jia Li

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.

45S rDNA Regions Are Chromosome Fragile Sites Expressed as Gaps In Vitro on Metaphase Chromosomes of Root-Tip Meristematic Cells in Lolium spp

Background In humans, chromosome fragile sites are regions that are especially prone to forming non-staining gaps, constrictions or breaks in one or both of the chromatids on metaphase chromosomes either spontaneously or following partial inhibition of DNA synthesis and have been well identified. So far, no plant chromosome fragile sites similar to those in human chromosomes have been reported. Methods and Results During the course of cytological mapping of rDNA on ryegrass chromosomes, we found that the number of chromosomes plus chromosome fragments was often more than the expected 14 in most cells for Lolium perenne L. cv. Player by close cytological examination using a routine chromosome preparation procedure. Further fluorescent in situ hybridization (FISH) using 45S rDNA as a probe indicated that the root-tip cells having more than a 14-chromosome plus chromosome fragment count were a result of chromosome breakage or gap formation in vitro (referred to as chromosome lesions) at 45S rDNA sites, and 86% of the cells exhibited chromosome breaks or gaps and all occurred at the sites of 45S rDNA in Lolium perenne L. cv. Player, as well as in L. multiflorum Lam. cv. Top One. Chromatin depletion or decondensation occurred at various locations within the 45S rDNA regions, suggesting heterogeneity of lesions of 45S rDNA sites with respect to their position within the rDNA region. Conclusions The chromosome lesions observed in this study are very similar cytologically to that of fragile sites observed in human chromosomes, and thus we conclude that the high frequency of chromosome lesions in vitro in Lolium species is the result of the expression of 45S rDNA fragile sites. Possible causes for the spontaneous expression of fragile sites and their potential biological significance are discussed.

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.

Trichostatin A and 5-azacytidine both cause an increase in global histone H4 acetylation and a decrease in global DNA and H3K9 methylation during mitosis in maize

BackgroundModifications of DNA and histones in various combinations are correlated with many cellular processes. In this study, we investigated the possible relationship between histone H4 tetraacetylation, DNA methylation and histone H3 dimethylation at lysine 9 during mitosis in maize root meristems.ResultsTreatment with trichostatin A, which inhibits histone deacetylases, resulted in increased histone H4 acetylation accompanied by the decondensation of interphase chromatin and a decrease in both global H3K9 dimethylation and DNA methylation during mitosis in maize root tip cells. These observations suggest that histone acetylation may affect DNA and histone methylation during mitosis. Treatment with 5-azacytidine, a cytosine analog that reduces DNA methylation, caused chromatin decondensation and mediated an increase in H4 acetylation, in addition to reduced DNA methylation and H3K9 dimethylation during interphase and mitosis. These results suggest that decreased DNA methylation causes a reduction in H3K9 dimethylation and an increase in H4 acetylation.ConclusionsThe interchangeable effects of 5-azacytidine and trichostatin A on H4 acetylation, DNA methylation and H3K9 dimethylation indicate a mutually reinforcing action between histone acetylation, DNA methylation and histone methylation with respect to chromatin modification. Treatment with trichostatin A and 5-azacytidine treatment caused a decrease in the mitotic index, suggesting that H4 deacetylation and DNA and H3K9 methylation may contain the necessary information for triggering mitosis in maize root tips.

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.

Fluorescence in situ hybridization (FISH) on maize metaphase chromosomes with quantum dot-labeled DNA conjugates

Semiconductor nanocrystals, also called quantum dots (QDs), are novel inorganic fluorophores which are brighter and more photostable than organic fluorophores. In the present study, highly dispersive QD-labeled oligonucleotide (TAG)8 (QD-deoxyribonucleic acid [DNA]) conjugates were constructed via the metal-thiol bond, which can be used as fluorescence in situ hybridization (FISH) probes. FISH analysis of maize metaphase chromosomes using the QD-DNA probes showed that the probes could penetrate maize chromosomes and nuclei and solely hybridized to complementary target DNAs. Compared with the conventional organic dyes such as Cy3 and fluorescein isothiocyanate, this class of luminescent labels bound with oligonucleotides is brighter and more stable against photobleaching on the chromosomes after FISH. These results suggest that QD fluorophores may be a more stable and useful fluorescent label for FISH applications in plant chromosome mapping considering their size-tunable luminescence spectra.

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.