Roger Deltour

The Involvement of Respiration in Free Radical Processes during Loss of Desiccation Tolerance in Germinating Zea mays L. (An Electron Paramagnetic Resonance Study).

When germinating Zea mays L. seeds are rapidly desiccated, free radical-mediated lipid peroxidation and phospholipid de-esterification is accompanied by a desiccation-induced buildup of a stable free radical associated with rapid loss of desiccation tolerance. Comparison of the electron paramagnetic resonance and electron nuclear double resonance properties of this radical with those of the radical in dried, desiccation-intolerant moss showed that the two were identical. At the subcellular level, the radical was associated with the hydrophilic fraction resulting from lipid extraction. Isolated mitochondria subjected to drying were also found to accumulate an identical radical in vitro. When increasing concentrations of cyanide were used, a significant positive correlation was shown between rates of respiration and the accumulation of the radical in desiccation-intolerant tissues. Another positive correlation was found when rates of O2 uptake by radicles at different stages of germination were plotted against free radical content following desiccation. This indicates that free radical production is closely linked to respiration in a process likely to involve the desiccation-induced impairment of the mitochondrial electron transport chain to form thermodynamically favorable conditions to induce accumulation of a stable free radical and peroxidized lipids. Modulation of respiration using a range of inhibitors resulted in broadly similar modulation of the buildup of the stable free radical. One site of radical generation was likely to be the NADH dehydrogenase of complex I and probably as a direct consequence of desiccation-impaired electron flow at or close to the ubiquinone pool.

DNA methylation as a key process in regulation of organogenic totipotency and plant neoplastic progression

SummaryProgressive loss of organogenic totipotency appears to be a common event in long-term plant tissue culture. This loss of totipotency, which has been proposed to be a typical trait of plant neoplastic progression, is compared to some mechanisms that occur during the establishment of animal differentiation-resistant cancer lines in vitro. Evidence is presented that alteration in DNA methylation patterns and expression of genes occur during long-term callus culture. An effect of the auxin, 2,4-dichlorophenoxyacetic acid, in the progressive methylation, is moreover suggested. Methylation of genes relevant to cell differentiation and progressive elimination of cells capable of differentiation is proposed as being responsible for this progressive loss of organogenic potential. Finally, the epigenetic alteration (DNA methylation) that occurs during prolonged periods of culture may induce other irreversible genetic alterations that ultimately make the loss of totipotency irreversible.

Nuclear bodies and compartmentalization of pre-mRNA splicing factors in higher plants

We studied the fine structural organization of nuclear bodies in the root meristem during germination of maize and Arabidopsis thaliana using electron microscopy (EM). Cajal bodies (CBs) were observed in quiescent embryos and germinating cells in both species. The number and distribution of CBs were investigated. To characterize the nuclear splicing domains, immunofluorescence labelling with antibodies against splicing factors (U2B″ and m3G-snRNAs) and in situ hybridisation (with U1/U6 antisense probes) were performed combined with confocal microscopy. Antibodies specific to the Arabidopsis SR splicing factor atRSp31 were produced. AtRSp31 was detected in quiescent nuclei and in germinating cells. This study revealed an unexpected speckled nuclear organization of atRSp31 in root epidermal cells where micro-clusters of interchromatin granules were also observed by EM. Therefore, we examined the distribution of green fluorescent protein (GFP)-tagged atRSp31 in living cells after Agrobacterium -mediated transient expression. When expressed transiently, atRSp31-GFP exhibited a speckled distribution in leaf cells. Treatments with α-amanitin, okadaic acid, staurosporine or heat shock induced the speckles to reorganize. Furthermore, we generated stable Arabidopsis transgenics expressing atRSp31-GFP. The distribution of the fusion protein was identical to that of endogenous atRSp31. Three-dimensional time-lapse confocal microscopy showed that speckles were highly dynamic domains over time.

The frequency of plasmodesmata increases early in the whole shoot apical meristem of Sinapis alba L. during floral transition

Abstract. The frequency of plasmodesmata increases in the shoot apical meristem of plants of Sinapis alba L. induced to flower by exposure to a single long day. This increase is observed within all cell layers (L1, L2, L3) as well as at the interfaces between these layers, and it occurs in both the central and peripheral zones of the shoot apical meristem. The extra plasmodesmata are formed only transiently, from 28 to 48 h after the start of the long day, and acropetally since they are detectable in L3 4 h before they are seen in L1 and L2. These observations indicate that (i) in the Sinapis shoot apical meristem at floral transition, there is an unfolding of a single field with increased plasmodesmatal connectivity, and (ii) this event is an early effect of the arrival at this meristem of the floral stimulus of leaf origin. Since (i) the wave of increased frequency of plasmodesmata is 12 h later than the wave of increased mitotic frequency (A. Jacqmard et al. 1998, Plant cell proliferation and its regulation in growth and development, pp. 67–78; Wiley), and (ii) the increase in frequency of plasmodesmata is observed in all cell walls, including in walls not deriving from recent divisions (periclinal walls separating the cell layers), it is concluded that the extra plasmodesmata seen at floral transition do not arise in the forming cell plate during mitosis and are thus of secondary origin.

Cytological study on water stress during germination of Zea mays.

SummaryKernels of Zea mays were subjected to dehydration treatment at various times during germination. Embryos from kernels dehydrated during the first 36 h of germination are resistant to dehydration and subsequently germinate earlier than controls. Dehydration of kernels germinated during 72h leads to an irreversible arrest of growth of the embryos. However, autoradiographic observations showed that these embryos are still able to incorporate [3H] uridine and probably [4-5-3H] lysine. Incorporation of [3H] thymidine does not occur. The effect of dehydration on root ultrastructure was studied. In embryos dehydrated after 24 h and 72 h of germination, condensation of chromatin is seen and association of elements of rough endoplasmic reticulum with vacuoles and glyoxysomes can be noted. These changes are reversible in drought-resistant embryos and irreversible in drought-sensitive embryos. However, more notable changes than those seen after 24 h can be observed in embryos dehydrated after 72 h of germination: mitochondria and proplastids can not be distinguished with certainty, glyoxysomes fuse and preferably dispose at the periphery of the cell. Rehydration of drought-sensitive embryos causes breakdown in plasma and nuclear membranes, which leads to the loss of cellular compartimentalization. Moreover, the chromatin remains definitively condensed and has lost its function of genetic regulation.

Three-dimensional electron microscopy of ribosomal chromatin in two higher plants : a cytochemical, immunocytochemical, and in situ hybridization approach

We report the 3-D arrangement of DNA within the nucleolar subcomponents from two evolutionary distant higher plants, Zea mays and Sinapis alba. These species are particularly convenient to study the spatial organization of plant intranucleolar DNA, since their nucleoli have been previously reconstructed in 3-D from serial ultra-thin sections. We used the osmium ammine-B complex (a specific DNA stain) on thick sections of Lowicryl-embedded root fragments. Immunocytochemical techniques using anti-DNA antibodies and rDNA/rDNA in situ hybridization were also applied on ultra-thin sections. We showed on tilted images that the OA-B stains DNA throughout the whole thickness of the section. In addition, very low quantities of cytoplasmic DNA were stained by this complex, which is now the best DNA stain used in electron microscopy. Within the nucleoli the DNA was localized in the fibrillar centers, where large clumps of dense chromatin were also visible. In the two plant species intranucleolar chromatin forms a complex network with strands partially linked to chromosomal nucleolar-organizing regions identified by in situ hybridization. This study describes for the first time the spatial arrangement of the intranucleolar chromatin in nucleoli of higher plants using high-resolution techniques.

Appearance and repair of apurinic/apyrimidinic sites in DNA during early germination of Zea mays

Abstract The number of AP (apurinic or apyrimidinic) sites found in DNA of radicle cells of Zea mays quiescent embryos after 2 years of storage is low; the low rate of spontaneous base loss is probably due to the low water content of the seed. But this number increases 4-fold during the first 20 h of germination, to decrease between 20 and 25 h, and increase again afterwards. These variations may well be due to competition between the formation of AP sites and their repair during early germination. Formation must be due to DNA glycosylases removing bases which have been damaged during the storage of the seeds. The first increase in the number of AP sites would be due to DNA glycosylases which have survived the storage period; the second increase might be the result of the synthesis of new DNA glycosylase molecules. The nuclear DNA-repair synthesis monitored by autoradiography closely follows the number of AP sites during early germination, suggesting that the repair of most of the damage which has accumulated in DNA during storage has AP sites as an intermediary step.

Somatic embryogenesis in pearl millet (Pennisetum glaucum): Strategies to reduce genotype limitation and to maintain long-term totipotency

Three genotypes of Pearl millet were screened in vitro for induction of embryogenic callus, somatic embryogenesis and regeneration. Shoot apices excised from in vitro germinated seedlings or immature embryos isolated from green house established plants were used as primary explants. The frequency of embryogenic callus initiation was significantly higher in shoot apices in comparison with immature zygotic embryos. Moreover, differences between genotypes were minimal when using shoot apices. Friable embryogenic calli (type II) developed on the initial nodular calli after 1 to 3 months of culture. The frequency of type II callus is related to the composition of the maintenance medium and they were more often found in ageing cultures. The transfer of embryogenic calli onto auxin-free medium was sufficient for inducing somatic embryo development in short-term culture (3 months) while a progressive loss in regeneration potential was observed with increasing time of subcultures. Maturation of embryogenic calli on medium supplemented with activated charcoal, followed by germination of somatic embryos on medium supplemented with gibberellic acid, restored regeneration in long-term cultures.

Root cell ultrastructure of Zea mays embryo during early stages of germination.

The ultrastructure of root cells of the germinating corn embryo has been studied during the first 72 hours of soaking. The most spectacular ultrastructural modifications occur in the nucleus. In the dry seed, the chromatin is heavily condensed and complete dispersion occurs during the first 8 hr of germination. The nucleolus appears as a compact structure in the dormant embryo, and as a uniform granular structure after 3 hr. At the 8th hour, large nucleolar vacuoles appear filled with material structurally similar to chromatin. Later on, the nucleolus is composed of a central, fibrillo-granular region surrounded by a thin, peripheral, granular region and fewer nucleolar vacuoles are found.In a previous autoradiographic study (Deltour, 1970), it was shown that the onset of RNA synthesis in these cells occurs 4 hr after soaking. From that time to the 8th hour, uridine-(3)H is incorporated exclusively into the chromatin. Incorporation of radioactive uridine into the nucleolus begins only after the 8th hour.It is interesting that the onset of RNA synthesis in the chromatin occurs simultaneously with the dispersion of this cell component, and that the appearance of vacuoles in the nucleolus is correlated with the beginning if uridine incorporation into this organelle.The following ultrastructural changes take place in the cytoplasm; (a) the lamellae system of proplastids increases slightly; (b) phytoferritin granules present in the proplastids of the dry seed disappear very rapidly; (c) polysomes appear 72 hr after soaking; (d) the spherosomes which are essentially localized in the vicinity of the wall in the dormant embryo become uniformly distributed throughout the cytoplasm at the 72nd hr.SummaryThe ultrastructure of root cells of the germinating corn embryo has been studied during the first 72 hours of soaking. The most spectacular ultrastructural modifications occur in the nucleus. In the dry seed, the chromatin is heavily condensed and complete dispersion occurs during the first 8 hr of germination. The nucleolus appears as a compact structure in the dormant embryo, and as a uniform granular structure after 3 hr. At the 8th hour, large nucleolar vacuoles appear filled with material structurally similar to chromatin. Later on, the nucleolus is composed of a central, fibrillo-granular region surrounded by a thin, peripheral, granular region and fewer nucleolar vacuoles are found.In a previous autoradiographic study (Deltour, 1970), it was shown that the onset of RNA synthesis in these cells occurs 4 hr after soaking. From that time to the 8th hour, uridine-3H is incorporated exclusively into the chromatin. Incorporation of radioactive uridine into the nucleolus begins only after the 8th hour.It is interesting that the onset of RNA synthesis in the chromatin occurs simultaneously with the dispersion of this cell component, and that the appearance of vacuoles in the nucleolus is correlated with the beginning if uridine incorporation into this organelle.The following ultrastructural changes take place in the cytoplasm; (a) the lamellae system of proplastids increases slightly; (b) phytoferritin granules present in the proplastids of the dry seed disappear very rapidly; (c) polysomes appear 72 hr after soaking; (d) the spherosomes which are essentially localized in the vicinity of the wall in the dormant embryo become uniformly distributed throughout the cytoplasm at the 72nd hr.

Three-dimensional electron microscopy of the nucleolus and nucleolus-associated chromatin (NAC) during early germination of Zea mays L.

Nucleoli and nucleolus‐associated chromatin (NAC) of radicle cells have been three‐dimensionally reconstructed from serial ultrathin sections during early germination of Zea mays. As a preliminary, the effect of 5 methods of fixation on the ultrastructure of the active NAC were tested qualitatively and quantitatively. It appeared that paraformaldehyde best preserved the fibrillar centres (FCs) and was consequently used for the 3‐D reconstructions. In quiescent cells, the NAC forms either 2 short internal strands about 0.7 μm thick running within the nucleolus or 2 peripheral knobs of the same diameter. Whatever its morphology, the NAC was composed of one clear zone, i.e., secondary constriction (SC) of the nucleolar organizer region (NOR), and one electron‐opaque zone, i.e., heterochromatic segment (HS). During germination the NAC was always connected to the nuclear envelope (NE) by a bridge of dense chromatin. The NAC strands or knobs of the quiescent cells are likely to be the counterpart of the 2 NORs of this species. 10–12 hr after onset of germination, one or several networks of nucleolar vacuoles were formed within which the whole NAC was located. Chromatin fibers about 12 nm thick emerged from unfolding portions of the NAC within these “nucleolar chromatin dispersal vacuoles” (NCDV). At 24 hr, the NAC appeared as 2 dichotomous strands. Seventy‐two hr after germination both the stretching out and branching of the NAC were more pronounced. After 120 hr, the transcribing ribosomal genes for each NAC strand together with the newly synthesized RNP transcripts formed a layer of dense fibrillar component surrounding a thin axis which was composed mainly of pale material. Together these formed a typical plant nucleolonema.