lycophytes are an ancient lineage of vascular plants that diverged from the seed plant lineage about 400 Myr ago. Although the lycophytes occupy an important phylogenetic position for understanding the evolution of plants and their genomes, no genomic resources exist for this group of plants.ResultsHere we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from the lycophyte Selaginella moellendorffii. Based on cell flow cytometry, this species has the smallest genome size among the different lycophytes tested, including Huperzia lucidula, Diphaiastrum digita, Isoetes engelmanii and S. kraussiana. The arrayed BAC library consists of 9126 clones; the average insert size is estimated to be 122 kb. Inserts of chloroplast origin account for 2.3% of the clones. The BAC library contains an estimated ten genome-equivalents based on DNA hybridizations using five single-copy and two duplicated S. moellendorffii genes as probes.ConclusionThe S. moellenforffii BAC library, the first to be constructed from a lycophyte, will be useful to the scientific community as a resource for comparative plant genomics and evolution.

ABAP1 is a novel plant Armadillo BTB protein involved in DNA replication and transcription

In multicellular organisms, organogenesis requires a tight control of the balance between cell division and cell differentiation. Distinct signalling pathways that connect both cellular processes with developmental cues might have evolved to suit different developmental plans. Here, we identified and characterized a novel protein that interacts with pre‐replication complex (pre‐RC) subunits, designated Armadillo BTB Arabidopsis protein 1 (ABAP1). Overexpression of ABAP1 in plants limited mitotic DNA replication and decreased cell proliferation in leaves, whereas ABAP1 downregulation increased cell division rates. Activity of ABAP1 in transcription was supported by its association with the transcription factor AtTCP24. The ABAP1–AtTCP24 complex bound specifically to the promoters of AtCDT1a and AtCDT1b in vitro and in vivo. Moreover, expression levels of AtCDT1a and AtCDT1b were reduced in ABAP1‐overexpressing plants and they were increased in plants with reduced levels of ABAP1. We propose that ABAP1 participates in a negative feedback loop regulating mitotic DNA replication during leaf development, either by repressing transcription of pre‐RC genes and possibly by regulating pre‐RC utilization through direct association with pre‐RC components.

A systemic gene silencing method suitable for high throughput, reverse genetic analyses of gene function in fern gametophytes

BackgroundCeratopteris richardii is a useful experimental system for studying gametophyte development and sexual reproduction in plants. However, few tools for cloning mutant genes or disrupting gene function exist for this species. The feasibility of systemic gene silencing as a reverse genetics tool was examined in this study.ResultsSeveral DNA constructs targeting a Ceratopteris protoporphyrin IX magnesium chelatase (CrChlI) gene that is required for chlorophyll biosynthesis were each introduced into young gametophytes by biolistic delivery. Their transient expression in individual cells resulted in a colorless cell phenotype that affected most cells of the mature gametophyte, including the meristem and gametangia. The colorless phenotype was associated with a 7-fold decrease in the abundance of the endogenous transcript. While a construct designed to promote the transient expression of a CrChlI double stranded, potentially hairpin-forming RNA was found to be the most efficient in systemically silencing the endogenous gene, a plasmid containing the CrChlI cDNA insert alone was sufficient to induce silencing. Bombarded, colorless hermaphroditic gametophytes produced colorless embryos following self-fertilization, demonstrating that the silencing signal could be transmitted through gametogenesis and fertilization. Bombardment of young gametophytes with constructs targeting the Ceratopteris filamentous temperature sensitive (CrFtsZ) and uroporphyrin dehydrogenase (CrUrod) genes also produced the expected mutant phenotypes.ConclusionA method that induces the systemic silencing of target genes in the Ceratopteris gametophyte is described. It provides a simple, inexpensive and rapid means to test the functions of genes involved in gametophyte development, especially those involved in cellular processes common to all plants.

Computational identification and analysis of novel sugarcane microRNAs.

BackgroundMicroRNA-regulation of gene expression plays a key role in the development and response to biotic and abiotic stresses. Deep sequencing analyses accelerate the process of small RNA discovery in many plants and expand our understanding of miRNA-regulated processes. We therefore undertook small RNA sequencing of sugarcane miRNAs in order to understand their complexity and to explore their role in sugarcane biology.ResultsA bioinformatics search was carried out to discover novel miRNAs that can be regulated in sugarcane plants submitted to drought and salt stresses, and under pathogen infection. By means of the presence of miRNA precursors in the related sorghum genome, we identified 623 candidates of new mature miRNAs in sugarcane. Of these, 44 were classified as high confidence miRNAs. The biological function of the new miRNAs candidates was assessed by analyzing their putative targets. The set of bona fide sugarcane miRNA includes those likely targeting serine/threonine kinases, Myb and zinc finger proteins. Additionally, a MADS-box transcription factor and an RPP2B protein, which act in development and disease resistant processes, could be regulated by cleavage (21-nt-species) and DNA methylation (24-nt-species), respectively.ConclusionsA large scale investigation of sRNA in sugarcane using a computational approach has identified a substantial number of new miRNAs and provides detailed genotype-tissue-culture miRNA expression profiles. Comparative analysis between monocots was valuable to clarify aspects about conservation of miRNA and their targets in a plant whose genome has not yet been sequenced. Our findings contribute to knowledge of miRNA roles in regulatory pathways in the complex, polyploidy sugarcane genome.

Integrated RNA-seq and sRNA-seq analysis identifies novel nitrate-responsive genes in Arabidopsis thaliana roots.

BackgroundNitrate and other nitrogen metabolites can act as signals that regulate global gene expression in plants. Adaptive changes in plant morphology and physiology triggered by changes in nitrate availability are partly explained by these changes in gene expression. Despite several genome-wide efforts to identify nitrate-regulated genes, no comprehensive study of the Arabidopsis root transcriptome under contrasting nitrate conditions has been carried out.ResultsIn this work, we employed the Illumina high throughput sequencing technology to perform an integrated analysis of the poly-A + enriched and the small RNA fractions of the Arabidopsis thaliana root transcriptome in response to nitrate treatments. Our sequencing strategy identified new nitrate-regulated genes including 40 genes not represented in the ATH1 Affymetrix GeneChip, a novel nitrate-responsive antisense transcript and a new nitrate responsive miRNA/TARGET module consisting of a novel microRNA, miR5640 and its target, AtPPC3.ConclusionsSequencing of small RNAs and mRNAs uncovered new genes, and enabled us to develop new hypotheses for nitrate regulation and coordination of carbon and nitrogen metabolism.