Dmc1 is a candidate for temperature tolerance during wheat meiosis.

The meiotic recombination gene Dmc1 on wheat chromosome 5D has been identified as a candidate for the maintenance of normal chromosome synapsis and crossover at low and possibly high temperatures. We initially assessed the effects of low temperature on meiotic chromosome synapsis and crossover formation in the hexaploid wheat (Triticum aestivum L.) variety 'Chinese Spring'.

Genome-wide identification of physically clustered genes suggests chromatin-level co-regulation in male reproductive development in Arabidopsis thaliana.

Co-expression of physically linked genes occurs surprisingly frequently in eukaryotes. Such chromosomal clustering may confer a selective advantage as it enables coordinated gene regulation at the chromatin level. We studied the chromosomal organization of genes involved in male reproductive development in Arabidopsis thaliana.

An NPF transporter exports a central monoterpene indole alkaloid intermediate from the vacuole.

Plants sequester intermediates of metabolic pathways into different cellular compartments, but the mechanisms by which these molecules are transported remain poorly understood. Monoterpene indole alkaloids, a class of specialized metabolites that includes the anticancer agent vincristine, antimalarial quinine and neurotoxin strychnine, are synthesized in several different cellular locations. However, the transporters that control the movement of these biosynthetic intermediates within cellular compartments have not been discovered.

Isolation of Nuclei and Nucleoli.

Here we describe methods for producing nuclei from Arabidopsis suspension cultures or root tips of Arabidopsis, wheat, or pea. These methods could be adapted for other species and cell types. The resulting nuclei can be further purified for use in biochemical or proteomic studies, or can be used for microscopy.

Immunolabeling and In Situ Labeling of Isolated Plant Interphase Nuclei.

Specific labeling of proteins and nucleic acids by immunofluorescence or in situ techniques is an important adjunct to microscopical analysis for cell biology. Labeling of nuclear structures in intact complex tissues is often hampered by problems of penetration of the macromolecular labeling reagents needed. Here we describe a method of labeling isolated plant nuclei that we have found to be a useful approach that can help to overcome these problems.

Cell differentiation and development in Arabidopsis are associated with changes in histone dynamics at the single-cell level.

The mechanism whereby the same genome can give rise to different cell types with different gene expression profiles is a fundamental problem in biology. Chromatin organization and dynamics have been shown to vary with altered gene expression in different cultured animal cell types, but there is little evidence yet from whole organisms linking chromatin dynamics with development. Here, we used both fluorescence recovery after photobleaching and two-photon photoactivation to show that in stem cells from Arabidopsis thaliana roots the mobility of the core histone H2B, as judged by exchange dynamics, is lower than in the surrounding cells of the meristem.

Immunofluorescence detection of callose deposition around plasmodesmata sites.

Accumulation of callose (β-1,3 glucans) at the plasmodesmata (PD) neck region dynamically regulates symplastic intercellular transport. Here we describe a 2-3-day immuno-labelling protocol to determine callose levels in the cell wall region at PD. The method relies on exposure of internal cell walls by hand-sectioning of the sample and digestion of the cell wall with enzymes in order to improve antibody penetration to deep tissue layers.

Physical clustering of FLC alleles during Polycomb-mediated epigenetic silencing in vernalization.

Vernalization, the promotion of flowering by cold, involves Polycomb-mediated epigenetic silencing of FLOWERING LOCUS C (FLC). Cold progressively promotes cell-autonomous switching to a silenced state. Here, we used live-cell imaging of FLC-lacO to monitor changes in nuclear organization during vernalization.

Symplastic intercellular connectivity regulates lateral root patterning.

Cell-to-cell communication coordinates the behavior of individual cells to establish organ patterning and development. Although mobile signals are known to be important in lateral root development, the role of plasmodesmata (PD)-mediated transport in this process has not been investigated. Here, we show that changes in symplastic connectivity accompany and regulate lateral root organogenesis in Arabidopsis.

The Ph1 locus suppresses Cdk2-type activity during premeiosis and meiosis in wheat.

Despite possessing multiple sets of related (homoeologous) chromosomes, hexaploid wheat (Triticum aestivum) restricts pairing to just true homologs at meiosis. Deletion of a single major locus, Pairing homoeologous1 (Ph1), allows pairing of homoeologs. How can the same chromosomes be processed as homologs instead of being treated as nonhomologs? Ph1 was recently defined to a cluster of defective cyclin-dependent kinase (Cdk)-like genes showing some similarity to mammalian Cdk2.

Plant U13 orthologues and orphan snoRNAs identified by RNomics of RNA from Arabidopsis nucleoli.

Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs whose main function in eukaryotes is to guide the modification of nucleotides in ribosomal and spliceosomal small nuclear RNAs, respectively. Full-length sequences of Arabidopsis snoRNAs and scaRNAs have been obtained from cDNA libraries of capped and uncapped small RNAs using RNA from isolated nucleoli from Arabidopsis cell cultures. We have identified 31 novel snoRNA genes (9 box C/D and 22 box H/ACA) and 15 new variants of previously described snoRNAs.

Aberrant mRNA transcripts and the nonsense-mediated decay proteins UPF2 and UPF3 are enriched in the Arabidopsis nucleolus.

The eukaryotic nucleolus is multifunctional and involved in the metabolism and assembly of many different RNAs and ribonucleoprotein particles as well as in cellular functions, such as cell division and transcriptional silencing in plants. We previously showed that Arabidopsis thaliana exon junction complex proteins associate with the nucleolus, suggesting a role for the nucleolus in mRNA production. Here, we report that the plant nucleolus contains mRNAs, including fully spliced, aberrantly spliced, and single exon gene transcripts.

Dynamic behavior of Arabidopsis eIF4A-III, putative core protein of exon junction complex: fast relocation to nucleolus and splicing speckles under hypoxia.

Here, we identify the Arabidopsis thaliana ortholog of the mammalian DEAD box helicase, eIF4A-III, the putative anchor protein of exon junction complex (EJC) on mRNA. Arabidopsis eIF4A-III interacts with an ortholog of the core EJC component, ALY/Ref, and colocalizes with other EJC components, such as Mago, Y14, and RNPS1, suggesting a similar function in EJC assembly to animal eIF4A-III. A green fluorescent protein (GFP)-eIF4A-III fusion protein showed localization to several subnuclear domains: to the nucleoplasm during normal growth and to the nucleolus and splicing speckles in response to hypoxia.

Preparation of Arabidopsis nuclei and nucleoli.

We describe a method for isolating nuclei from cultured Arabidopsis cells. The same method can be used to further isolate nucleoli. Cell walls are first digested to yield protoplasts, which are purified by flotation on a Percoll gradient.

A distant coilin homologue is required for the formation of cajal bodies in Arabidopsis.

Cajal bodies (CBs) are subnuclear bodies that are widespread in eukaryotes, being found in mammals, many other vertebrates and in all plant species so far examined. They are mobile structures, moving, fusing, and budding within the nucleus. Here we describe a screen for Arabidopsis mutants with altered CBs and describe mutants that have smaller Cajal bodies (ncb-2, ncb-3), lack them altogether (ncb-1), have increased numbers of CBs (pcb) or have flattened CBs (ccb).

Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions.

The eukaryotic nucleolus is involved in ribosome biogenesis and a wide range of other RNA metabolism and cellular functions. An important step in the functional analysis of the nucleolus is to determine the complement of proteins of this nuclear compartment. Here, we describe the first proteomic analysis of plant (Arabidopsis thaliana) nucleoli, in which we have identified 217 proteins.