A proline-, threonine-, and glycine-rich protein down-regulated by drought is localized in the cell wall of xylem elements.

A cDNA clone encoding a proline-, threonine-, and glycine-rich protein (PTGRP) was isolated from a wild tomato species (Lycopersicon chilense) (L.X. Yu, H. Chamberland, J.G. Lafontain, Z. Tabaeizadeh [1996] Genome 39: 1185-1193). Northern-blot analysis and in situ hybridization studies revealed that PTGRP is down-regulated by drought stress. The level of the mRNA in leaves and stems of 8-d drought-stressed plants decreased 5- to 10-fold compared with that in regularly watered plants. The mRNA re-accumulated when drought-stressed plants were rewatered. Antibodies raised against a glutathione S-transferase/PTGRP fusion protein were used to elucidate the subcellular localization of the protein by immunogold labeling. In regularly watered L. chilense plants, PTGRP protein was found to be localized in xylem pit membranes and disintegrated primary walls. Examination of sections from drought-stressed plants revealed a significant decrease in the levels of labeling. In these samples, only a few scattered gold particles were detected in the same areas. In the leaf tissues of plants that had been rewatered for 3 d following an 8-d drought stress, the labeling pattern was similar to that of the regularly watered plants. To our knowledge, PTGRP is the first drought-regulated protein that has been precisely localized in the cell wall.

Detection of a homologue of bcl-2 in plant cells.

An emerging family of bcl-2-like genes has been identified from nematode to humans. These genes play a role in the maintenance of homeostasis. Its members have highly conserved domains that are important for their dimerization. Since nothing is known about the importance of these genes in plant cells, we have investigated their presence in an alga as well as in three higher plants both by Western analysis and by immunocytochemistry. Immunoblots revealed the presence of a protein immunoreacting with the anti-bcl-2 polyclonal antibody in leaves of tobacco plants. Furthermore, immunocytochemical localization has shown that this protein is mainly associated with mitochondria, plastids, and nuclei of plant cells. Taken together, our results suggest that bcl-2 is a protein highly conserved throughout evolution.

The enzyme involved in sulfation of the turgorin, gallic acid 4-O-(beta-D-glucopyranosyl-6'-sulfate) is pulvini-localized in Mimosa pudica.

A sulfotransferase (ST) which catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to gallic acid glucoside was characterized from microsomal preparations of Mimosa pudica. The product of the reaction was found to co-elute on HPLC with the periodic leaf movement factor 1 (PLMF-1)(gallic acid beta-D-gluco-pyranosyl-6'-sulfate). The distribution of the enzyme activity was restricted to plasma membrane preparations from primary, secondary and tertiary pulvini. The M. pudica ST activity was inhibited in a dose-dependent manner in the presence of an antibody raised against the flavonol 3-sulfotransferase of Flaveria chloraefolia, suggesting structural similarities between the two proteins. Western blot analysis of M. pudica protein extracts using these antibodies indicated the presence of a cross-reactive polypeptide with an apparent molecular mass of 42,000 Da whose distribution correlates with the presence of the gallic acid glucoside ST activity. Indirect immunogold labeling of resin-embedded sections from tertiary pulvini showed a specific localization of gold particles on the sieve-tube plasma membranes. The label distribution was uniform and other cellular organelles and membrane systems displayed little or no labeling. The results of the Western blot and immunocytochemical studies are consistent with the detection of the gallic acid glucoside ST activity in plasma membrane preparations of M. pudica pulvini cells. The specific tissue distribution of the ST in motor organ phloem cells suggests that this is the site of synthesis and/or accumulation of PLMF-1 and supports the proposed hypothesis that PLMF-1 may be acting as a chemical signal during the seismonastic response of M. pudica.

Negative regulation of gene expression of a novel proline-, threonine-, and glycine-rich protein by water stress in Lycopersicon chilense.

We have isolated a full length cDNA clone (designated PTGRP) encoding a proline-rich protein from leaves of Lycopersicon chilense. Sequence analysis of the 552-bp insert revealed that the open reading frame encodes a 12.6-kDa protein. The deduced amino acid sequence of PTGRP consists of a C-terminal proline-rich domain with two identical repeat motifs Phe-Pro-Met-Pro-Thr-Thr-Pro-Ser-Thr-Gly-Gly-Gly-Phe-Pro-Ser. The N terminus lacks proline and is hydrophobic. Unlike other proline-rich proteins this protein contains five glycine-rich repeat motifs (Gly-X)n representative of glycine-rich proteins. Southern blot analysis showed that PTGRP is a member of a small gene family within the L. chilense genome. Northern blot experiments revealed that the PTGRP gene is significantly down regulated by water stress. PTGRP mRNA transcription decreased 5- to 10-fold in leaves and stems after 4-8 days of water stress. The mRNA reaccumulated when the drought-stressed plants were rewatered. The in situ hybridization experiments also revealed that PTGRP mRNAs were more abundant in leaf sections of plants watered regularly compared with those of plants submitted to water stress. Down regulation of the PTGRP gene was also observed in desiccated cell suspensions of L. chilense and in those treated with abscisic acid, mannitol, and NaCl. Based on the common features of proline-rich proteins (high proline content, repeated motifs, and a putative signal peptide) and their involvement in the cell wall, it is likely that the PTGRP protein is targeted to the cell wall. Its down regulation by drought could be correlated with the remodeling of the plant cell wall in response to water stress.

Relationship of nucleolus-associated bodies with the nucleolar organizer tracks in plant interphase nuclei (Pisum sativum).

Nucleolus-associated bodies (NABs) have long been noted in interphase nuclei of a wide variety of plant species. We have recently shown that these bodies consist largely of snRNPs and that they are located on the nucleolar surface in the immediate vicinity of the nucleolar organizer tracks. The present study revealed that, following exposure of roots to KCN, an agent that induces nucleolar segregation, NABs were intimately associated with intranucleolar chromatin. Although immunocytochemical tests with anti-DNA indicated that NABs contained no demonstrable amounts of DNA, our observations nevertheless add further support to the notion that these bodies are somehow related to the nucleolar chromosomes.

Far upstream activating promoter regions are responsible for expression of the BnC1 cruciferin gene from Brassica napus.

Cruciferin is the major seed storage protein in Brassica napus. As much as 1.9 kbp of the BnC1 cruciferin gene promoter have been sequenced and analyzed. Promoter fragments with 5' deletions from -2500 to -v202 were fused with the ß-glucuronidase reporter gene and used for Nicotiana tabacum transformation. ß-glucuronidase could be specifically expressed in transgenic tobacco seeds under the control of the BnC1 promoter and regulatory elements were found to be dispersed over 1903 bp. An almost 5-fold increase in ß-glucuronidase expression was obtained when the promoter length was increased from -379 to -498, and another 10-fold increase was observed when sequences between -1266 and -1903 were added. Histochemical analysis shows that the region between -844 and -1266 directs the expression of the chimeric gene specifically to the root apical meristem.

Ultrastructural localization of L-fucose residues in nuclei of root primordia of the green pea Pisum sativum.

Sugars have been demonstrated in animal cell nuclei, but only a few studies have mentioned their presence in plant cell nuclei. In this study L-fucose residues were localized at the ultrastructural level, using Ulex europeaus agglutinin I lectin, during the early stages of germination of Pisum sativum and in mature root tip cells. This sugar was present after 1 h of germination, and its concentration was found to vary during 3 to 6 h inhibition; after 72h of inhibition its concentration had more than doubled. Furthermore, labelling was particularly abundant in the nucleolus, nucleolus-associated bodies and dense nuclear bodies. The possibility that some of the L-fucose residues are associated with proteins is discussed.

Ultrastructural localization of DNA and RNA in Allium porrum interphase cells by means of nuclease-gold complexes.

Nucleic acids have been localized in Allium porrum interphase meristematic cells by means of labelling with nuclease-gold complexes, a technique which provides high resolution and improved specificity. DNase-gold labelling was observed over dense chromatin and to a lesser extent over dispersed chromatin. Nucleolar labelling was restricted to the dense fibrillar component, very few particles being located over the fibrillar centres. Labelling by the RNase-gold complex was present over both the cytoplasm and the nucleoplasm. Cytoplasm labelling was intense over the rough endoplasmic reticulum but absent over vacuoles. In the nucleoplasm many gold particles were located at the border between the condensed and the dispersed chromatin. Nucleolar labelling was intense over the granular zones but many gold particles were also seen over the dense fibrillar component. Fibrillar centres showed, however, no labelling with the RNase-gold complex. These results are consistent with previous autoradiographic and cytochemical observations carried out on the same plant material.

Changes in protein and RNA during asexual differentiation of Physarum polycephalum.

Some of the events during the growth and asexual differentiation of Physarum polycephalum amoebae are described. Encysted amoebae contain low levels of protein and RNA. When these cells are mixed with bacteria and inoculated onto agar plates, there is an increase in cellular RNA content followed by an increase in protein content. The cellular RNA and protein contents of all strains tested decrease during the subsequent cell divisions. In nondifferentiating cells (strain Cl at 30 degrees C and strain LU648), RNA and protein contents continue to decrease, and the cell eventually encyst. In the asexually differentiating strain Cl grown at 26 degrees C, the cellular RNA and protein contents stop decreasing and begin to increase when the first amoebae become committed to form plasmodia. At early stages of differentiation a new 36,000 molecular weight polypeptide appears. In fully formed plasmodia another polypeptide of 38,000 molecular is observed. These two plasmodial-specific polypeptides are among the most abundant plasmodial proteins.

An ultracytochemical study of nucleolar organization in meristematic plant cells (Allium porrum).

The interphase nucleolus in Allium porrum, as in many of the plant species studied so far, is highly heterogeneous in ultrastructure owing to the presence of coarse, contorted, thread-like structures, or nucleolonemata. Each nucleolonema appears to be sharply twisted and to give rise to a skein within the nucleolar mass. In order to characterize further these nucleolar components, a variety of cytochemical techniques were exploited. For that purpose, specimens were mostly fixed in 4% formaldehyde and stained in the block according to procedures known to reveal the presence of nucleic acids or proteins. Certain specimens were also digested with deoxyribonuclease, ribonuclease or proteinase K before staining. By staining with phosphotungstic acid or bismuth oxynitrate, the presence of a high concentration of proteins can be demonstrated within thin (0.15 micrometer), filamentous structures which are believed to correspond to the outer region of the nucleolonema. Such convoluted formations disappear upon sufficiently long extraction with proteinase K. Using Bernhard's regressive staining technique for chromatin, the distribution of this substance throughout the nucleolar mass was found to match closely that of the nucleolonemata as revealed by several other procedures. As a last test for investigating the cytochemical make-up of the nucleolus, blocks of tissues were stained with 3,3'-diaminobenzidine, a substance known to react specifically with nucleic acids. When such specimens are digested with ribonuclease for 1 h, there persist within the nucleolus, fibrillogranular zones the localization of which is highly reminiscent of that of the nucleolonemata. Combination of ribonuclease hydrolysis with subsequent treatment with proteinase K (30 min) induces the extraction of a large proportion of the nucleolar material, the persisting loose and rather evenly distributed fibrils exhibiting a diamter of 3-5 nm. The possibility is considered that these units may correspond to chromatin fibrils although they have most likely been displaced from their original localization during the extraction procedures. Our cytochemical data suggest that, in Allium porrum, the nucleolonema is approximately 0.3 micrometer in diameter and may consist of a central axis from which chromatin loops project radially. A possible interpretation for the presence of protein-rich, 0.1 micrometer-thick, annular structures throughout the nucleolonemal skein is that the newly synthesized RNP products are accumulated transiently at the extremities of these loops before migrating to the immediately adjacent granular nucleolar zones.

A cytochemical and radioautographic study of the ultrastructural organization of puff-like fibrillar structures in plant interphase nuclei (Allium porrum).

Loose, fibrillar, spherical structures have been observed during recent years in interphase nuclei of both animal and plant cells. These nuclear formations have been referred to as karyosomes, fibrillar bodies, micropuffs and centromeres. In order to gain further information on the nature of these structures, a cytochemical and radioautographic investigation was undertaken using plant meristematic cells (Allium porrum). For that purpose roots were fixed with either formaldehyde or glutaraldehyde in order to carry out cytochemical tests for DNA, RNA and proteins. Certain of the preparations were also first digested with DNase, RNase or proteinase K and then stained according to different procedures. Other specimens were labelled with thymidine for high-resolution radioautographic observations. Staining with diaminobenzidine (DAB) revealed that these nuclear puff-like formations consisted partly of a loose fibrillar meshwork containing nucleic acids. Part of this fine fibrillar reticulum persisted whether the preparations were digested with DNase or RNase before staining with DAB, thus indicating that these nuclear structures contained both DNA and RNA. The fact that these formations incorporate thymidine furnished additional support for the view that they correspond to specific chromosome segments. Staining with ethanolic phosphotungstic acid or digestion of specimens with proteinase K showed that these loose fibrillar structures also consisted of proteins. Judging from their ultrastructure, their association with the chromatin reticulum as well as from their cytochemical characteristics, these nuclear formations most likely correspond to centromeres. In view of the presence of DNA within these structures, it is possible to distinguish them from other equally spherical nuclear formations, observed in certain plant species, that have generally been referred to as karyosomes or micronucleoli and that appear to consist of ribonucleoproteins.

Analysis of plant genomes. V. Comparative study of molecular properties of DNAs of seven Allium species.

The genomes of seven plant species belonging to the genus Allium and exhibiting a threefold variation in their nuclear DNA content were analyzed by studying their reassociation kinetics, equilibrium centrifugation behavior in neutral CsCl gradients, and melting properties. The reassociation kinetics experiments revealed the presence of 44-65% repeated DNA sequences. A comparison between DNA contents and the proportion of repeated DNA sequences indicated that, in Allium, increase in the genome size is not exclusively due to variations in the proportions of repetitive DNA. The total DNA as well as the various repetitive DNA fractions in all the Allium species examined exhibited, in spite of a few differences, a gross similarity in their behavior in neutral CsCl gradients and in their melting properties.

Analysis of plant genomes. III. Denaturation and reassociation properties of cryptic satellite DNAs in barley (Hordeum vulgare) and wheat (Triticum aestivum).

A cryptic satellite fraction was isolated from barley and wheat by preparatory ultracentrifugation of total DNA in Ag+-Cs2SO4 density gradients and was characterized by studying its denaturation-reassociation properties. Wheat satellite DNA underwent thermal denaturation as a single component with a Tm of 81 degrees C while barley satellite DNA consisted of one major (Tm = 82.5 degrees C) and one minor (Tm = 91 degrees C) component. When the barley and wheat satellites were reassociated and then melted, the Tm values were found to be 6--7 degrees C lower than those of the corresponding native DNA preparations. Examination of the C0t curves of these two satellite DNAs revealed the presence of a major, fast reassociating and a minor, slow reassociating fraction. The fast reassociating DNA fraction of barley was found to have a complexity of 9.7 . 10(5) daltons while that of wheat satellite was 5.8 . 10(5) daltons. Since these satellites reassociated with about 4--5% base mismatching, as judged by their deltsTm (6--7 degrees C), they each appear to consist of rather similar base sequences.

Analysis of plant genomes. IV. Isolation and characterization of satellite DNA components from two dicotyledons cucumber (Cucumis sativus) and radish (Raphanus sativus).

Satellite DNA fractions from cucumber and radish, two plants having low DNA contents and relatively small chromosomes, were isolated and characterized. Reassociation studies of satellite and total nuclear DNA showed that the satellite fractions in these two plants contain most of the rapidly reassociating DNA. Cucumber satellite I was found to contain one major component (70% of the total satellite) having a density of 1.706 g/cm3 and a Tm of 90.5 degrees C and a minor component with a density of 1.712 g/cm3 and a Tm of 93.5 degrees C. The complexity of the major component was estimated to be 3.8 X 10(5) daltons while that of the minor one was 12.9 X 10(7) daltons. Although cucumber satellite II banded as a single peak at a density of 1.700 g/cm3 in neutral CsCl gradients, it was observed to have a rather broad denaturation profile with a Tm of 86.5 degrees C. Its Cot curve was also broader than that of satellite I and one of its components (40% of the total) had a complexity of 5.8 X 10(5) daltons. Two satellite fractions were also observed in the case of radish DNA but only satellite I was isolated in a pure form and characterized. This radish satellite formed a sharp, symmetrical peak at a density of 1.698 g/cm3 in neutral CsCl gradients and underwent denaturation in a narrow temperature range of 6 to 7 degrees C. An analysis of the optical reassociation kinetics showed that this satellite contained a major and a minor component. The major component, which comprised 80% of the satellite, had a complexity of 12.9 X 10(5) daltons. Hybridization experiments revealed that the ribosomal DNA was present in satellite II.

Comparative study of rye and thymus histones: amino acid analysis and tryptic fingerprinting.

Amino acid composition and tryptic fingerprints of rye (Secal cereale) H1, H2B (PH1), and H2A(PHII) histones indicate the presence of major differences between these and the corresponding calf or rabbit fractions. In addition to variations for other amino acids, fraction H1 from rye contains twice as much arginine as the corresponding animal fraction; the plant H2B (PHI) and H2A (PHII) histones show lysine to arginine ratios greater than those of their animal counterparts. The tryptic maps of the same proteins appear to differ between plants and animals by the number and the general pattern of the peptides, as well as by the quantity and distribution of the arginine-containing peptides. Such results suggest the presence of differences in the primary structure of the calf and rye lysine-rich and moderately lysine-rich histones. Furthermore, the possibility is ruled out that each of these plant histones consists of an animal-like protein with an additional segment of 20--30 amino acid residues. On the other hand, the rye and calf arginine-rich fractions H3 and H4 show similar amino acid compositions and tryptic peptides maps.

Characterization of ribonucleoprotein particles from the protozoa Astasia longa.

Ribonucleoprotein particles released from nucleoli of Astasia longa by treatment with heparin were characterized biochemically. When centrifuged in a sucrose gradient containing an appropriate buffer (Tris/KCl/Mg2+), four populations of particles were obtained, sedimenting at 90 S, 75 S, 60 C, and 44 S, respectively. The first type of particles contained the high molecular weight (3.5-10(6) ribosomal RNA precursor. The RNAs present as the major components in the 75-S and 44-S particles had molecular weights of 1.35-10(6) and 0.85-10(6), respectively, whereas the 60-S particles contained a mixture of 0.85-10(6) and 1.35-10(6) RNA. After a brief labeling, the radioactivity was found in the RNA constituent of the 90-S particles; following a 90 min chase, the label disappeared from this latter fraction and accumulated in the 75-S, 60-S and 44-S particles. This indicates a precursor-product relationship between the RNA of the 90-S particles and that of the three other ribonucleoprotein particles, consistent with the conversion:: 3.5-10(6) RNA LEADS TO 1.35-10(6) RNA+0.85-10(6) RNA.

Ultrastructural and radioautographic investigation of the nucleolar cycle in Physarum polycephalum. Characterization of DNA-containing subunits.

The present study has been mainly focused on the nucleolar cycle in the slime mould Physarum polycephalum. The ultrastructural characteristics of the interphase nucleolus, in this species, are quite similar to those of nucleoli in other organisms: it is essentially constituted of large particulate zones surrounding denser regions which are predominantly fibrillar in texture. The latter nucleolar zones, following fixation with osmium tetroxide, are characterized by the presence of opaque granules approximately 25 nm in diameter. Contrary to the situation which generally prevails in other eukaryotes, the late prophase nucleolus fragments into numerous globular bodies which are recognizable by the presence of opaque particles. These fibrillogranular nucleolar fragments persist during mitosis and are observed to become incorporated in the newly formed nucleolus. High-resolution radioautographic observations reveal that these nucleolar remnants contain DNA. The present observations together with recent biochemical data from other authors on the characteristics and mode of duplication of nucleolar DNA in P. polycephalum have led us to the hypothesis that the nucleolus, in this organism, contains several distinct globular subunits each containing ribosomal DNA as a key component. The existence of such morphological subunits appears to account for the unusual behaviour of the nucleolus during the cell cycle.

A light- and electron-microscope study of nuclear structure throughout the cell cycle in the euglenoid Astasia longa (Jahn).

The structure of nuclei of Astasia longa in synchronized cultures was examined at the light- and electron-microscope levels. Three types of nuclei, differing mainly in chromatin conformation, were observed during interphase and were tentatively classed in the G1, S and G2-periods. The fibrillar nucleolar regions exhibited a most complex organization and appeared to consist of convoluted, coarse filaments or nucleolonemata approximately 0.15 micrometer in diameter. Chromosome condensation was evidenced first by the longer, thicker profiles of chromatin observed in late prophase. Furthermore, the nucleolus, that persists throughout mitosis, began to elongate at late prophase. Furthermore, the nucleolus, that persists thorughout mitosis, began to elongate at this stage, simultaneously with the appearance of short, unoriented profiles of intranuclear microtubules. Chromosome condensation was complete by mid-metaphase and the nucleolus was elongated into a cylindrical shape with irregular extremities. Microtubule profiles were longer than in prophase; they were now oriented parallel to the nucleolus and frequently lay closely appressed to its sides. In anaphase, the chromosomes segregated into 2 groups, one towards each extremity of the dumb-bell-shaped nucleolus. The telophase chromosomes assumed a random orientation with respect to the still intact nucleolus. Throughout the division stages the persiting nucleolus maintained its ultrastructural organization and consisted partly of conspicuous nucleolonemal profiles which tended to be oriented along the major axis of this organelle. Nucleolar separation into 2 fragments occurred late in telophase and was followed by a reformation of daughter nuclei and initiation of cell fission during cytokinesis.

An ultrastructural and radioautographic study of the chromocentric interphase nucleus in plant meristematic cells (Raphanus sativus).

In Raphanus sativus, the mitotic chromosomes are quite short and, on reaching the cell poles, soon undergo extensive unravelling. By late telophase and early interphase, only a few chromosome segments, believed to correspond to the centromeric regions, are still visible in the form of chromocentres closely associated with the nuclear envelope. Although interphase nuclei show little internal structural differentiation, high-resolution radioautography has permitted us to establish which of them have reached the early, mid and late S periods. In early S nuclei, only the nucleolus and the euchromatin which pervades the nuclear cavity become labelled. By the mid S-period, the diffuse chromatin and nucleolus incorporate less thymidine and DNA synthesis is initiated within the peripheral chromocentres. Subsequently, the radioautographic grains become restricted to the chromocentres. The finding that certain late S nuclei exhibit loosely organized chromocentres strongly suggests that these heterochromatic chromosome segments undergo important conformational modifications during DNA replication. Finally, the presence of radioautographic grains over the lacunar regions of the nucleolus in early and mid S nuclei demonstrates that intranucleolar DNA replicates during the earlier portion of the S-period.