Induction of a long-lived fusogenic state in viable plant protoplasts permeabilized by electric fields.

Electropermeabilized tobacco mesophyll protoplasts are shown to fuse by creating cell contact several minutes after electropulsation. Electropermeabilization was analysed by measuring calcein uptake. Experiments were performed at low temperature to avoid resealing of protoplast transient permeation structures. These results confirm that the long-lived permeabilized state induced by the electric field is associated to a fusogenic state, under viability conditions. This is indicative that as for mammalian cells, the electric field-induced membrane modifications, which give the permeable state, are such as to decrease the magnitude of the intercellular repulsive forces between plant protoplasts. Such a fusion method may be useful for somatic hybrids production with protoplasts showing morphological and physiological differences.

Studies on ion channel antagonist-binding sites in sunflower protoplasts.

The cytological location of ion channel antagonist-binding sites was studied in sunflower protoplasts using the fluorescent probes DM-Bodipy-PAA and DM-Bodipy-DHP. The binding specificity of the probes was established by competition experiments with Bepridil, phenylalkylamine (Verapamil) and dihydropyridine (Nifedipine) which are known as calcium and potassium channel antagonists. Quantitative image analysis of the fluorescence emitted by the protoplasts showed the existence of interactions between PAA- and DHP-binding sites. Moreover, studies on the cytolocalization of the PAA receptors by confocal imaging showed that in freshly isolated protoplasts, DM-Bodipy-PAA binds exclusively at sites located in the cortical region of the cell.

Protocols for efficient repetitive and secondary somatic embryogenesis in Helianthus maximiliani (Schrader).

Indirect somatic embryogenesis was induced on leaf explants of greenhouse-grown Helianthus maximiliani plants. Leaves of the regenerated plants were used as starting explants for the induction of direct somatic embryogenesis. Another cycle of somatic embryogenesis was induced on the leaves of regenerated plants. In both cases, leaf explants were cultured on media containing different auxin/cytokinin ratios. The auxin/cytokinin ratio had an influence on the intensity of embryo formation, germination and the capability to regenerate plants. Somatic embryogenesis was generally more intensive on the medium with lower concentrations of 6-benzylamino-purine. Further, the percentage of regenerated plants was higher when embryos were induced on high-cytokinin, low-auxin medium. Secondary somatic embryogenesis was induced on embryos by culture in liquid hormone-free medium. Similar to direct embryogenesis the efficiency of secondary embryogenesis depended on the medium used for the induction of the primary embryos. In contrast to the mostly low frequencies of conversion of secondary embryos into plants that has been observed in other species, the percentage of regenerated plants from secondary embryos of H. maximiliani was quite high, although slightly lower than that obtained in primary embryos.

Effect of atmospheric pressure on sunflower (Helianthus annuus L.) protoplast division.

Sunflower protoplasts were cultured in liquid medium under high atmospheric pressure (0.2 to 0.6 MPa) and the plating efficiency, cell wall synthesis and microtubule organization were assessed. In 7-day-old cultures under a pressure of 0.4 MPa and above, the division rate was strongly reduced by more than 60% as compared to the control. Although most of the protoplasts had begun to regenerate a new cell wall they were unable to complete this process. Pressure also had an inhibitory effect on microtubule synthesis. The percentage of protoplasts showing a disassembled cortical network of microtubules was significantly increased up to 60% of the population. These effects were reversible: when protoplasts were transferred to normal pressure most of them rapidly recovered their capacity to divide and afterwards developed normally. Culturing protoplasts under a pressurized atmosphere revealed to be a good model system for studying cortical microtubule dynamics.

Recherches sur les enzymes catalysant la biosynthese des acides phénoliques chez Quercus pedunculata (EHRH.): I - Formation des premiers termes des series cinnamique et benzöique.

Occurence of PAL, cinnamate-hydroxylase and p-coumarate-hydroxylase, is found in cell-free extracts from Quercus pedunculata roots; moreover, an enzyme system which catalyzes benzoïc acid formation from cinnamic acid is caracterized for the first time. Role of these enzymes and their interactions within the same organ are discussed.

Reversible phosphorylation of tonoplast proteins involves tonoplast-bound calcium-calmodulin-dependent protein kinase(s) and protein phosphatase(s).

In highly purified tonoplast fractions from Acer pseudoplatanus cells, the in vitro reversible phosphorylation of proteins affected only a restricted set of polypeptides. The phosphorylation process has been shown to be dramatically stimulated by calcium via the mediation of calmodulin as the transducer. The protein kinase(s) was totally inhibited by micromolar concentrations of a calmodulin antagonist. Tonoplast appears to be potentially a good experimental system for the evaluation of the effects of protein phosphorylation on membrane properties in plants.

Haploid regeneration from tetraploid wheat using maize pollen.

Crosses were made between 21 tetraploid wheat genotypes (6 parents, 15 F1 hybrids) and a single F1 hybrid of maize that was used as the male parent. Plants were grown under controlled greenhouse conditions (daylength, 16 h; temperature, 25 degrees C days and 15 degrees C nights). To enhance embryo survival, 2,4-D (10 mg/L) was applied to spikes 24 h after pollination with maize. Embryos were recovered from the tetraploid wheat genotypes at a rate of 2.34-14.14/100 developed ovaries. Sixty-nine haploid plants were obtained from 3 parents and 12 F1, hybrids. Fifty-six of these were successfully doubled. General combining ability was significant for the two traits studied, indicating that additive genetic control is important for the number of developed ovaries and haploid embryo production in tetraploid wheat x maize crosses. In this report, we demonstrate the potential of using maize pollen to produce haploid plants from tetraploid wheat genotypes.

Auxin reduces the synthesis of major vacuolar proteins in tobacco mesophyl protoplast.

We have established that polypeptides whose synthesis is reduced by 2,4-dichlorophenoxyacetic acid during in vitro culture of tobacco mesophyll protoplasts are secreted into the vacuole where they constitute the bulk of labeled proteins. In addition, these proteins continue to be synthesized in protoplast-derived cultured cells and their synthesis is strictly correlated with the size of the cell, i.e. with vacuolar size.

Studies on plant regeneration from protoplasts in the genus Helianthus.

Protoplasts were produced from 7-day-old hypocotyls of two cultivated sunflower genotypes and three wild sunflowers. When included in agarose droplets and cultured in TL medium supplemented with 0.1 mg/l 2,4-dichlorophenoxyacetic acid, the protoplasts gave rise to loose colonies and to "embryoids". After two months the small calli emerging from the agarose were transferred to a regeneration medium on which they grew and began to differentiate. A second transfer to the same medium 40 days later induced shoot formation on one callus of H. petiolaris. Several shoots were successfully rooted and transferred to soil where they flowered. This is the first documented report, in the genus Helianthus. of regeneration from protoplasts to fully soil-adapted plants.

Relationship between protoplast size and critical field strength in protoplast electropulsing and application to reliable DNA uptake in Brassica.

Protoplast diameter, a physical parameter controlling the susceptibility to electropermeation, was observed to vary between different batches of Brassica napus and Brassica campestris protoplasts. In order to control the permeation field strength associated with the protoplast size, we investigated the mathematical relationship between permeant field strength and protoplast size by measuring electroloading of the calcein dye under conditions that did not alter protoplast survival. A roughly linear relationship was obtained when the permeant field strength was plotted against the reciprocal of the radius. The plot characteristics were found to be tissue specific (mesophyll, hypocotyl and cotyledon), species specific (turnip, rapeseed) and modulated by the pulse duration. These plots were used as reference curves to determine accurate electrical conditions for DNA transfer whatever the size distributions of different protoplast batches.

Regeneration of fertile plants from protoplasts of sunflower (Helianthus annuus L.).

Sunflower hypocotyl protoplasts (Helianthus annuus L.) from 5 PIONEER genotypes (PT024, SMF3, EMIL, HA300*PT024, VK5F) and 1 public line (RHa 274) formed colonies at frequencies of up to 60% when plated in 0.25ml agarose beads in a modified L4 medium (Lenée and Chupeau 1986) containing 3mg/l NAA, 1mg/l BA and 0.1mg/l 2,4-D, and 1000mg/l casamino acids. Protoplast-derived colonies grew slowly into calli. Organogenesis was obtained from callus of PT024 on a MS medium containing NAA and BA at 1mg/l and GA at 0.1mg/l. Freshly excised shoots were induced to root by an IAA treatment. Regenerated plants were transferred to the greenhouse and seed was harvested within 7 months of the initial protoplast isolation.

Isolation and Characterization of Vacuoles from Melilotus alba Mesophyll.

Methods for the preparation of protoplasts and vacuoles from mesophyll tissues of sweet clover (Melilotus alba Desr.) are described. Vacuoles are obtained using a new procedure which involves lysis of the plasmalemma during a brief centrifugation of protoplasts through a diethylaminoethyl dextran layer. This method combines the release of vacuoles and their purification in one step. The contamination of vacuole preparations was found to be low, as judged by enzymic markers and microscopic inspection. The method described is rapid and gives a good yield of vacuoles without causing changes in osmotic pressure. Several hydrolases were found to be located in vacuoles from sweet clover, which were also examined for their amino acid content.

Photoregulation of the incorporation of guaiacyl units into lignins.

When fed with [(14)C] phenylalanine in the light, xylem tissues isolated from poplar stems were able to incorporate part of the radioactivity into both the guaiacyl and the syringyl residues of lignins. In the dark, only syringyl units were integrated into the polymer whereas the guaiacyl residues remained unlabeled.When a membrane perturber (isopropanol) was added to the incubation mixture, the label was incorporated into the guaiacyl units either in the light or in the dark. Conversely, a membrane stabilizer (CaCl2) prevented the labeling of the guaiacyl units even when the tissues were illuminated. These results suggest that light acts through the modification of membrane permeabilities, altering specifically the synthesis and the transport or the polymerization of guaiacyl-type units during the process of lignification.

Hydrolysis of Intracellular Proteins in Vacuoles Isolated from Acer pseudoplatanus L. Cells.

Acer pseudoplatanus cell suspension cultures were used to examine the ability of vacuoles isolated from protoplasts to hydrolyze their endogenous proteins. Total cell proteins were labeled by addition of [(3)H]leucine to the culture medium. After preparation of the protoplasts, vacuoles were isolated and were shown to be essentially free from other cellular components. Up to 30% of the [(3)H]leucine-labeled newly synthesized proteins were recovered in the vacuoles. When incubated for 6 hours at 20 degrees C, the vacuoles degraded half of these proteins. The protein breakdown was temperature and pH dependent. Analysis by electrophoresis, in denaturing polyacrylamide gels, revealed that most of the vacuolar proteins were degraded. However, some vacuolar proteins were unaffected during a 6-hour incubation period. The results indicate that vacuoles are able to acquire and degrade intracellular proteins.

Rapid Degradation of Abnormal Proteins in Vacuoles from Acer pseudoplatanus L. Cells.

In Acer pseudoplatanus cells, the proteins synthesized in the presence of an amino acid analog ([(14)C]p-fluorophenylalanine), were degraded more rapidly than normal ones ([(14)C]phenylalanine as precursor). The degradation of an important part of these abnormal proteins occurred inside the vacuoles. The degradation process was not apparently associated to a specific proteolytic system but was related to a preferential transfer of these aberrant proteins from the cytoplasm to the vacuole.

Genetic analysis of organogenesis in the cotyledons of zygotic embryos of sunflower (Helianthus annuus L.).

Crosses were made between five cytoplasmic male-sterile and five restorer sunflower inbred lines. Twenty-five F1 hybrids and their parents were studied for their organogenesis ability in a randomized block design with four replications. Each replication per genotype consisted of ten petri dishes with four expiants. Regeneration medium consisted of full MS medium modified by the addition of hormones and solidified with 6 g/l agar. Statistical analysis showed that both general and specific combining abilities were significant for all of the organogenesis parameters studied, and both showed several significant positive or negative values. General combining ability values were usually higher than those of specific combining ability, indicating the importance of additive genetic control for organogenesis parameters in sunflower. Narrow-sense heritability for the number of explants producing shoots and roots was 65.8%, which suggests that organogenesis of currently inferior inbred lines in sunflower should be improved in a crossing program.

Optimisation of DNA transfer and transientß-glucuronidase expression in electroporated maize (Zea mays L.) microspores.

The ability to deliver free DNA into microspores of a highly androgenic hybrid of maize was assessed by electroporation, using a square wave pulse discharge apparatus. The electroporation medium was chosen according to its ability to maintain a high level of regeneration. Nuclease activities were analyzed and were inhibited by the addition of 100 mM KNO3 and MgSO4 in the electroporation medium. Seven expression vectors withUid A as the reporter gene under the control of cauliflower mosaic virus 35S, Lat 52-7, Zmg 13, Emu, Ubiq-1, Al, or Actl promoters were tested in relation to the level of ß-glucuronidase expression in maize microspores. The highest level of expression was obtained when theUid A gene was driven by the Actl promoter. Therefore, this vector was further used to define optimal conditions leading to highest levels of ß-glucuronidase expression. The parameters determined in this study could provide an ideal starting point for the obtention of transgenic maize plants from electroporated microspores.

Effects of monocerin on cell cycle progression in maize root meristems synchronized with aphidicolin.

Monocerin is a benzopyran fungal toxin with broad activity on plants, fungi and insects. Its effect upon cell cycle progression has been analyzed in maize roots. Meristematic cells were synchronized by treatment with aphidicolin. Flow cytometric DNA analysis and mitotic indices indicated durations of 1.5 h, 5 h, 2 h and 1 h for respectively G1, S, G2 and M phases of the normal cell cycle at 25°C. Treatment of these synchronized meristems with 0.5 mM monocerin during release after an aphidicolin block produced a short delay in S phase and then a more important delay (about 2.5 h) in entry into mitosis. Treatments for similar durations (3 h) during progression through the cycle revealed two periods of action of monocerin. The first appears to be mid to late S and the second one G2, before the transition point between G2 and M. Action on either one of these target periods could lead to a delay in the G2/M transition, but these two responses did not appear to be additive.

Intracellular Sites of Synthesis and Storage of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid in Acer pseudoplatanus Cells.

Vacuoles were isolated from Acer pseudoplatanus cells that were incubated with [(14)C]1-aminocyclopropane-1-carboxylic acid (ACC). The kinetics of [(14)C]1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) formation are consistent with the interpretation that MACC is synthesized in the cytosol, transported through the tonoplast, and accumulated in the vacuole. Twenty hours after chasing the labeled ACC with unlabeled ACC and adding 1 millimolar unlabeled MACC, all the [(14)C]MACC synthesized is located in the vacuole. Whole cells preloaded with [(14)C]MACC and then submitted to a continuous washing out, readily release their cytosolic MACC until complete exhaustion. The half-time of MACC efflux from the cytosol, calculated by the technique of compartmental analysis, is about 22 minutes. In contrast, vacuolar MACC remains sequestered within the vacuole. The transport of labeled MACC into the vacuole is stimulated by the presence of unlabeled MACC in the suspension medium, probably as a result of a reduced efflux of the labeled MACC from the cytosol into the suspending medium.