A cDNA encoding a nucleolar protein was selected from a pea (Pisum sativum) plumule library, cloned, and sequenced. l h e translated sequence of the cDNA has significant percent identity t o Xenopus Iaevis nucleolin (31 %), the alfalfa (Medicago safiva) nucleolin homolog (66%), and the yeast (Saccbaromyces cerevisiae) nucleolin homolog (NSR1) (28%). It also has sequence patterns in its primary structure that are characteristic of all nucleolins, including an N-terminal acidic motif, RNA recognition motifs, and a C-terminal Gly-and Arg-rich domain. By immunoblot analysis, the polyclonal antibodies used t o select the cDNA bind selectively to a 90-kD protein in purified pea nuclei and nucleoli and t o an 88-kD protein i n extracts of Escberichia coli expressing the cDNA. I n immunolocalization assays of pea plumule cells, the antibodies stained primarily a region surrounding the fibrillar center of nucleoli, where animal nucleolins are typically found. Southern analysis indicated that the pea nucleolin-like protein is encoded by a single gene, and northern analysis showed that the labeled cDNA binds to a single band of RNA, approximately the same size as the cDNA.After irradiation of etiolated pea seedlings by red light, the mRNA level in plumules decreased during the 1 st hour and then increased t o a peak of six times the o-h level at 12 h. Far-red light reversed this effect of red light, and the mRNA accumulation from red/far-red light irradiation was equal t o that found in the dark control. This indicates that phytochrome may regulate the expression of this gene.Nuclear rRNA synthesis occurs in the nucleolus and is the first step in ribosome biogenesis. A number of nucleolar proteins are thought to play important roles in rRNA synthesis, early rRNA processing, and ribosome assembly. Among the best characterized of these is nucleolin, a multifunctional protein typically concentrated in the transition zone between the fibrillar center and the dense fibrillar component of nucleoli (Scheer et al., 1993).Most of 'what is known about the structure and function of nucleolin comes from animal studies. The most recent study of a plant nucleolin-like protein is that of Bogre et al. (1996), who cloned several nucleolin-like cDNAs in alfalfa and identified a 95-kD nucleolin-like protein by immuno-

We analysed the presence and localization of spectrin-like proteins in nuclei of various plant tissues, using several anti-erythrocyte spectrin antibodies on isolated pea nuclei and nuclei in cells. Western blots of extracted purified pea nuclei show a cross-reactive pair of bands at 220-240 kDa, typical for human erythrocyte spectrin, and a prominent 60 kDa band. Immunolocalization by means of confocal laser scanning microscopy reveals spectrin-like proteins in distinct spots equally distributed in the nucleoplasm and over the nuclear periphery, independent of the origin of the anti-spectrin antibodies used. In some nuclei tracks of spectrin-like proteins are also observed. No signal is present in nucleoli. The amount and intensity of signal increases when nuclei were extracted, successively, with detergents, DNase I and RNase A, and high salt, indicating that the spectrin-like protein is associated with the nuclear matrix. The labelling is similar in nuclei of various plant tissues. These data are the first that show the presence and localization of spectrin-like epitopes in plant nuclei, where they may stabilize specific interchromatin domains.

To obtain intbrination on plant microtubuic stability to low temperature and Ca^*, the regulatory domain of polymerized tubulin from maize {Zea mays cv. Black Mexican Sweet) was dissected by limited proteolysis with snbtilisin. Tubulin in taxol-stabilized microtubules was cleaved in a subtilisin concentration-and time-dependent manner. Immunoblotting of microtubules with antibodies having mapped epitopes on a-and /^tubnlins revealed that eleavage initially removed <15 residues IVom the /3-tubulin carboxyl terminus to prodnec a/i^-microtubules. Subsequent cleavage occurred at an extreme site and an internal site within the a-tubniin carboxyl terminus. Electron microscopy revealed that a/3s-microtnbules were ultrastructurally indistinguishable from uncleaved control a^microtubules. Quantitative polymer sedimentation showed that low temperature treatment (0 °C) caused significant depolymcrization of a/J-microtnbules, but little depolymerization of a/Jj-microtubules. Ca^* enhanced the cold-induced depolymerization of both aft-and a/Js-microtubnles. However, a/iJ^-microtubules were significantly more stable to depolymerization by cold and Ca^* than were a/iJ-niicrotubules. The results showed that maize microtubules containing shortened /^tubiilin carboxyl termini are relatively resistant to the combined depolymerizing effects of cold and Ca^*. Thus, the extreme carboxyl terminus of /3-tubulin is a crucial element of the plant tubulin regulatory domain and may be involved in the modulation of microtubule stability during the chilling response in plants.

In immunoblot assays, at least three putative nuclear intermediate filament (NIF) proteins were detected in nuclear envelope-matrix (NEM) and lamin (L1) fractions of nuclei from plumules of dark-grown pea (Pisum sativum L.) seedlings. These NIF proteins had apparent molecular masses of ca. 65, 60, and 54 kDa (also referred to as p65, p60, and p54), and appeared as multiple isoelectric forms, with pIs ranging from ca. 4.8 to 6.0. Polyclonal and monoclonal antibodies were raised to the 65-kDa NIF protein bands excised from gels after electrophoresis. These anti-pea antibodies were specifically cross-reactive with the pea nuclear p65, p60, and p54 proteins and also with chicken lamins. Sequence alignment of peptide fragments obtained from the 65- and 60-kDa pea NIF proteins showed similarity with animal intermediate filament proteins such as lamins and keratins and with certain plant proteins predicted to have long coiled-coil domains. These pea NIF proteins were further purified and enriched from the NEM fraction using methods similar to those used for isolating animal lamins. When negatively stained and viewed by transmission electron microscopy, the filaments in the pea lamin (L1) fraction appeared to be 6-12 nm in diameter. As assayed by immunofluorescence cytochemistry using a confocal laser-scanning microscope, fixed pea plumule cells displayed uniform as opposed to peripheral nuclear staining by several of the antibody preparations, both polyclonal and monoclonal. This report describes the biochemical and immunological properties of these pea NIF proteins.

Plant cells have a well organized nucleus and nuclear matrix, but lack orthologues of the main structural components of the metazoan nuclear matrix. Although data is limited, most plant nuclear structural proteins are coiled-coil proteins, such as the NIFs (nuclear intermediate filaments) in Pisum sativum that cross-react with anti-intermediate filament and anti-lamin antibodies, form filaments 6-12 nm in diameter in vitro, and may play the role of lamins. We have investigated the conservation and features of NIFs in a monocot species, Allium cepa, and compared them with onion lamin-like proteins. Polyclonal antisera against the pea 65 kDa NIF were used in 1D and 2D Western blots, ICM (imunofluorescence confocal microscopy) and IEM (immunoelectron microscopy). Their presence in the nuclear matrix was analysed by differential extraction of nuclei, and their association with structural spectrin-like proteins by co-immunoprecipitation and co-localization in ICM. NIF is a conserved structural component of the nucleus and its matrix in monocots with Mr and pI values similar to those of pea 65 kDa NIF, which localized to the nuclear envelope, perichromatin domains and foci, and to the nuclear matrix, interacting directly with structural nuclear spectrin-like proteins. Its similarities with some of the proteins described as onion lamin-like proteins suggest that they are highly related or perhaps the same proteins.