Nuclei Of Plants Research Articles

Global nuclear reorganization during heterochromatin replication in the giant-genome plant Nigella damascena L.

Among flowering plants, genome size varies remarkably, by >2200-fold, and this variation depends on the loss and gain of noncoding DNA sequences that form distinct heterochromatin complexes during interphase. In plants with giant genomes, most chromatin remains condensed during interphase, forming a dense network of heterochromatin threads called interphase chromonemata. Using super-resolution light and electron microscopy, we studied the ultrastructure of chromonemata during and after replication in root meristem nuclei of Nigella damascena L. During S-phase, heterochromatin undergoes transient decondensation locally at DNA replication sites. Due to the abundance of heterochromatin, the replication leads to a robust disassembly of the chromonema meshwork and a general reorganization of the nuclear morphology visible even by conventional light microscopy. After replication, heterochromatin recondenses, restoring the chromonema structure. Thus, we show that heterochromatin replication in interphase nuclei of giant-genome plants induces a global nuclear reorganization.

Cloning and functional characterization of lignan glycosyltransferase gene IiUGT349 in Isatis indigotica

Based on the transcriptome data of Isatis indigotica, a total of 110 putative glycosytransferases were identified. Through prokaryotic expression and enzymic activity assay in vitro, a novel lignan glycosyltransferase gene was screened out and named IiUGT349, which catalyzed lariciresinol into lariciresinol-4-O-β-D-glucoside and lariciresinol-4'-O-β-D-glucoside. Bioinformatics analysis suggested that IiUGT349 contained an open reading frame(ORF) of 1 401 bp encoding a protein of 467 amino acids. A protein analysis indicated that IiUGT349 have a predecited molecular weight of 52.77 kDa and pI of 5.96. Phylogenetic analysis showed that IiUGT349 belonging to UGT90 family shared low amino acid sequence identity with the reported lignan glycosyltransferases, which may represent a novel type of lignan glycosyltransferases. Quantitative real-time PCR(qRT-PCR) analysis showed that IiUGT349 was expressed in roots, stems, young leaves and leaves, with the highest expression level in stems. Further biochemical analysis showed that the optimal reaction time of IiUGT349 recombinant protein was 12 h and the optimal temperature was 45 ℃. Subcellular localization demonstrated that IiUGT349 was located in the cytoplasm and nucleus of plants. In this study, a new glucosyltransferase gene IiUGT349 from I. indigotica belonging to the UGT90 family was cloned, which laid a foundation to further investigate its' function and elucidate the lignan glycosides biosynthesis pathway and plays an important role for great significance for the synthetic biology of active lignan glycosides.

Songs in the Key of Life

Songs in the Key of Life

Comprehensive Effects of Flowering Locus T-Mediated Stem Growth in Tobacco.

In flowering plants, Flowering locus T (FT) encodes a major florigen. It is a key flowering hormone in controlling flowering time and has a wide range of effects on plant development. Although the mechanism by which FT promotes flowering is currently clearly understood, comprehensive effects of the FT gene on plant growth have not been evaluated. Therefore, the effects of FT on vegetative growth need to be explored for a complete understanding of the molecular functions of the FT gene. In this study, the Jatropha curcas L. FT gene was overexpressed in tobacco (JcFTOE) in order to discover multiple aspects and related mechanisms of how the FT gene affects plant development. In JcFTOE plants, root, stem, and leaf development was strongly affected. Stem tissues were selected for further transcriptome analysis. In JcFTOE plants, stem growth was affected because of changes in the nucleus, cytoplasm, and cell wall. In the nucleus of JcFTOE plants, the primary effect was to weaken all aspects of DNA replication, which ultimately affected the cell cycle and cell division. The number of stem cells decreased significantly in JcFTOE plants, which decreased the thickness and height of tobacco stems. In the cell wall of JcFTOE plants, hemicellulose and cellulose contents increased, with the increase in hemicellulose associated with up-regulation of xylan synthase-related genes expression. In the cytoplasm of JcFTOE plants, the primary effects were on biogenesis of ribonucleoprotein complexes, photosynthesis, carbohydrate biosynthesis, and the cytoskeleton. In addition, in the cytoplasm of JcFTOE plants, there were changes in certain factors of the core oscillator, expression of many light-harvesting chlorophyll a/b binding proteins was down-regulated, and expression of fructose 1,6-bisphosphatase genes was up-regulated to increase starch content in tobacco stems. Changes in the xylem and phloem of JcFTOE plants were also identified, and in particular, xylem development was affected by significant increases in expression of irregular xylem genes.

Ran-GTP/-GDP-dependent nuclear accumulation of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 and TGACG-BINDING FACTOR2 controls salicylic acid-induced leaf senescence.

Leaf senescence is the final stage of leaf development and can be triggered by various external factors, such as hormones and light deprivation. In this study, we demonstrate that the overexpression of the GTP-bound form of Arabidopsis (Arabidopsis thaliana) Ran1 (a Ras-related nuclear small G-protein, AtRan1) efficiently promotes age-dependent and dark-triggered leaf senescence, while Ran-GDP has the opposite effect. Transcriptome analysis comparing AtRan1-GDP- and AtRan1-GTP-overexpressing transgenic plants (Ran1T27Nox and Ran1G22Vox, respectively) revealed that differentially expressed genes (DEGs) related to the senescence-promoting hormones salicylic acid (SA), jasmonic acid, abscisic acid, and ethylene (ET) were significantly upregulated in dark-triggered senescing leaves of Ran1G22Vox, indicating that these hormones are actively involved in Ran-GTP/-GDP-dependent, dark-triggered leaf senescence. Bioinformatic analysis of the promoter regions of DEGs identified diverse consensus motifs, including the bZIP motif, a common binding site for TGACG-BINDING FACTOR (TGA) transcription factors. Interestingly, TGA2 and its interactor, NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), which are two positive transcriptional regulators of SA signaling, differed in their extent of accumulation in the nucleus versus cytoplasm of Ran1T27Nox and Ran1G22Vox plants. Moreover, SA-induced, Ran-GTP-/-GDP-dependent functions of NPR1 included genome-wide global transcriptional reprogramming of genes involved in cell death, aging, and chloroplast organization. Furthermore, the expression of AtRan1-GTP in SA signaling-defective npr1 and SA biosynthesis-deficient SA-induction deficient2 genetic backgrounds abolished the effects of AtRan1-GTP, thus retarding age-promoted leaf senescence. However, ET-induced leaf senescence was not mediated by Ran machinery-dependent nuclear shuttling of ETHYLENE-INSENSITIVE3 and ETHYLENE-INSENSITIVE3-LIKE1 proteins. We conclude that Ran-GTP/-GDP-dependent nuclear accumulation of NPR1 and TGA2 represents another regulatory node for SA-induced leaf senescence.

Homologous chromosome pairing in meiosis of higher eukaryotes-still an enigma?

Meiosis is the basis of the generative reproduction of eukaryotes. The crucial first step is homologous chromosome pairing. In higher eukaryotes, micrometer-scale chromosomes, micrometer distances apart, are brought together by nanometer DNA sequences, at least a factor of 1000 size difference. Models of homology search, homologue movement, and pairing at the DNA level in higher eukaryotes are primarily based on studies with yeast where the emphasis is on the induction and repair of DNA double-strand breaks (DSB). For such a model, the very large nuclei of most plants and animals present serious problems. Homology search without DSBs cannot be explained by models based on DSB repair. The movement of homologues to meet each other and make contact at the molecular level is not understood. These problems are discussed and the conclusion is that at present practically nothing is known of meiotic homologue pairing in higher eukaryotes up to the formation of the synaptonemal complex, and that new, necessarily speculative models must be developed. Arguments are given that RNA plays a central role in homology search and a tentative model involving RNA in homology search is presented. A role of actin in homologue movement is proposed. The primary role of DSBs in higher eukaryotes is concluded to not be in paring but in the preparation of Holliday junctions, ultimately leading to chromatid exchange.

Cajal body dynamics in soybean root meristem cells under chilling stress and recovery

Cajal bodies (CBs) are nuclear domains widely present in eukaryotic cells. Their main functions include processing and modifications of small nuclear RNAs (snRNAs), including those participating in mRNA splicing. CBs are dynamic structures in terms of their mobility, fusion and split, which results in their variable number and size during cell cycle. CBs are supposed to play a role in response to various stresses or at least the cell reactions to these stressors is related with changing CB number and size. That is why the number and size of CBs were analyzed in primary root meristematic cells of soybean, a subtropical plant, whose seedlings were grown at 23 °C (the control), subjected to chilling stress (10 °C) and recovered at 23 °C after chilling. These parameters were examined in three classes of cell size which corresponded to the cells in G1, S and G2 phases. The general number of CBs in nuclei of the chilled plants was halved in comparison to the control cells, however this parameter returned to the control value during recovery. Such a result suggests that chilling may hinder formation of CBs or it may provoke their fusion. Moreover, the number of small CBs decreased and the number of large CBs increased during interphase progress, while the general number of CBs was constant in each treatment group. The constant number of CBs from G1 to G2 phase proves that in soybean cells they enlarge as a result of component accumulation rather than their fusion. The reasons of such a state of soybean CBs are discussed.

Physiological and biochemical aspects of the functioning of Arg-X proteolytic system in the cell nuclei of plants

Plants are structural and biochemical systems in which at each moment of ontogenesis are established inherent only to this plant, the ratio of growth, biorhythmic and morphogenetic processes. The conjugacy of circadian rhythm and the activity of chromatin-modifying enzymes is becoming increasingly important. In this aspect, histones play an important role, since they are able to fine-tune transcriptional programs through a series of accurately organized post-translational modifications that combinatorially regulate chromatin function. It is proved that almost all histones are proteolytically modified. It was shown that differences in the exhibition of activity Arg-X proteolysis between spring and winter seedlings are observed mainly in the fractions of core histones at the level of chromatin tightly bound. The preferential localization of Arg-X protease activity was found in the histone (H3 + H4) block as spring and winter varieties. In addition, activity Arg-X proteolysis at the level of nuclear matrix correlated with the growth characteristics of spring and winter seedlings in coleoptiles and roots growing by cell stretching. Results can be viewed as an illustration of a multi-layered regulatory proteinase network involved in proteolytic processing of nuclear proteins, taking part in circadian spatial and temporal structural changes of chromatin states.

LIPID COMPOSITION OF CALLUSES CELL NUCLEI OF PLANTS OF DIFFERENT SYSTEMATIC GROUPS

The study revealed the results of the comparative analysis of the deoxyribonucleoproteid complex (chromatin) lipid composition in the callus and explant nuclei of plants, belonging to different systematic groups (winter rye, potatoes, peas). It provided the data on the phospho- and neutral lipid composition of the investigated plants and showed that the phospholipid content in the explants from dormant winter rye seed embryos and potato stems, having low metabolic activity, is significantly higher than in the calluses in the stage of active proliferation. Both winter rye and potato calluses tend to have a lower share of neutral lipids, including sterols, in their composition. The revealed factors can be used as markers of metabolic activity of the plant cell biology objects (callus and cell cultures).

Parasites structuring ecological communities: The mistletoe footprint in Mediterranean pine forests

Abstract The capacity of parasitic plants in structuring natural communities is increasingly recognized. These plants can affect the structure, composition and productivity of plant communities by modifying the competitive balance between hosts and non‐host species and by altering the quantity and quality of resources entering the soil. Despite the progress made in this field, there is still a lack of integrative studies showing the structuring capacity of parasitic plants in forest ecosystems, where their effect may be less detectable due to the long life span of the system. In this study we evaluate the long‐term impact of Viscum album subsp. austriacum on the woody‐plant community of a Mediterranean pineland. This mistletoe remains several years on the same host, exerting long‐lasting, spatially concentrated effects on community and ecosystem characteristics. Mistletoe concentrates zoochorous seeds and induces changes in the soil fertility and light availability beneath the canopy of parasitized trees, which have the potential to facilitate zoochorous–plant colonization, recruitment, and growth at the same time as it weakens the host. Here, we analyse whether mistletoe‐driven changes could result in a nucleus of zoochorous woody plants nourished by the abundant organic detritus accumulated under the host. We also analyse whether mistletoe effects can expand after host death. We selected unparasitized, parasitized, and dead parasitized Pinus nigra trees, in which we studied the joint effect of mistletoe‐mediated changes in soil nutrient and light availability, with the seed rain, seed predation, seedling establishment, plant recruitment, and plant growth. Light‐ and soil‐nutrient resources were greater under parasitized trees, and intensified after host death. The seed rain was maximum under parasitized trees, where seedling recruitment proved more likely. Sapling density, richness, and growth increased with the development of parasitism. Our findings show that V. album exerts a strong and lasting impact on the structure and dynamics of Mediterranean pinelands, with parasitized trees acting as centres for the establishment and growth of colonizing fleshy‐fruited woody species, which, over the long term, promote vegetation shifts by limiting dominant pine trees and facilitating less represented fleshy‐fruited shrubs. A plain language summary is available for this article.

Ultrastructural organization of the domains in the cell nucleus of dicotyledonous and monocotyledonous plants under abiotic stress

The transformation of the structural organization of interphase nuclei of a plant cell, depending on the type of tissue, ploidy, and the action of abiotic factors of the medium, has been studied by light and transmission electron microscopy. It is shown that the location and the quantitative relationship between condensed and decondensed chromatin, the presence and localization of nuclear bodies in the plant nucleus, and the presence of invaginations of the nuclear membrane and inclusions depend on the tissue type and cell age as well as the intensity, time, and type of exposure to abiotic factors. Examples of different degrees of chromatin condensation in one plant organism are given. It has been established that the degree of chromatin condensation and decondensation, the state of other domains and nonspecific inclusions of the cell nucleus can be artificially modeled for research purposes or subsequent modification. The issue of application of such technologies for the creation and selection of stable forms of agricultural plants taking into account the controlled modification of their genome is discussed. A possible mechanism for incorporation of nonspecific inclusions in a nuclear compartment is proposed.

Cell Biology of the Plant Nucleus.

The eukaryotic nucleus is enclosed by the nuclear envelope, which is perforated by the nuclear pores, the gateways of macromolecular exchange between the nucleoplasm and cytoplasm. The nucleoplasm is organized in a complex three-dimensional fashion that changes over time and in response to stimuli. Within the cell, the nucleus must be viewed as an organelle (albeit a gigantic one) that is a recipient of cytoplasmic forces and capable of morphological and positional dynamics. The most dramatic reorganization of this organelle occurs during mitosis and meiosis. Although many of these aspects are less well understood for the nuclei of plants than for those of animals or fungi, several recent discoveries have begun to place our understanding of plant nuclei firmly into this broader cell-biological context.

Abstract IA23: Targeting nuclear noncoding RNAs

Abstract RNA interference (RNAi) is well known as a mechanism for controlling mammalian mRNA translation in the cytoplasm, but what would be the consequences if it also functioned in cell nuclei? Although RNAi has also been found in nuclei of plants, yeast, and other organisms, there has been relatively little progress towards understanding the potential involvement of mammalian RNAi factors in nuclear processes including transcription and splicing. When RNAi factors bind small RNAs, they form ribonucleoprotein complexes that can be selective for target sequences within different classes of nuclear RNA substrates. The versatility of nuclear RNAi may supply a previously underappreciated layer of regulation to transcription, splicing, and other nuclear processes. This talk focuses on recent work targeting noncoding RNA at the frataxin and C9orf72 loci as examples for how nuclear RNAi can be used to control gene expression. Citation Format: David Corey. Targeting nuclear noncoding RNAs. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr IA23.

Three-Dimensional, Live-Cell Imaging of Chromatin Dynamics in Plant Nuclei Using Chromatin Tagging Systems.

In plants, chromatin dynamics spatiotemporally change in response to various environmental stimuli. However, little is known about chromatin dynamics in the nuclei of plants. Here, we introduce a three-dimensional, live-cell imaging method that can monitor chromatin dynamics in nuclei via a chromatin tagging system that can visualize specific genomic loci in living plant cells. The chromatin tagging system is based on a bacterial operator/repressor system in which the repressor is fused to fluorescent proteins. A recent refinement of promoters for the system solved the problem of gene silencing and abnormal pairing frequencies between operators. Using this system, we can detect the spatiotemporal dynamics of two homologous loci as two fluorescent signals within a nucleus and monitor the distance between homologous loci. These live-cell imaging methods will provide new insights into genome organization, development processes, and subnuclear responses to environmental stimuli in plants.

Regulation of mammalian transcription and splicing by Nuclear RNAi.

RNA interference (RNAi) is well known as a mechanism for controlling mammalian mRNA translation in the cytoplasm, but what would be the consequences if it also functions in cell nuclei? Although RNAi has also been found in nuclei of plants, yeast, and other organisms, there has been relatively little progress towards understanding the potential involvement of mammalian RNAi factors in nuclear processes including transcription and splicing. This review summarizes evidence for mammalian RNAi factors in cell nuclei and mechanisms that might contribute to the control of gene expression. When RNAi factors bind small RNAs, they form ribonucleoprotein complexes that can be selective for target sequences within different classes of nuclear RNA substrates. The versatility of nuclear RNAi may supply a previously underappreciated layer of regulation to transcription, splicing, and other nuclear processes.

SYBR Green-activated sorting of Arabidopsis pollen nuclei based on different DNA/RNA content.

Key message: Purification of pollen nuclei. Germ cell epigenetics is a critical topic in plants and animals. The male gametophyte (pollen) of flowering plants is an attractive model to study genetic and epigenetic reprogramming during sexual reproduction, being composed of only two sperm cells contained within, its companion, vegetative cell. Here, we describe a simple and efficient method to purify SYBR Green-stained sperm and vegetative cell nuclei of Arabidopsis thaliana pollen using fluorescence-activated cell sorting to analyze chromatin and RNA profiles. The method obviates generating transgenic lines expressing cell-type-specific fluorescence reporters and facilitates functional genomic analysis of various mutant lines and accessions. We evaluate the purity and quality of the sorted pollen nuclei and analyze the technique's molecular basis. Our results show that both DNA and RNA contents contribute to SYBR Green-activated nucleus sorting and RNA content differences impact on the separation of sperm and vegetative cell nuclei. We demonstrate the power of the approach by sorting wild-type and polyploid mutant sperm and vegetative cell nuclei from mitotic and meiotic mutants, which is not feasible using cell-type-specific transgenic reporters. Our approach should be applicable to pollen nuclei of crop plants and possibly to cell/nucleus types and cell cycle phases of different species containing substantially different amounts of DNA and/or RNA.

Further observations on the activity of Y chromosome heterochromatin in Rumex thyrsiflorus

Observation of premeiotic nuclei of plants containing different numbers (from one to five) of Y chromosomes reveal that is such plants prior to meiosis all Y chromosomes are in fuzzy state, while in nuclei of tapetal cells or nuclei from anther stalk the number of big chromocentres is directly correlated with the number of Y chromosomes in given plants. This finding provides a further indication of genetic activity of Y chromosomes in this particular stage of the life cycle (premeiosis). Another indication of genetical activity of Y chromosomes was obtained from analysis of the rate of RNA synthesis in premeiosis. It was found that PMC's in the last premeioitc interphase exhibit a high rate of 3H-uridine incorporation. Thus in PMC's the fuzzy appearance of Y chromosomes coincides with intensive RNA synthesis.

Sense Transgene-Induced Post-Transcriptional Gene Silencing in Tobacco Compromises the Splicing of Endogenous Counterpart Genes

Sense transgene-induced post-transcriptional gene silencing (S-PTGS) is thought to be a type of RNA silencing in which ARGONAUTE1 directs the small interfering RNA (siRNA)-mediated cleavage of a target mRNA in the cytoplasm. Here, we report that the altered splicing of endogenous counterpart genes is a main cause for the reduction of their mature mRNA levels. After the S-PTGS of a tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) gene, 3′-truncated, polyadenylated endo-NtFAD3 transcripts and 5′-truncated, intron-containing endo-NtFAD3 transcripts were detected in the total RNA fraction. Although transcription proceeded until the last exon of the endogenous NtFAD3 gene, intron-containing NtFAD3 transcripts accumulated in the nucleus of the S-PTGS plants. Several intron-containing NtFAD3 transcripts harboring most of the exon sequences were generated when an endogenous silencing suppressor gene, rgs-CaM, was overexpressed in the S-PTGS plants. These intron-containing NtFAD3 splice variants were generated in the presence of NtFAD3 siRNAs that are homologous to the nucleotide sequences of these splice variants. The results of this study indicate that the inhibition of endo-NtFAD3 gene expression is primarily directed via the alteration of splicing and not by cytoplasmic slicer activity. Our results suggest that the transgene and intron-containing endogenous counterpart genes are differentially suppressed in S-PTGS plants.

Localization of Daucus carota NMCP1 to the nuclear periphery: the role of the N-terminal region and an NLS-linked sequence motif, RYNLRR, in the tail domain

Recent ultrastructural studies revealed that a structure similar to the vertebrate nuclear lamina exists in the nuclei of higher plants. However, plant genomes lack genes for lamins and intermediate-type filament proteins, and this suggests that plant-specific nuclear coiled-coil proteins make up the lamina-like structure in plants. NMCP1 is a protein, first identified in Daucus carota cells, that localizes exclusively to the nuclear periphery in interphase cells. It has a tripartite structure comprised of head, rod, and tail domains, and includes putative nuclear localization signal (NLS) motifs. We identified the functional NLS of DcNMCP1 (carrot NMCP1) and determined the protein regions required for localizing to the nuclear periphery using EGFP-fused constructs transiently expressed in Apium graveolens epidermal cells. Transcription was driven under a CaMV35S promoter, and the genes were introduced into the epidermal cells by a DNA-coated microprojectile delivery system. Of the NLS motifs, KRRRK and RRHK in the tail domain were highly functional for nuclear localization. Addition of the N-terminal 141 amino acids from DcNMCP1 shifted the localization of a region including these NLSs from the entire nucleus to the nuclear periphery. Using this same construct, the replacement of amino acids in RRHK or its preceding sequence, YNL, with alanine residues abolished localization to the nuclear periphery, while replacement of KRRRK did not affect localization. The sequence R/Q/HYNLRR/H, including YNL and the first part of the sequence of RRHK, is evolutionarily conserved in a subclass of NMCP1 sequences from many plant species. These results show that NMCP1 localizes to the nuclear periphery by a combined action of a sequence composed of R/Q/HYNLRR/H, NLS, and the N-terminal region including the head and a portion of the rod domain, suggesting that more than one binding site is implicated in localization of NMCP1.

Ago1 Interacts with RNA Polymerase II and Binds to the Promoters of Actively Transcribed Genes in Human Cancer Cells

Argonaute proteins are often credited for their cytoplasmic activities in which they function as central mediators of the RNAi platform and microRNA (miRNA)-mediated processes. They also facilitate heterochromatin formation and establishment of repressive epigenetic marks in the nucleus of fission yeast and plants. However, the nuclear functions of Ago proteins in mammalian cells remain elusive. In the present study, we combine ChIP-seq (chromatin immunoprecipitation coupled with massively parallel sequencing) with biochemical assays to show that nuclear Ago1 directly interacts with RNA Polymerase II and is widely associated with chromosomal loci throughout the genome with preferential enrichment in promoters of transcriptionally active genes. Additional analyses show that nuclear Ago1 regulates the expression of Ago1-bound genes that are implicated in oncogenic pathways including cell cycle progression, growth, and survival. Our findings reveal the first landscape of human Ago1-chromosomal interactions, which may play a role in the oncogenic transcriptional program of cancer cells.