I. Claparols

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.

Influence of some exogenous amino acids on the production of maize embryogenic callus and on endogenous amino acid content

The effects of four exogenous amino acids (proline, glycine, asparagine and serine) on the production of maize embryogenic callus and on its endogenous amino acid content have been investigated. For this purpose, an established embryogenic line of Type 1 callus from the inbred W64Ao2 has been used. From the results it may be concluded that a concentration of proline exceeding 6 mM is negative for the production of embryogenic callus. When proline is eliminated from the medium, other amino acids tested in certain concentrations yield a percentage of embryogenic callus production that exceeds or equals that of proline. The endogenous free proline content in embryogenic callus is significantly higher than that in non-embryogenic callus regardless of proline presence in the medium. The only exception are the glycine-containing media, in which endogenous free alanine of embryogenic callus increases at the expense of endogenous free proline. This study suggest a positive role of endogenous free proline or alanine accumulation in the embryogenic callus production which might be related to an adaptation to the metabolic changes produced by in vitro culture and embryogenesis induction. Furthermore, these results indicate that treatments with amino acids that are different from proline can be used to improve the efficiency of embryogenic callus production from well established maize callus cultures.

Influence of exogenous proline on embryogenic and organogenic maize callus subjected to salt stress

The effect of exogenous proline (6 mM) and increasing NaCl doses (from 0.4 to 1.2% w/v) on the maintenance of organogenic and embryogenic callus lines derived from the salt-sensitive maize inbred W64Ao2 were studied. To this end, total protein, free amino acid and polyamine content were analyzed. The demand of exogenous nitrogen and especially of proline, even in the presence of salt, differed in the two types of morphogenic calluses. The total protein content of embryogenic calluses was higher in the presence of proline than in its absence, in all the cases studied. An opposite effect of proline was observed in organogenic calluses: the presence of proline and salt decreased significantly their protein content. With respect to amino acid and polyamine contents, the organogenic calluses showed physiological characteristics of salt-adaptation, whereas the embryogenic calluses were more sensitive to NaCl. Although endogenous proline increased in the organogenic calluses cultured in the presence of salt, in embryogenic calluses it only rose at the lowest salt concentration. Furthermore, the endogenous arginine content under saline conditions was higher in organogenic calluses. A compensatory effect between proline and polyamine metabolism related to the endogenous arginine content in response to salt stress was also observed. This effect differed in the two types of calluses.

Producing transglutaminases by molecular farming in plants: Minireview article

Summary.Transglutaminases have a range of catalytic activities, most of which concern the post-translational modification of proteins. The most important of these activities, both in terms of biology and biotechnology, is the cross-linking of proteins into large supramolecular networks. The widespread use of transglutaminases in research, medicine and industry has increased the demand for an inexpensive, efficient and safe source of recombinant enzymes. We describe initial results concerning the production of a mammalian transglutaminase in transgenic rice plants as a first step towards the large-scale molecular farming of this enzyme.

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.

Role of putrescine metabolic pathways in the differentiation process of maize meristematic callus

The role of putrescine and its interaction with auxin in modulating the differentiation of maize meristematic callus was studied. The effect of exogenous putrescine on differentiation depends on the endogenous polyamine level of the callus and the stage at which putrescine was applied. Plant regeneration was highest in the 1 mM putrescine-pretreated calluses that initiated the differentiation process with a higher endogenous putrescine content versus control. The enzymatic activities of argi- nine and ornithme decarboxylase (responsible for putrescine synthesis) were considerably lower in the presence of exogenous putrescine, indiscating a feed-back or a final product inhibition, respectively. The rise observed in the perchloric acid-insoluble polyamine fraction, especially putrescine, could be related to the initiation of the differentiation process. Low doses of auxin in differentiation medium induced a general decrease in plant regeneration. However, activation of the differentiation process was observed in calluses cultured in the presence of auxin and an excess of putrescine. In these calluses, the perchloric acid-soluble conjugated fraction increased significantly. These data indicate that putrescine is involved in the endogenous regulation of callus morphogenesis. 0 Elsevier, Paris

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.

Polyamines and Aging: Effect of Polyamine Biosynthetic Inhibitors on Plant Regeneration in Maize Callus Cultured in Vitro

Culture age is an important factor in expressing the genetic potential of plant cells and tissues in vitro, since it is well known that many cultures lose their morphogenetic capacity as they aged (Vasil et al., 1984). Although the precise biochemical and molecular mechanisms underlying the gradual loss of totipotency are not known, some compounds such as polyamines (PAs) are involved in these phenomena. Thus, the diamine putrescine (Put), the triamine spermidine (Spd), the tetraamine spermine (Spm), and their biosynthetic enzyme arginine decarboxylase (ADC) are biochemical markers of both plant cell aging and plant cell differentiation. In senescing leaves of cereals incubated in darkness both the ADC activity and the endogenous PA levels progressively decrease, while exogenous application of PAs, especially Spd and Spm, inhibits or retards the symptoms of senescence (Kaur-Sawhhey et al., 1979; 1982). On the other hand, using tobacco thin cell layer cultures (TCL; Tran Thanh Van, 1973) we have demonstrated that Spd is a marker of floral differentiation (Tiburcio et al., 1987; 1988; Kaur-Sawhney et al., 1988), while the Put formed via ADC is a marker of root differentiation (Tiburcio et al., 1989a).

Fundamental and Applied Aspects of Somatic Embryogenesis in Araujia sericifera

Araujia sericifera belongs to the Asclepiadaceae family. It has twining stems up to 10 m high, and the leaves are ovate-oblong, acurate, truncate at the base and white-tomentose beneath. Cymes are few-flowered with long peduncles. The flowers which are in axillary cymes with single corola and five segments, are white and pink striped and give off a delicate fragance. Flowers are always insect pollinated. The fruit is oblong and pruinose, containing many seeds with terminal filaments. More botanical information can be attained from Cabrera and Zardini (1978), Fiori (1925) or Flora Europea, vol.III.