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Abstract Background Animal and yeast proteins containing long coiled-coil domains are involved in attaching other proteins to the large, solid-state components of the cell. One subgroup of long coiled-coil proteins are the nuclear lamins, which are involved in attaching chromatin to the nuclear envelope and have recently been implicated in inherited human diseases. In contrast to other eukaryotes, long coiled-coil proteins have been barely investigated in plants. Results We have searched the completed Arabidopsis genome and have identified a family of structurally related long coiled-coil proteins. Filament-like plant proteins (FPP) were identified by sequence similarity to a tomato cDNA that encodes a coiled-coil protein which interacts with the nuclear envelope-associated protein, MAF1. The FPP family is defined by four novel unique sequence motifs and by two clusters of long coiled-coil domains separated by a non-coiled-coil linker. All family members are expressed in a variety of Arabidopsis tissues. A homolog sharing the structural features was identified in the monocot rice, indicating conservation among angiosperms. Conclusion Except for myosins, this is the first characterization of a family of long coiled-coil proteins in plants. The tomato homolog of the FPP family binds in a yeast two-hybrid assay to a nuclear envelope-associated protein. This might suggest that FPP family members function in nuclear envelope biology. Because the full Arabidopsis genome does not appear to contain genes for lamins, it is of interest to investigate other long coiled-coil proteins, which might functionally replace lamins in the plant kingdom.

Tomato MAF1 (LeMAF1) is a plant-specific, nuclear envelope (NE)-associated protein. It is the founding member of a group of WPP domain-containing, NE-associated proteins. This group includes the Arabidopsis WPP family, which is involved in cell division, as well as plant RanGAPs. In addition to its NE localization, LeMAF1 accumulates in speckles in the cytoplasm. Here, we show that the LeMAF1-containing speckles are components of the Golgi apparatus. A novel tomato coiled-coil protein was identified that specifically binds to LeMAF1. Tomato WPP domain-associated protein (LeWAP) interacts in yeast and in vitro through its coiled-coil domain with several WPP-domain containing proteins, including AtRanGAP1 and the WPP family (LeMAF, WPP1 and WPP2). Like LeMAF1, LeWAP is localized at the Golgi. Moreover, we present data showing that Arabidopsis WAP is necessary for the existence of a multi-protein complex containing WPP2.

A cDNA for a novel plant protein was isolated from tomato. Nuclear Matrix Protein 1 (NMP1) is a ubiquitously expressed 36 kDa protein, which has no homologues in animals and fungi, but is highly conserved among flowering and non-flowering plants, including gymnosperms, moss, and the liverwort Marchantia polymorpha. NMP1 is predominantly alpha-helical with multiple stretches of short amphipathic regions. Cell fractionation, immunofluorescence, and GFP localization experiments showed that NMP1 is located both in the cytoplasm and nucleus and that the nuclear fraction is associated with the nuclear matrix. NMP1 is a candidate for a plant-specific structural protein with a function both in the nucleus and cytoplasm.

Several repetitive sequences of the genome of Beta procumbens Chr. Sm., a wild beet species of the section Procumbentes of the genus Beta have been isolated. According to their genomic organization, the repeats were assigned to satellite DNA and families of dispersed DNA sequences. The tandem repeats are 229-246 bp long and belong to an AluI restriction satellite designated pAp11. Monomers of this satellite DNA form subfamilies which can be distinguished by the divergence or methylation of an internal restriction site. The satellite is amplified in the section Procumbentes, but is also found in species of the section Beta including cultivated beet (Beta vulgaris). The existence of the pAp11 satellite in distantly related species suggests that the AluI sequence family is an ancient component of Beta genomes and the ancestor of the diverged satellite subfamily pEV4 in B. vulgaris. Comparative fluorescent in-situ hybridization revealed remarkable differences in the chromosomal position between B. procumbens and B. vulgaris, indicating that the pAp11 and pEV4 satellites were most likely involved in the expansion or rearrangement of the intercalary B. vulgaris heterochromatin. Furthermore, we describe the molecular structure, and genomic and chromosomal organization of two repetitive DNA families which were designated pAp4 and pAp22 and are 1354 and 582 bp long, respectively. The families consist of sequence elements which are widely dispersed along B. procumbens chromosomes with local clustering and exclusion from distal euchromatic regions. FISH on meiotic chromosomes showed that both dispersed repeats are colocalized in some chromosomal regions. The interspersion of repeats of the pAp4 and pAp22 family was studied by PCR and enabled the determination of repeat flanking sequences. Sequence analysis revealed that pAp22 is either derived from or part of a long terminal repeat (LTR) of an Athila-like retrotransposon. Southern analysis and FISH with pAp4 and pAp22 showed that both dispersed repeats are species-specific and can be used as DNA probes to discriminate parental genomes in interspecific hybrids. This was tested in the sugar beet hybrid PRO1 which contains a small B. procumbens chromosome fragment.

Background: Animal and yeast proteins containing long coiled-coil domains are involved in attaching other proteins to the large, solid-state components of the cell. One subgroup of long coiled-coil proteins are the nuclear lamins, which are involved in attaching chromatin to the nuclear envelope and have recently been implicated in inherited human diseases. In contrast to other eukaryotes, long coiled-coil proteins have been barely investigated in plants., Results: We have searched the completed Arabidopsis genome and have identified a family of structurally related long coiled-coil proteins. Filament-like plant proteins (FPP) were identified by sequence similarity to a tomato cDNA that encodes a coiled-coil protein which interacts with the nuclear envelope-associated protein, MAF1. The FPP family is defined by four novel unique sequence motifs and by two clusters of long coiled-coil domains separated by a non-coiled-coil linker. All family members are expressed in a variety of Arabidopsis tissues. A homolog sharing the structural features was identified in the monocot rice, indicating conservation among angiosperms., Conclusion: Except for myosins, this is the first characterization of a family of long coiled-coil proteins in plants. The tomato homolog of the FPP family binds in a yeast two-hybrid assay to a nuclear envelope-associated protein. This might suggest that FPP family members function in nuclear envelope biology. Because the full Arabidopsis genome does not appear to contain genes for lamins, it is of interest to investigate other long coiled-coil proteins, which might functionally replace lamins in the plant kingdom.

We have constructed a sugar beet bacterial artificial chromosome (BAC) library of the chromosome mutant PRO1. This Beta vulgaris mutant carries a single chromosome fragment of 6-9 Mbp that is derived from the wild beet Beta procumbens and is transmitted efficiently in meiosis and mitosis. The library consists of 50,304 clones, with an average insert size of 125 kb. Filter hybridizations revealed that approximately 3.1% of the clones contain mitochondrial or chloroplast DNA. Based on a haploid genome size of 758 Mbp, the library represents eight genome equivalents. Thus, there is a greater than 99.96% probability that any sequence of the PROI genome can be found in the library. Approximately 0.2% of the clones hybridized with centromeric sequences of the PRO1 minichromosome. Using the identified BAC clones in fluorescence in situ hybridization experiments with PRO1 and B. procumbens chromosome spreads, their wild-beet origin and centromeric localization were demonstrated. Comparative Southern hybridization of pulsed-field separated PROI DNA and BAC inserts indicate that the centromeric region of the minichromosome is represented by overlapping clones in the library. Therefore, the PRO1 BAC library provides a useful tool for the characterization of a single plant centromere and is a valuable resource for sugar beet genome analysis.

The interaction of chromatin with the nuclear matrix via matrix attachment region (MAR) DNA is considered to be of fundamental importance for chromatin organization in all eukaryotic cells. MAR binding filament-like protein 1 (MFP1) from tomato is a novel plant protein that specifically binds to MAR DNA. Its filament protein-like structure makes it a likely candidate for a structural component of the nuclear matrix. MFP1 is located at nuclear matrix-associated, specklelike structures at the nuclear envelope. Here, we report the identification of a novel protein that specifically interacts with MFP1 in yeast two-hybrid and in vitro binding assays. MFP1 associated factor 1 (MAF1) is a small, soluble, serine/threonine-rich protein that is ubiquitously expressed and has no similarity to known proteins. MAF1, like MFP1, is located at the nuclear periphery and is a component of the nuclear matrix. These data suggest that MFP1 and MAF1 are in vivo interaction partners and that both proteins are components of a nuclear substructure, previously undescribed in plants, that connects the nuclear envelope and the internal nuclear matrix.

Recently, it has been suggested that nuclear processes, such as replication, transcription, and splicing, are spatially organized and associated with a nuclear framework called the nuclear matrix, a structure of unknown molecular composition. It has been shown that chromatin is attached to the nuclear matrix via specific DNA fragments called matrix attachment regions (MARs). We have begun to dissect the plant nuclear matrix by isolating a DNA binding protein with specific affinity for MARs. Here, it is shown that MAR binding filament-like protein 1 (MFP1) is associated with specklelike structures at the nuclear periphery that are part of isolated nuclei and the nuclear matrix. A predicted N-terminal transmembrane domain is necessary for the specific targeting of MFP1 to the speckles, indicating an association with the nuclear envelope-endoplasmic reticulum continuum. In addition, it is shown that a marker protein for plant microtubule organizing centers, which has been shown to be localized on the outside of the plant nuclear envelope, is also part of the nuclear matrix. These findings indicate a close and previously undescribed connection in plants between the nuclear envelope and the internal nuclear matrix, and they suggest a function for MFP1 in attaching chromatin to specific sites at the nuclear periphery.