Shibin He

Trichostatin A Selectively Suppresses the Cold-Induced Transcription of the ZmDREB1 Gene in Maize

Post-translational modifications of histone proteins play a crucial role in responding to environmental stresses. Histone deacetylases (HDACs) catalyze the removal of an acetyl group from histones and are generally believed to be a transcriptional repressor. In this paper, we report that cold treatment highly induces the up-regulation of HDACs, leading to global deacetylation of histones H3 and H4. Treatment of maize with the HDAC inhibitor trichostatin A (TSA) under cold stress conditions strongly inhibits induction of the maize cold-responsive genes ZmDREB1 and ZmCOR413. However, up-regulation of the ZmICE1 gene in response to cold stress is less affected. The expression of drought and salt induced genes, ZmDBF1 and rab17, is almost unaffected by TSA treatment. Thus, these observations show that HDACs may selectively activate transcription. The time course of TSA effects on the expression of ZmDREB1 and ZmCOR413 genes indicates that HDACs appear to directly activate the ZmDREB1 gene, which in turn modulates ZmCOR413 expression. After cold treatment, histone hyperacetylation and DNA demethylation occurs in the ICE1 binding region, accompanied by an increase in accessibility to micrococcal nuclease (MNase). The two regions adjacent to the ICE1 binding site remain hypoacetylated and methylated. However, during cold acclimation, TSA treatment increases the acetylation status and accessibility of MNase and decreases DNA methylation at these two regions. However, TSA treatment does not affect histone hyperacetylation and DNA methylation levels at the ICE1 binding regions of the ZmDREB1 gene. Altogether, our findings indicate that HDACs positively regulate the expression of the cold-induced ZmDREB1 gene through histone modification and chromatin conformational changes and that this activation is both gene and site selective.

Single-walled carbon nanotubes selectively influence maize root tissue development accompanied by the change in the related gene expression

The inconsistent impact of nanomaterials on different plant species has been reported, but little is known about this effect at the cellular and genetic levels. Here we report that single-walled carbon nanotubes (SWCNTs) accelerate maize seminal root growth, but display little effect on the primary root growth. In contrast, root hair growth inhibition by SWCNTs is observed. Further gene transcription analysis shows that SWCNTs could increase the expression of seminal root associated genes whereas decrease root hair associated gene expression. Their effect is on both tissue and gene selectiveness since both enhanced and inhibited gene expression and tissue growth are observed during root development. Microscopy images reveal the distribution of SWCNTs inside the root and mainly in the intercellular space in cortex tissues. We also find that SWCNT-treatment dynamically and selectively induces the up-regulation of epigenetic modification enzyme genes, leading to global deacetylation of histone H3, similar to the response of plants to other stress. Our results suggest that the nanoparticle-root cell interaction could cause the change in gene expression, and consequently affect relative root growth and development.

ABA treatment of germinating maize seeds induces VP1 gene expression and selective promoter‐associated histone acetylation

Seed germination commences from a low metabolic state to a bioactive state and is associated with changes in the pattern of gene expression. Recent studies have revealed that epigenetic processes are involved in abscisic acid (ABA)-regulated seed germination processes. In this study, we showed that the expression of both histone acetyltransferases (HATs) and histone deacetylases (HDACs) was increased gradually during seed germination accompanying an increase in overall acetylation level of histone H3. Application of exogenous ABA repressed the expression of HATs as well as HDACs and delayed histone acetylation. Suppressing HDAC by treatment with an HDAC inhibitor, trichostatin A (TSA), led to an increase in global histone acetylation and inhibited seed germination and growth. However, ABA and TSA both delayed the downregulation of the embryogenesis-related gene viviparous1 (VP1) during seed germination. The further chromatin immunoprecipitation experiments showed that the promoter region of the VP1 gene was deacetylated during seed germination, and this deacetylation event was inhibited by both ABA and TSA. These results suggested that a balance of the two enzymes HATs and HDACs affected the acetylation status of the VP1 gene and ABA selectively activated its transcription by an accumulation of acetylated histone H3 associated with the promoter region during seed germination.

Cold stress selectively unsilences tandem repeats in heterochromatin associated with accumulation of H3K9ac

Knobs are cytologically observable major interstitial heterochromatin present on maize nuclei, which consist of highly tandem-repetitive elements that are always silenced. Here we investigated the genome-wide change of H3K9ac, an active chromatin mark, during cold stress using chromatin immunoprecipitation sequencing (ChIP-Seq) and identified differential cold-induced H3K9ac enrichment at repetitive sequences in maize. More detailed analysis of two knob-associated tandem-repetitive sequences, 180-bp and TR-1, demonstrated that cold activated their transcription and this cold-induced transcriptional activation of repetitive sequences is selective, transient, and associated with an increase in H3K9ac and a reduction in DNA methylation and H3K9me2. Furthermore, knob sequence expression is accompanied by localized chromatin remodelling and silencing is recovered upon prolonged treatment. In addition, no evidence of copy number change and rearrangement of these repetitive elements are found in plants subjected to cold stress. These results suggest that cold-mediated unsilencing of heterochromatic tandem-repeated sequences, accompanied with epigenetic regulation, might play an important role in the adaptation of plants to cold stimuli.

Maximizing specificity and yield of PCR by the quantum dot itself rather than property of the quantum dot surface.

We found that semiconductor quantum dots (QDs) dramatically improved both product yield and specificity of PCR. The concentration of QDs is important for improving PCR amplification. In the presence of appropriate concentration of mercaptoacetic acid (MAA)-coated QDs, specificity and yield of PCR were enhanced. Also, strong nonspecific bands and weaker smeared bands were eliminated. At lower annealing temperatures (25-45 degrees C), addition of MAA-coated QDs into the PCR reagent produced specific PCR products without nonspecific sequence amplification. MAA alone did not improve PCR amplification. Streptavidin (SA) surface modified QDs with different size also effectively improved the specificity of PCR, demonstrating that the observed effect was not due to property of the QD surface but instead due to the QD itself. Bovine Serum Albumin (BSA) could relieve Taq polymerase from MAA-coated QDs in PCR by interaction with QDs and therefore imply that QDs improve specificity of PCR by interaction with Taq polymerase. These results demonstrate that QDs, added to reaction mixes at appropriate concentrations, can increase PCR yield and improve PCR specificity, even at low annealing temperatures. We assume that many different surface modified polymeric nanoparticles might have similar effects.

One-to-one quantum dot-labeled single long DNA probes

Quantum dots (QDs) have been received most attention due to their unique properties. Constructing QDs conjugated with certain number of biomolecules is considered as one of the most important research goals in nanobiotechnology. In this study, we report polymerase chain reaction (PCR) amplification of primer oligonucleotides bound to QDs, termed as QD-based PCR. Characterization of QD-based PCR products by gel electrophoresis and atomic force microscopy showed that QD-labeled long DNA strands were synthesized and only a single long DNA strand was conjugated with a QD. The QD-based PCR products still kept fluorescence properties. Moreover, the one-to-one QD-labeled long DNA conjugates as probes could detect a single-copy gene on maize chromosomes by fluorescence in situ hybridization. Labeling a single QD to a single long DNA will make detection of small single-copy DNA fragments, quantitative detection and single molecule imaging come true by nanotechnology, and it will promote medical diagnosis and basic biological research as well as nano-material fabrication.

High efficiency transport of quantum dots into plant roots with the aid of silwet L-77.

Quantum dots (QDs) are a novel type of small, photostable and bright fluorophores that have been successfully applied to mammalian and human live cell imaging. In this study, highly dispersive water-soluble mercaptoacetic acid (MAA)-coated CdSe/ZnS QDs were synthesized, which were suitable for investigation as fluorescent probe labels. The treatment of maize seedling roots with QDs showed that the surfactant silwet L-77 aided the efficient transport of QDs into maize roots. Under a concentration ranging from 0.128 to 1.28 microM, QDs caused very low cytotoxicity on maize seed germination and root growth. The addition of mercuric chloride to the Hoagland solution resulted in a decrease of QD content in root tissues, and this decrease was reversed upon the addition of beta-mercaptoethanol, which suggests that mercury-sensitive processes play a significant role in regulating QD flow in the maize root system. We speculate that the apoplastic pathway can contribute substantially to the total quantity of QDs reaching the stele. Therefore, based on this transport approach, MAA-coated QDs can be utilized for live imaging in plant systems to verify known physiological processes.

Plant 45S rDNA clusters are fragile sites and their instability is associated with epigenetic alterations.

Our previous study demonstrated that 45S ribosomal DNA (45S rDNA) clusters were chromosome fragile sites expressed spontaneously in Lolium. In this study, fragile phenotypes of 45S rDNA were observed under aphidicolin (APH) incubation in several plant species. Further actinomycin D (ActD) treatment showed that transcriptional stress might interfere with chromatin packaging, resulting in 45S rDNA fragile expression. These data identified 45S rDNA sites as replication-dependent as well as transcription-dependent fragile sites in plants. In the presence of ActD, a dramatic switch to an open chromatin conformation and accumulated incomplete 5′ end of the external transcribed spacer (5′ETS) transcripts were observed, accompanied by decreased DNA methylation, decreased levels of histone H3, and increased histone acetylation and levels of H3K4me2, suggesting that these epigenetic alterations are associated with failure of 45S rDNA condensation. Furthermore, the finding that γ-H2AX was accumulated at 45S rDNA sites following ActD treatment suggested that the DNA damage signaling pathway was associated with the appearance of 45S rDNA fragile phenotypes. Our data provide a link between 45S rDNA transcription and chromatin-packaging defects and open the door for further identifying the molecular mechanism involved.

ABA-mediated inhibition of seed germination is associated with ribosomal DNA chromatin condensation, decreased transcription, and ribosomal RNA gene hypoacetylation

Seed germination is a highly organized biological process accompanied by many cellular and metabolic changes. The ribosomal RNA (rRNA) gene, which forms the nucleolus at interphase and is transcribed for ribosome production and protein synthesis, has an important role during seed germination. In this study, we report that there is a decondensation of ribosomal DNA (rDNA) chromatin during seed germination accompanied with increased rRNA gene expression and overall genomic hyperacetylation. Analysis of the rRNA gene promoter region by using chromatin immunoprecipitation (ChIP) shows that there is an increase in acetylation levels at the rRNA gene promoter region. Application of seed germination inhibitor abscisic acid (ABA) suppresses rDNA chromatin decondensation, the expression of rRNA genes and global genomic acetylation. The further ChIP experiments show that ABA treatment hinders the elevation of acetylation levels in the promoter region of the rRNA gene. The data together indicate that ABA treatment inhibits seed germination, which is associated with rDNA chromatin condensation, decreased transcription and rRNA gene hypoacetylation.

Comparative Analysis of Genome-Wide Chromosomal Histone Modification Patterns in Maize Cultivars and Their Wild Relatives

Recent advances demonstrate that epigenome changes can also cause phenotypic diversity and can be heritable across generations, indicating that they may play an important role in evolutionary processes. In this study, we analyzed the chromosomal distribution of several histone modifications in five elite maize cultivars (B73, Mo17, Chang7-2, Zheng58, ZD958) and their two wild relatives (Zea mays L. ssp. parviglumis and Zea nicaraguensis) using a three-dimensional (3D) epigenome karyotyping approach by combining immunostaining and 3D reconstruction with deconvolution techniques. The distribution of these histone modifications along chromosomes demonstrated that the histone modification patterns are conserved at the chromosomal level and have not changed significantly following domestication. The comparison of histone modification patterns between metaphase chromosomes and interphase nuclei showed that some of the histone modifications were retained as the cell progressed from interphase into metaphase, although remodelling existed. This study will increase comprehension of the function of epigenetic modifications in the structure and evolution of the maize genome.