Hélène David

A Basic Chitinase-like Protein Secreted by Embryogenic Tissues of Pinus caribaea acts on Arabinogalactan Proteins Extracted from the same Cell Lines

Summary Embryogenic cell lines of Pinus caribaea consist of a high number of somatic embryos. We have previously characterized the embryogenic state by studying the proteins and glycoproteins ionically bound to the cell surfaces of preglobular somatic embryos. The embryogenic tissues and nonembryogenic calli produce proteins of 48 kDa or 56 and 25 kDa, respectively. All of these proteins cross-react with several classes of tobacco chitinases (CHs). These CH-like proteins express a potential chitinase activity on SDS-PAGE gels overlaid with glycol chitin as a synthetic substrate. When an arabinogalactan protein (AGP) fraction from embryogenic tissues substitutes for glycol chitin on gels, only the 48 kDa embryo-specific CH-like protein acts on this substrate, indicating that an interaction between this protein and a specific set of AGPs might exist within embryogenic tissues of Carribean pine.

cDNA sequence, genomic organization and differential expression of three Arabidopsis genes for germin/oxalate oxidase-like proteins

Wheat germin is a protein expressed during germination which possesses an oxalate oxidase activity. Germin-type oxalate oxidases have been extensively studied in monocotyledons (wheat and barley) where they are thought to have important functions for development, stress response and defence against pathogens. In contrast, almost nothing is known about the germin-like proteins found in dicotyledons, gymnosperms and myxomycetes. In this work, cDNA clones for three genes (ATGER1, ATGER2 and ATGER3) encoding germin-like proteins, initially characterized as expressed sequence tags (ESTs), from Arabidopsis thaliana cDNA libraries were further characterized. In addition, we isolated and sequenced a Brassica napus cDNA which was strongly homologous to the cDNA for ATGER1. Sequence analysis and secondary structure predictions of the proteins encoded by these cDNAs showed that they possess all the characteristic features of members of the germin family and of the germin/seed globulins/sucrose binding protein superfamily. Sequence comparisons and mapping demonstrated the existence of at least two different gene families in the A. thaliana genome encoding a minimum of three genes for germins. These three genes have been mapped in three different location on the Arabidopsis genome. By northern blot hybridizations we found that these genes are differentially regulated. ATGER1 was expressed during germination, like wheat germin, but also in leaves whereas ATGER2 transcripts were exclusively found in developing embryos, like wheat pseudo-germin. ATGER3 mRNAs were found in leaves and flowers and their abundance was shown to vary during the circadian cycle.

Cryopreservation does not affect the expression of a foreign sam gene in transgenic Papaver somniferum cells

Abstract Transgenic cell lines of Papaver somniferum have been obtained via Agrobacterium tumefaciens. Papaver somniferum is known to be genetically unstable in in vitro culture conditions. Cryopreservation at ultra-low temperature is an appropriate strategy for long-term preservation of germplasm. We have studied the effects of osmotic stress due to cryoprotectants during pretreatment and of storage at –196 °C on the stability and the expression of the foreign sam1 gene from Arabidopsis thaliana. We established that the integrity, transcription of the transgene and enzymatic activity of its product were not affected by cryopreservation in liquid nitrogen

Pectins in walls of protoplast-derived cells imbedded in agarose and alginate beads

SummaryFlax hypocotyl protoplasts were embedded in agarose and alginate beads. The pectin molecules of the formed colonies were observed in electron microscopy using 2F4 antibody specific of a calcium-induced supramolecular conformation of homopolygalacturonic acid. Little pectin, mostly methylesterified, was present in agarose-entrapped colonies. The regenerating cells immobilized in alginate secreted much higher amounts of methylesterified pectins in their walls. De-esterification of the pectins was clearly seen after 6 days of culture. These results illustrate the importance of the external matrix on wall differentiation.

How plant regeneration from Mentha × piperita L. and Mentha × citrata Ehrh. Leaf protoplasts affects their monoterpene composition in field conditions

Summary A procedure to regenerate plants from leaf protoplasts of two micropropagated hybrid species of mint, Mentha × piperita L. and Mentha X citrata Ehrh., has been developed in order to determine whether the in vitro treatment could influence the monoterpene composition. Purified protoplasts were first plated in liquid media containing 3.5 μmol/L BA, 1.25 μmol/L zeatin, 2.5μmol/L NAA and 2.25 or 5.5μmol/L 2,4-D as growth regulators to induce initial divisions. In these conditions, high percentages of protoplast-derived cells divided, especially for M. × piperita clones (ADF ranging from 17 % to 31 % at day 6). Reduction of both medium osmolality and 2,4-D concentration resulted in a sustained development of microcalli for both species. Calli were then transferred onto solidified regeneration media. The first regenerated shoots of M. × piperita were observed 3 months after protoplast isolation on media containing 8.9μmol/L and 13.2μmol/L BA or 1.8μmol/L and 4.5 μmol/L TDZ. Regeneration frequency did not exceed 4%. Regeneration medium sequences were required for shoot regeneration on M. × citrata calli. At first, they were cultured on a 1.8 μmol/L TDZ containing-medium for 1 week. They were then transferred onto media supplemented with various concentrations of TDZ or BA. The highest frequencies of shoot regeneration were around 10 %. Shoot morphology was affected by TDZ for both species, but not by BA. In a field trial, the amount of menthone, menthol and carvone in the regenerated plants of M. × piperita vulgaris was compared with that of the control. Results showed a decrease in the amount of menthone and menthol and an increase of carbone levels in all protoplast-derived plants.

Supporting matrix influences protoplast-derived colony formation: Structural analysis

SummaryFlax (Linum usitatissimum) protoplasts were immobilized in agarose and in Ca-alginate, medium (MV) and high viscosity (HV) grades. Protoplast viability was markedly decreased, probably as a result of the entrapment procedure itself. On the other hand, mitotic activity of surviving protoplasts was not influenced by the type of matrix agarose or alginate HV grade. Light and electron microscopical observations revealed compact heterogeneous cell colonies in agarose surrounded by cells in a more or less advanced state of lysis. In Ca-alginate (MV and HV) cell colonies were compact and spherical with poorly vacuolated cells. In this matrix, cell walls were intensely stained and sinuous, separated from the plasma membrane by a large periplasmic space.

Cell Wall Composition and Morphogenic Response in Callus Derived from Protoplasts of two Fibre flax (Linum usitatissimum L.) Genotypes

Summary Cotyledonary protoplasts of two French fibre flax ( Linum usitatissimum ) cultivars (Ariane and Viking) were isolated and cultured. The optimal conditions for high yield were from 4-5 day old seedlings, where 6-6.5x10 6 and 6.7-7.4x10 6 protoplasts were released from cv. Ariane and cv. Viking respectively. Culture in glutamine (20mM) containing medium led to first divisions after 2 days with an efficiency of 19% or 21.9% (cv. Ariane) and 49.3% or 44% (cv. Viking) of divided cells, when plating densities were 10 5 versus 5 x 10 4 protoplasts per mL. Rhizogenesis occurred on protoplast derived calli at high frequency 4 weeks after transfer onto solid medium supplemented with 0.5 μM 2.4 dichlorophenoxyacetic acid. Variations of cell wall sugar composition during culture on inducing and non-inducing media expressed some significant differences. The percentage of galacturonic acid increased on non-inducing medium, but remained constant on root-inducing medium. Glucose presented a peak of accumulation concomitant of the emergence of the root meristems 4 weeks after transfer onto 2,4-D containing medium. On the contrary galactose and arabinose variations were not characteristic of the rhizogenic process.

Rice salT promoter is activated in Papaver somniferum and Nicotiana tabacum transgenic cells in the absence of exogenous ABA.

With the aim of modifying secondary metabolism in Opium poppy (Papaver somniferum) and tobacco (Nicotiana tabacum) cells, gene transfer was performed using the sam1 gene from Arabidopsis thaliana under the control of the salT promoter. This promoter is induced by ABA in rice and in tobacco and we have shown that it is also induced in poppy cells (gus gene). Putatively transformed poppy and tobacco cell lines with the sam1 gene were obtained. In the absence of exogenous inducer we noticed the expression of the transgene resulting in a significant increase of SAM-S activity in all tested transformants of poppy and in half the transgenic tobacco cell lines tested. Addition of ABA to the culture medium failed to enhance the expression of the transgene in both species and resulted in a decrease of the sam1 gene expression in some cell lines. Since the salT promoter is induced by exogenous ABA in both species (gus reporter gene), we suggest a partial sam1 transgene inactivation in certain cell lines. These results show that the efficiency of a regulatory sequence may be different when fused with a reporter gene (gus) compared to fusion with a gene belonging to the housekeeping family (sam1).