Somatic embryogenesis in Araujia sericifera

This work presents the preliminary results of in vitro studies with Araujia sericifera, which is cultivated for ornamental purposes. Immature seeds from wild plants were used to start the cultures. Somatic embryos and friable embryogenic calluses were obtained from white cotyledons in media containing naphthaleneacetic acid and benzyladenine or 2,4-dichlorophenoxyacetic acid. Plants were regenerated from these somatic embryos. Cell suspensions obtained from friable calluses cultured in M1 modified medium showed a considerable growth capacity. The packed cell volume was doubled in about 15 days of culture at the exponential phase. the results obtained may be used to design further experiments with the aim of improving somatic embryogenesis.This work was supported by a grant from CICYT (Bio 88-0215).Peer reviewe

Effect of Exogenous Arginine, Ornithine, Methionine and γ-Amino Butyric Acid on Maize (Zea Mays L.) Embryogenesis, and Polyamine Content

The influence of four exogenous amino acids related to polyamine metabolism (γ-aminobutyric acid, arginine, methionine and ornithine) on maize (Zea mays L.) somatic embryogenesis was investigated. The endogenous polyamine contents of the treated calli were analyzed, and arginine and ornithine decarboxylase activities were determined. An established embryogenic callus (Type 1) of the inbred W64Ao2 was used. The endogenous polyamine content of calli was increased by addition of all four amino acids tested and the levels of spermidine plus spermine were higher than those of putrescine in all cases. Upon the addition of 2 mM arginine to the culture medium a 25% increase in embryogenesis was observed. Moreover, arginine decarboxylase (ADC) activity was significantly improved and ornithine decarboxylase (ODC) activity was also raised. The addition of 1.5 mM ornithine also increased embryogenic callus production, and ODC and ADC activities. Nevertheless, this increase was not as marked as in the case of arginine. This study indicates that the addition of amino acids, which are precursors of polyamine synthesis (especially Arg but also Orn), may be used to improve the rate of embryogenic callus production in an auxin-established maize culture system. A possible explanation of this effect is also dicussed.Peer reviewe

Effect of DL-Alpha-Difluoromethylornithine Pretreatments in Maize Callus Differentiation

The effect of pretreatments with DL-alpha-difluoromethylornithine (DFMO), an irreversible suicide inhibitor of the ornithine decarboxylase (ODC) activity, in plant differentiation, polyamine (PA) and amino acid contents of maize callus cultures was investigated. This study indicates that DFMO pretreatments can be used to improve regenerative response from maize callus cultures. These findings may also be useful in other recalcitrant cultures.Peer reviewe

AtPng1p. The first plant transglutaminase

Studies have revealed in plant chloroplasts, mitochondria, cell walls, and cytoplasm the existence of transglutaminase (TGase) activities, similar to those known in animals and prokaryotes having mainly structural roles, but no protein has been associated to this type of activity in plants. A recent computational analysis has shown in Arabidopsis the presence of a gene, AtPng1p, which encodes a putative N-glycanase. AtPng1p contains the Cys-His-Asp triad present in the TGase catalytic domain. AtPng1p is a single gene expressed ubiquitously in the plant but at low levels in all light-assayed conditions. The recombinant AtPng1p protein could be immuno-detected using animal TGase antibodies. Furthermore, western-blot analysis using antibodies raised against the recombinant AtPng1p protein have lead to its detection in microsomal fraction. The purified protein links polyamines—spermine (Spm) > spermidine (Spd) > putrescine (Put)—and biotin-cadaverine to dimethylcasein in a calcium-dependent manner. Analyses of the γ-glutamyl-derivatives revealed that the formation of covalent linkages between proteins and polyamines occurs via the transamidation of γ-glutamyl residues of the substrate, confirming that the AtPng1p gene product acts as a TGase. The Ca2+- and GTP-dependent cross-linking activity of the AtPng1p protein can be visualized by the polymerization of bovine serum albumine, obtained, like the commercial TGase, at basic pH and in the presence of dithiotreitol. To our knowledge, this is the first reported plant protein, characterized at molecular level, showing TGase activity, as all its parameters analyzed so far agree with those typically exhibited by the animal TGases.This work was supported by Ministero dell’Universita e della Ricerca Scientifica e Tecnologica (FIRB 2001), by the ESF Transglutaminases Program and the COST 844 Transglutaminases in Apoptosis (grant to M.D.M.), and by the Spanish Government (Secretaria de Estado de Educacion, Universidades e Investigacion, support to D.S.-F.).Peer reviewe

Modulator effect of DL-α-difluoromethylarginine treatments on differentiation processes of young maize calluses

The effect of pretreatments with 0.5, 1, 3, 5 and 10 mM of DL-α-difluoromethylarginine (DFMA), an irreversible suicide inhibitor of the arginine decarboxylase (ADC) activity, on the differentiation process of young maize calluses was studied. Callus protein, total polyamine content and ADC activity decreased versus control in all the assayed treatments. Furthermore, ornithine decarboxylase (ODC) activity was significantly lower in the treated calluses, which was probably due to the arginase activity detected in them. Short tratments at high doses of DFMA significantly increased the number of regenerated buds versus the control (four times more in 10 mM and almost two times more in 5 mM). By contrast, long treatments at low doses (0.5, 1 and 3 mM) reduced the number of plantlets. Conjugated putrescine seems to be implicated in the regeneration response of control and high DFMA-treated calluses.This study was financed by a grant from DGICYT (PB92-0087).Peer reviewe

Embryogenesis induction in petals of Araujia sericifera

The embryogenic capacity of Araujia sericifera petals and some of the factors involved in the induction of embryos was investigated. The influence of 6-benzyladenine and α-naphthalene acetic acid, light intensity (90 or 5 μmol m-2 s-1) and silver thiosulphate (inhibitor of ethylene action) were studied. It was found that petals are an easy system in which to induce somatic embryogenesis. Plants were recovered from somatic embryos. Although 6-benzyladenine is essential for inducing an efficient response, a high dosage increased callogenesis and reduced embryogenesis. The highest rate of embryogenesis is induced with high light intensity (90-100 μmol m-2 s-1), even though the presence of silver thiosulphate in the medium markedly reduced embryo induction.Research work was supported by a grant from DGICYT: PB92-0087.Peer reviewe

Immunogold localization of a transglutaminase related to grana development in different maize cell types

A comparative study of the subcellular localization of a plant transglutaminase (TGase; EC 2.3.2.13) in various in vivo and in vitro maize cell types was carried out with a polyclonal antibody raised against a 58 kDa TGase purified from Helianthus tuberosus leaves. Immunocytochemical staining, followed by electron microscopy, showed that this enzyme was markedly present in the grana-appressed thylakoids of mature chloroplasts of the light-exposed cells. Moreover, during embryogenic callus chloroplast differentiation, the abundance of TGase in the grana-appressed thylakoids depended on the degree of grana development and was greater than in mature leaf chloroplasts. In addition to the 58 kDa form, two other forms of the protein (of 77 and 34 kDa) were obtained by Western blot. The 77 kDa form might correspond to the inactive form and was immunodetected in dense vesicles observed in dark-grown embryogenic callus cells. In adult leaves, the enzyme was also markedly present in the grana-appressed thylakoids of the mesophyll cell chloroplasts, though very scarce and dispersed in the bundle-sheath cell chloroplasts (which do not contain grana). The concordance of these localizations with those described for the light-harvesting antenna proteins of the photosystem II suggests that it is possible that this TGase has a functional role in photosynthesis, perhaps modulating the photosynthetic efficiency and the absorption of excess light by means of polyamine conjugation to the antenna proteins.This work was supported by a Spanish grant DGICYT PB-97 1138.Peer reviewe

Changes in polyamine content, arginine and ornithine decarboxylases and transglutaminase activities during light/dark phases (of initial differentiation) in maize calluses and their chloroplasts

Maize calluses and their isolated chloroplasts were analysed to study the changes in polyamine content, arginine and ornithine decarboxylases and transglutaminase activities during light/dark phases of the first day after subculture in maintenance medium (containing 2,4-D) and differentiation medium (without 2,4-D). Free polyamine content changed significantly in both differentiating calluses and chloroplasts showing a maximum during light phase and also increasing after mid-dark phase. Acid-insoluble polyamines showed a similar trend. In whole cells from the callus cultured in maintenance medium, the changes were not significant, except for free putrescine which increased in the dark phase. In chloroplasts of both types of calluses, the trend was similar. Arginine decarboxylase activity in vitro assayed in optimal conditions was not affected by hormone deprivation either in whole cells from the callus or in chloroplasts. The formation of putrescine by arginine decarboxylase activity gradually increased in the light until 9-12 h after subculture, whereas at the onset of the dark phase, a significant decrease was observed. Ornithine decarboxylase activity in vitro always showed slight changes, except in growing callus where putrescine synthesis increased abruptly at 8 h and decreased thereafter. Transglutaminase was immunodetected in whole cells from the callus and in isolated chloroplasts by western blot. In the entire cells, protein substrates were found which were not present in isolated chloroplast. Transglutaminase activity was light sensitive and also affected by hormone deprivation. This enzyme was more active in differentiation than in maintenance medium, in both callus and chloroplasts, in light and dark phases. These data indicate that, the parameters studied here are not only light affected but also regulated by a daily rhythm.This work was supported by a Spanish grant DGICYT PB-94-0082 and two short term stages of I. Claparols and E. Bernet in the laboratory of Dr Serafini-Fracasini (Bologna University) financed by CIRIT and Unesco grants, respectively. Part of the work was also supported by an Italian C.N.R. grant (contributo 97.04274.CT04) to Serafini-FracassiniPeer reviewe