J. G. Lafontaine

Nuclear genes encoding chloroplast hemoglobins in the unicellular green alga Chlamydomonas eugametos.

When the green unicellular alga Chlamydomonas eugametos is grown under light/dark regimes, nuclear genes are periodically activated in response to the changes in light conditions. These genetic responses are dependent upon the activation of genes associated with photosynthesis (LI616 and LI637), nonphotosynthetic photoreceptors (LI410 and LI818) and the biological clock (LI818). We report here that the LI410 and LI637 genes are part of a small gene family encoding hemoglobins (Hbs) related to those from two unicellular eukaryotes, the ciliated protozoa Paramecium caudatum and Tetrahymena pyriformis, and from the cyanobacterium Nostoc commune. Investigations of the intracellular localization of C. eugametos Hbs by means of immunogold electron microscopy indicate that these proteins are predominantly located in the chloroplast, particularly in the pyrenoid and the thylakoid region. To our knowledge, this constitutes the first evidence for the presence of Hbs in chloroplasts. Alignment of the LI637 cDNA nucleotide sequence with its corresponding genomic sequence indicates that the L1637 gene contains three introns, the positions of which are compared with those in the Hb genes of plants, animals and the ciliate P. caudatum. Although the LI637 gene possesses a three-intron/four-exon pattern similar to that of plant leghemoglobin genes, introns are inserted at different positions. Similarly the position of the single intron in the P. caudatum gene differs from the intron sites in the LI637 gene. The latter observations argue against the current view that all eukaryotic Hbs have evolved from a common ancestor having a gene structure identical to that of plant or animal Hbs.

In vivo studies on the nuclear behavior of the arbuscular mycorrhizal fungusGigaspora rosea grown under axenic conditions

SummaryThe distribution and fate of nuclei of the arbuscular-my-corrhizal fungusGigaspora rosea during late stages of axenic cultures were studied in fixed cultures by transmitted light, conventional and confocal laser scanning microscopy, and in live cultures with two-photon fluorescence microscopy. Mature specimens not yet showing apical septation displayed oval-shaped nuclei localized in lateral positions of the hypha all along the germ-tube length. Beside these, round-shaped nuclei were found to migrate along the central germ-tube core. Some (rare) germ-tube areas, delimited by septa and containing irregularly shaped, much brighter fluorescent nuclei were also found. Specimens that had just initiated the septation process after germ-tube growth arrest displayed round or oval-shaped nuclei in several portions of the germ tubes. These hyphal areas often alternated with other septa-delimited cytoplasmic clusters which contained distorted, brightly fluorescent nuclei. Completely septated specimens mostly lacked nuclei along their germ tubes. However, highly fluorescent chromatin masses appeared within remnants of cytoplasmic material, often compressed between close septa. Our results provide a first clear picture of the in vivo distribution of nuclei along arbuscular mycorrhizal fungal germ tubes issued from resting spores, and suggest that selective areas of their coenocytic hyphae are under specific, single nuclear control. They indicate as well that random autolytic processes occur along senescingG. rosea germ tubes, probably as a consequence of the absence of a host root signal for mycorrhizal formation. Finally, the data presented here allow us to envisage the fate of nuclei released by the germinating spore after nonsymbiotic fungal growth arrest.

Effect of Nikkomycin Z, a chitin-synthase inhibitor, on hyphal growth and cell wall structure of two arbuscular-mycorrhizal fungi

SummaryDifferent concentrations of Nikkomycin Z, a competitive inhibitor of chitin-synthase, were applied to the arbuscular-mycorrhizal fungi (AMF)Gigaspora margarita andGlomus intraradices under in vitro conditions. These two fungi are known to differ in the structure and composition of their cell wall. The two AMF were able to grow in the presence of a higher concentration of this antibiotic than so far reported for other fungi. Fluorescence, electron microscopic and cytochemical studies showed that the blocking of the fungal chitin-synthase activity induces alterations in hyphal morphology, a reduction in fungal wall thickness, and several other changes in the hyphal wall structure and organization. The possible role of chitin-synthase in the hyphal growth and morphogenesis of these symbiotic fungi and its putative regulation by the host plant during the symbiosis are discussed.

Chitinase: Differential induction of gene expression and enzyme activity by drought stress in the wild (Lycopersicon chilense Dun.) and cultivated (L. esculentum Mill.) tomatoes

Summary We have previously reported the isolation of a drought and abscisic acid (ABA)-induced chitinase gene from Lycopersicon chilense , a drought tolerant wild tomato (Chen et al., 1994. Mol. Gen. Genet. 245: 195–202). We have therefore carried out a comparative study in order to examine chitinase gene expression and enzyme activity in Lycopersicon chilense (LA2747 and LA1930) and L. esculentum (cultivar Starfire) during water stress. Both enzyme assay and northern blot analysis revealed that chitinase expression was differentially induced by drought in the two species. Higher induction of chitinase occurred in tolerant species compared to the sensitive one. Among the genotypes examined, L. chilense LA2747 which is the most drought tolerant, presented the highest level of chitinase induction, while the lowest level was found in L. esculentum , Starfire. Similar results were obtained using cell suspensions treated with ABA and mannitol. Both ABA treatment and osmotic stress induced chitinase gene expression in the cell suspensions and this induction was more pronounced in the wild tomato compared to the cultivated one. Drought induced accumulation of chitinase mRNA was investigated in L. chilense , LA2747 leaves, stems and roots. The results revealed that the chitinase gene was organ specifically expressed during drought stress. Leaves of drought stressed plants showed the highest expression and roots showed the lowest. The accumulation of chitinase during drought stress was also confirmed by in situ hybridization. Leaf water potentials (LWP) were determined in the three genotypes during drought stress. A correlation was found between the chitinase gene expression and leaf water potential during drought stress. The highest level of leaf water potential was maintained in L. chilense LA2747 which also demonstrates the highest level of chitinase expression among the three genotypes. Our results suggest that the L. chilense chitinase gene might be involved in drought tolerance of this species.

Identification and immunolocalization of a 65 kDa drought induced protein in cultivated tomatoLycopersicon esculentum

SummaryA 65 kDa protein with a pI value of 5.2 accumulated gradually in tomato leaves during water stress. Protein levels returned to those of the control upon rehydration of the plants. Antiserum raised against the protein, purified from two dimensional electrophoresis gels, was obtained and used as a probe to localize the protein in tomato leaves by immunofluorescence and immunogold labeling. The protein was found to be mainly localized in different areas of nuclei (peripheral chromatin masses, nucleoli and nucleoplasm), chloroplasts, and some leaf cell cytoplasmic regions. Quantification of the gold labeling clearly demonstrated that the amount of the protein increased significantly in nuclei and chloroplasts of cells in drought-stressed plants. Cytological changes occurring in leaf tissues during water stress are also reported.

Presence of small-nuclear-ribonucleoprotein-containing nuclear bodies in quiescent and early germinatingZea mays embryos

SummaryNucleolus-associated bodies (NABs) occur in interphase nuclei of many plant species. The present work shows that, inZea mays, NABs are present in dry seeds as well as in germinating tissues. The frequency of these nuclear bodies remains more or less constant during the first 24 h of imbibition but decreases significantly during the next 24 h. By the time the nucleolus reaches maturation and contains granular zones, these bodies are still found in close association with the surface of this organelle, as is the case in mature root meristematic cells. Immunocytochemical observations on both dry seeds and germinating tissues further revealed that NABs reacted positively with a monoclonal antibody (mAbK121) recognizing the m3G cap of sn(small nuclear)RNAs. It is, therefore, concluded that the NABs present in such tissues already contain components characterizing snRNPs (small nuclear ribonucleoproteins) in mature tissues. The possible function of NABs as storage deposits of snRNPs in dry seeds and early germinating tissues is discussed. In view of their many similarities with the coiled bodies described in both animal and plant cells, it is most likely that NABs correspond to those structures.

Immunocytochemical localization of nuclear antigens in the unicellular green alga,Chlamydomonas reinhardtii, processed by cryofixation and freeze-substitution

SummaryWe describe the preparation of monoclonal antibodies to nuclear antigens in the green alga,Chlamydomonas reinhardtii, and their localization at the light and electron microscope level. Supernatants from hybridomas were screened by the ELISA method and the four antibodies giving the strongest signal were subjected to further analysis. At the LM level immunogold silver staining was used on semi-thick resinless sections. We have examined at the EM level the distribution of these antigens by post-embedding immunocytochemical techniques on sections of conventionally fixed specimens compared to cryofixed and freeze-substituted ones. Enhanced ultrastructural preservation was observed in cells which were cryofixed, freeze-substituted and embedded at −35°C in Lowicryl K4M. Different preparative procedures involving cryofixation and substitution are described. Of the four antibodies three were localized under light and electron microscopy. All three were distributed in the interchromatin space. One of these antigens (QUL4D2, 54 kDa) is also found in the dense fibrillar component and fibrillar centers of the nucleolus.

Composition of nuclear dense bodies and nucleolus-associated bodies in interphase nuclei of the unicellular green alga Chlamydomonas reinhardtii

Summary— The interphase nucleus of the green alga, Chlamydomonas reinhardtii, displayed two types of bodies some of them, the dense bodies, lying apparently free in the nucleoplasm while the others were attached to the nucleolus and were, therefore, referred to as nucleolus‐associated bodies (NABs). The presence of DNA, RNA and histones in dense bodies was investigated by means of post‐embedding immunocytochemistry and cytochemistry using a monoclonal antibody to single and double stranded DNA, a polyclonal antibody to rye H3 histones and RNase A‐gold complexes. The dense bodies were shown to contain significant amounts of RNA but neither DNA nor histones were detected; their composition was thus similar to that of the dense bodies described in higher plant cells. We propose that dense bodies might be implicated in the assembly of the 25 to 45 nm granules observed throughout the nucleoplasm of Chalamydomonas interphase nuclei. The composition of NABs was found to be distinct from that of the dense bodies since they were labeled by the antibody to DNA, specially in cryofixed and cryosubstituted specimens. The presence of DNA in NABs together with their intimate association to the nucleolus suggest that they may correspond to specific segments of chromosomes.

Ultrastructural, cytochemical and immunocytochemical investigation of the interphase nucleus in the unicellular green alga Chlamydomonas reinhardtii

Summary— The ultrastructural organization of the interphase nucleus of the green alga Chlamydomonas reinhardtii was investigated and found to be largely dependent on the fixation conditions. In specimens stained with bismuth, densely contrasted granules ranging from 25 to 45 nm in diameter were localized throughout the interchromatin space and often formed clusters. These granules were labeled by RNase A‐gold complexes and may represent the counterparts of animal and higher plant cll interchromatin granules. Within the nucleolus the Ag‐NOR and pyroantimonate stains and, to a lesser extent, the bismuth stain reacted with the nucleolar dense fibrillar component (DFC). When cells were subjected to a heat shock at 42°C, the nucleolar DFC was found to progressively separate from the nucleolus and, after 3 h, appeared as a continuous meandering thread about 0.1 μm in width. Within the nucleolus, labeling on conventional preparations occurred as small clusters with antibodies to H3 histones or to DNA whereas RNase A‐gold complexes labeled most of it including fibrillar centers. Improved ultrastructural preservation in cryofixed, cryosubstituted specimens gently fixed in glutaraldehyde permitted to localize nucleolar DNA predominantly at the outer edge of fibrillar centers and to a lesser extent within the neighbouring DFC. Our results indicate that the structure and composition of Chlamydomonas interphase nuclei are comparable, despite particularities, to those of animal and higher plant nuclei.

An electron microscope thick section study of endomembrane organization in the myxamoebae ofPhysarum polycephalum

SummaryThe post-fixation ofPhysarum polycephalum myxamoebae with a zinc iodide-osmium tetroxide mixture resulted in the accumulation of an electron dense deposit in the lumen of the nuclear envelope, the endoplasmic reticulum, and most of the cisternae of the Golgi apparatus. In double-stained thin sections of impregnated myxamoebae the preservation of other cytoplasmic organelles was excellent. Examination of thick sections (0.25 μm) at 120 kV revealed the complexity of the endomembrane system and continuities between both the ER and the nuclear envelope, and the ER and the Golgi apparatus. No direct continuities were observed between the cisternae of the Golgi apparatus and the nuclear envelope. A three dimensional view of membrane organization was obtained from stereopairs, while tilting the sections at much steeper angles revealed whether any of the apparent continuities seen were real or were simply the result of overlap. A morphologically distinct region of the ER, which bears similarities to the GERL region described in other organisms, was found in continuity with the remaining ER, the Golgi apparatus, and the nuclear envelope.