Jacques Vasseur

Phenolic compounds and antioxidant activities of buckwheat (Fagopyrum esculentum Moench) hulls and flour

The interest of polyphenolics as therapeutic agents against diseases involving radical damage is growing. The phenolic contents of the hulls and flour from the seeds of Fagopyrum esculentum (French variety La Harpe) (total phenols, flavonoids, total flavanols, oligomeric proanthocyanidins) are compared with the antioxidative effects of the extracts against reactive oxygen species: hydrogen peroxide, hypochlorous acid, superoxide anion. The higher efficiency of the flour extract can be related to its higher flavanolic content rather than to flavonoids which are predominant in the hull extract.

Arabinogalactan-proteins in Cichorium somatic embryogenesis: effect of β-glucosyl Yariv reagent and epitope localisation during embryo development

Abstract.u2002Direct somatic embryogenesis was induced in root tissues of the Cichorium hybrid `474 (C. intybus L. var. sativum×C. endivia L. var. latifolia). Addition of β-d-glucosyl Yariv reagent (βGlcY), a synthetic phenylglycoside that specifically binds arabinogalactan-proteins (AGPs), to the culture medium blocked somatic embryogenesis in a concentration-dependent manner with complete inhibition of induction occurring at 250u2009μM βGlcY. The AGP-unreactive α-d-galactosyl Yariv reagent had no biological activity in this system. Upon transfer of 250 μM βGlcY-treated roots to control conditions, somatic embryogenesis was recovered with a time course similar to that of control roots. The βGlcY penetrated roots and bound abundantly to developing somatic embryos, to the root epidermis and the stele. Immunofluorescence and immunogold labelling using monoclonal antibodies (JIM13, JIM16 and LM2) revealed that AGPs were localised in the outer cell walls peripheral cells of the globular embryo. A spatio-temporal expression of AGPs appeared to be associated with differentiation events in the somatic embryo during the transition from the globular stage to the torpedo stage. To verify βGlcY specificity, molecules that bound βGlcY were extracted from treated conditioned medium and identified as AGPs by using the same monoclonal antibodies. In addition, AGPs were found to be abundantly present in the medium during embryogenic culture. All of these results establish the implication of AGPs in embryo development, and their putative role in somatic embryogenesis is discussed.

Extracellular β-1,3-glucanases are induced during early somatic embryogenesis in Cichorium

Abstract. In leaf tissues of the Cichorium hybrid clone `474 (C. intybus L. var. sativumu2009×u2009C. endivia L. var. latifolia), the acquisition and expression of embryogenic competence was characterised by the appearance of 15 polypeptides (Boyer et al., 1993, Plant Sci 93: 41–53). The 38-kDa proteins were found to be abundantly present in conditioned embryogenic medium after the first division of the induced cells. These proteins seemed to be glycosylated as indicated by general carbohydrate detection methods. Internal amino-acid sequences obtained after microsequencing tryptic peptides appeared to be 36–57% homologous with plant β-1,3-endoglucanases. In addition, these 38-kDa proteins were recognised by antibodies raised against the pathogenesis-related tobacco glucanase PR2a and their β-1,3-glucanase activity was demonstrated by direct detection in polyacrylamide gels after electrophoresis. These results strongly suggested that the 38-kDa somatic-embryogenesis-related (SER) polypeptides are β-1,3-glucanases. Moreover, the level of glucanase activity was nearly three times higher in the medium of the embryogenic `474 line than in the medium of a non-embryogenic line. The possible involvement of the extracellular 38-kDa proteins in callose degradation during somatic embryogenesis is discussed.

Cloning of β-1,3-glucanases expressed during Cichorium somatic embryogenesis.

Three different β-1,3-glucanase cDNA fragments, CG1, CG2 and CG3, were obtained by RT-PCR from RNA isolated from Cichorium hybrid `474 leaf fragments cultured for 11xa0days under somatic embryogenesis-inducing conditions. When expressed in Escherichia coli the proteins encoded by the three cDNAs were recognized by antibodies raised against 38xa0kDa extracellular β-1,3-glucanases studied previously (Helleboid etxa0al., Planta 205 (1998) 56–63). The CG2 and CG3 cDNAs may represent expressed alleles of one gene because their sequences showed a very high identity (98.5%) and are only 70% identical with CG1. Southern blot analysis revealed the presence of 3–4 genes coding for β-1,3-glucanases in the Cichorium genome. Expression analysis of the genes corresponding to the three clones analysed by semi-quantitative RT-PCR indicated that CG1 mRNAs were only detectable in Cichorium hybrid `474 leaf fragments from dayxa03 of somatic embryogenesis induction, whereas CG2-CG3 mRNAs were already present in non-induced leaf tissue of both the embryogenic hybrid `474 and a non-embryogenic genotype. The level of CG1 mRNAs was particularly high when embryogenic cells were dividing to produce embryos, and when the amount of callose deposited in cell walls surrounding embryogenic cells and young embryos decreased. These results indicate that expression of the CG1 gene is correlated to the somatic embryogenesis process and that it encodes a 38xa0kDa β-1,3-glucanase protein that may be involved in the degradation of callose localized around embryogenic cells and young embryos. A full-length CG1 cDNA clone was obtained using 3′ and 5′ RACE-PCR, and its sequence revealed that it encodes a β-1,3-glucanase that is equally homologous to both classxa0III and classxa0IV plant β-1,3-glucanases.

Inhibition of direct somatic embryogenesis by α-difluoromethylarginine in a Cichorium hybrid: effects on polyamine content and protein patterns

Direct somatic embryogenesis was induced in mesophyll cells of leaves of the hybrid Cichorium intybus L. var. sativum x C. endivia L. var. latifolia through a twostep procedure during 5 d in a M17 liquid medium supplemented with 330 mM glycerol. The lack of cell division during the induction step seems to be associated with a relatively stable low free-polyamine content. Synchronised divisions of competent cells occurred after transfer onto a glycerol-free M17 medium. Increased free-polyamine levels, especially putrescine, accompanied embryo differentiation. The addition of 0.1 to 10 mM α-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of polyamine biosynthesis in this Cichorium hybrid, to the medium caused a drastic decrease of polyamine content. Putrescine and spermidine rapidly decreased after 1 d incubation with DFMA and remained at ten times lower concentration than the controls. Embryogenesis was decreased (2 embryos per mm2 of leaf versus 12 embryos per mm2) in DFMA-incubated leaves. The addition of 5 mM putrescine to DFMA-treated cultures restored both the initial spermidine content and the embryogenesis. The effects of somatic-embryogenesis inhibition on protein biosynthesis were analysed using two-dimensional electrophoresis. In control samples, extracellular polypeptides were secreted into the culture medium. Two of these secreted polypeptides exhibited the same features as two tissue somatic-embryogenesis-related polypeptides. In the presence of 5 mM DFMA their contents in the medium were reduced, whereas they reached similar levels as in the control samples in the presence of DFMA and putrescine.

Removal of the fibrillar network surrounding Cichorium somatic embryos using cytoskeleton inhibitors: analysis of proteic components.

Abstract In Cichorium ‘474’, the embryo globular stage was characterised by a fibrillar network linking peripheral neighbouring cells. To test a putative connection between this fibrillar network and the cytoskeleton (CTK), we have used CTK disrupting agents (cold; colchicine; cytochalasine B) on Cichorium somatic embryos. Scanning electron microscopy observations showed that these three treatments induced the disappearance of the fibrillar network and suggested that this network could take part of the CTK-plasma membrane (PM)-extracellular matrix (ECM) continuum. The treatment media containing the removal fibrillar network were used to analyse its proteic component by 2D-PAGE. Using a Sun SPARCstation computer, the comparison of the gels corresponding to the different treatments allowed us to detect a group of 25 common proteins recovered in the medium after each treatment and in somatic embryogenesis conditioned medium. During the short treatments applied on somatic embryos, a large amount of high molecular weight glycoproteins corresponding to arabinogalactan proteins (AGPs) have been quantified with β- d -glucosyl Yariv reagent and identified with monoclonal antibodies raised against AGP epitopes (JIM13, JIM16, LM2). The implication of the fibrillar network and AGPs in the continuum CTK-PM-ECM are discussed in attempt to elucidate their possible function during Cichorium somatic embryogenesis.

Histological analysis of indirect somatic embryogenesis in the Marsh clubmoss Lycopodiella inundata (L.) Holub (Pteridophytes)

An efficient in vitro plant regeneration method was developed for Lycopodiella inundata (L.) Holub, an endangered medicinal Lycopod (Pteridophytes). Vegetative apices were used as explant material. Nodular calluses were established after three cycles (13 weeks each) on a medium containing a few minerals and organic compounds and supplemented with 0.05 µM 3-indolebutyric acid (IBA) and 1.4 µM kinetin (Kin). Propagation was achieved every 13 weeks on this callus medium (CM). When nodular calluses were transferred on a medium supplemented with 2.5 µM IBA and 0.33 µM gibberellic acid (GA(3)) designated as embryogenic medium (EM), organized structures appeared and developed into plantlets. Development phases were characterized by histological studies. Some phases of zygotic embryogenesis previously described for Lycopods were observed in L. inundata. Histological analyses established that an indirect somatic embryo was derived from a single embryogenic cell by following the zygotic developmental pathway. As this phenomenon has not previously been reported in Lycopods, a comparison between somatic and zygotic embryos is discussed based upon morphology and histology.

Changes in lipid composition during somatic embryogenesis in leaves of Cichorium

The present studies were conducted to investigate the changes in lipid and fatty acid composition during the earliest stages of somatic embryogenesis in leaf tissues of the Cichorium hybrid 474. The presence of glycerol in the culture medium during the induction step allowed to separate two phases in the embryogenic process. Firstly, cells are induced for morphogenetic competence (induction phase) and secondly, they express an embryogenic competence (expression phase). The analysis of fatty acid composition of total lipids showed that the percentage of linolenic acid (18:3) decreased while that of linoleic acid (18:2) increased throughout the culture period. A comparison with a non-embryogenic genotype of Cichorium indicated a higher increase of linoleate content in embryogenic genotype. The incorporation of [14C] glycerol into lipid classes was studied in leaf tissues. During the induction step, label was confined almost exclusively in polar lipids, particularly in phosphatidylcholine (PC). An important increase of labeled triacylglycerols (TAG) amounts was noted during the expression step. The accumulation of 18:2 was observed in PC and also in TAG. These results show that the early stages of somatic embryogenesis are associated with the increase of PC and TAG which are mainly enriched in 18:2.

Dormance et germination des microtubercules de Pomme de terre (Solanum tuberosum L.) produits in vitro: effets de la concentration en saccharose du milieu de tubérisation, de la durée de conservation à 4°C et d'un traitement avec de l'acide gibbérellique

Summary The results reported describe the effects of sucrose concentrations (80, 100 and 140 g.l−1), storage temperature at 4°C or gibberellic acid treatment (GA3) on in vitro potato (Solanum tuberosum L.) microtuber dormancy and their germination. High concentration of sucrose (140 g.l−1) and cold environment shorten microtuber dormancy period. On the other hand, germination is more rapid and homogenous with an increase in the 4°C storage period. GA3 does not allow suppression of dormancy but simply stimulates germination at the end of the dormancy period. At a concentration superior or equal to 10−4 M, GA3 increases the length of the plantlets.