M. M. Altamura

Polyamines and Morphogenesis in Normal and Transgenic Plant Cultures

Direct and indirect approaches have demonstrated that polyamines are involved in cell division and in other events connected with growth and morphogenesis in plant tissue and cell cultures and that they affect senescence and stress responses (Bagni, 1989; Galston and Flores, 1991). However since the early report of Bagni (1966) clearly indicated that, in the absence of exogenous hormones and with endogenous hormone concentrations at levels unable to sustain growth, 10 to 100 μM polyamines could support cell division in Helianthus tuberosus tuber expiants, very few reports have demonstrated that a specific morphogenic effect (i.e. embryogenesis or organogenesis) is induced by exogenous polyamines.

Two SERK genes are markers of pluripotency in Cyclamen persicum Mill

The genetic basis of stem cell specification in somatic embryogenesis and organogenesis is still obscure. SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes are involved in embryogenesis and organogenesis in numerous species. In vitro culture of Cyclamen persicum immature ovules provides a system for investigating stem cell formation and maintenance, because lines forming either organs or embryos or callus without organs/embryos are available for the same cultivar and plant growth regulator conditions. The present aim was to exploit this property of cyclamen cultures to understand the role of SERK(s) in stem cell formation and maintenance in somatic embryogenesis and organogenesis in vitro, in comparison with expression in planta. CpSERK1 and CpSERK2 were isolated from embryogenic callus. CpSERK1 and CpSERK2 levels by RT-PCR showed that expression is high in embryogenic, moderate in organogenic, and null in recalcitrant calli. in situ hybridizations showed that the expression of both genes started in clumps of pluripotent stem cells, from which both pre-embryogenic aggregates and organ meristemoids derived, and continued in their trans-amplifying, meristem-like, derivatives. Expression declined in organ meristemoids, in parallel with a partial loss of meristematization. In mature somatic embryos, and in shoot and root primordia, CpSERK1 and CpSERK2 were expressed in meristems, and similar patterns occurred in zygotic embryo and primary meristems in planta. The results point to SERK1 and SERK2 as markers of pluripotency in cyclamen. It is proposed that the high expression of these genes in the trans-amplifying derivatives of the stem cells maintains a pluripotent condition leading to totipotency and, consequently, somatic embryogenesis.

Ethylene involvement in vegetative bud formation in tobacco thin layers

SummaryThe role of ethylene in vegetative bud regeneration was studied in cultured tobacco (Nicotiana tabacum L. cvSamsun) thinlayer expiants. The experimental approach consisted in supplementing the bud-inducing medium with an inhibitor of ethylene biosynthesis, aminoethoxyvinylglycine (AVG), an ethylene antagonist, silver thiosulphate (STS), or an ethylene-releasing compound, 2-chloroethylphosphonic acid (CEPA), at various concentrations. The organogenic response was assessed both macroscopically (percentage of bud-forming expiants, final number of buds per expiant) and cytohistologically (number, characteristics, and localisation of meristemoids and bud primordia). The time course of ethylene production during culture was also evaluated. At the end of culture (day 27) all the expiants treated with these compounds had a lower number of buds compared to controls. STS was detrimental to meristemoid initiation at all the concentrations tested. In contrast, 0.5 μM AVG, which strongly inhibited ethylene production, provoked a large increase in the formation of meristemoids early in culture and the appearance of anomalous (“twin”) buds. CEPA reduced meristemoid formation but, at the lower concentrations (1 and 10 μM) speeded up bud emergence. On the whole it mainly favoured disorganised growth and xylogenesis. The results of this work highlight the contrasting effects of ethylene in relation to the two critical stages of the organogenic process, i.e., meristemoid formation and bud primordium development.

THE ROLD GENE FROM AGROBACTERIUM RHIZOGENES IS DEVELOPMENTALLY REGULATED IN TRANSGENIC TOBACCO

SummaryTherolD gene fromAgrobacterium rhizogenes has recently been shown to induce striking precocity of flowering in transgenic tobacco. A transcriptional fusion between 578 bp of therolD upstream regulating sequence and the GUS reporter gene has been transferred to tobacco plants and its expression analysed throughout the whole life cycle of the plant. A detailed histological analysis revealed thatrolD is strongly expressed in elongating and differentiating tissues of each organ, from the mature embryo to the adult plant. In organs with determinate growth, such as cotyledons, leaves and floral parts,rolD expression follows the age gradient of the tissues. In mature tissues,rolD expression is high, begins to decrease with ageing and is switched off when senescence occurs.RolD is also active in the vascular system, in the procambium, pith- and receptacular-meristems. Our data show that expression ofrolD is under developmental control as it correlates with, and may represent a molecular marker for, the elongation/expansion and maturation phases of the plant tissues.

Polyamines as markers in sexual reproduction of kiwifruit

Aliphatic polyamines are plant growth regulators that affect mainly the induction and sustenance of cell division (Bagni 1989). They are involved in all the steps of protein synthesis (replication, transcription and translation) through specific and aspecific interactions with DNA and different RNAs (Feuerstein and Marton 1989). In particular, the specific interactions of two molecules of spermine or spermidine, one located in the major groove at one end of the anticodon stem, the other near the variable loop of tRNA, are able to switch tRNA conformation from the inactive to the active form (which is then able to accept the specific aminoacid) (Quigley et al. 1978). This observation provides one of the most plausible explanations for the mode of action of these compounds in the cell.

Inhibition of Polyamine Biosynthesis Affects the Rhizogenic Response in Tobacco Thin Cell Layers

Aliphatic polyamines (PA) occur throughout the plant kingdom; they are polycations capable of binding with nucleic acids, cell wall polysaccharides and proteins (1, 2). Much evidence indicates that these substances may function as growth regulators or as second messengers in plant cells (1, 3). The need for PA in sustaining cell division is well established; in general, non-growing tissues have low levels of PA, as well as of other growth substances, while actively growing tissues have higher levels (3). Although less is known about their involvement in organogenesis, elevated levels of PA and of their biosynthetic enzymes have been associated with cells undergoing changes in their state of differentiation, for example during somatic embryogenesis and during in vitro bud or root formation (4, 5, 6, 7, 8).

Long-sized oligogalacturonides inhibit, whereas spermidine enhances, xylogenesis in tobacco leaf explants

Abstract Long-sized oligogalacturonides (OGs) are plant cell wall fragments involved in defence responses and developmental processes. A hormone/OG interaction in the control of different organogenic processes is known. However, hormones also modulate polyamine (PA) effects on organogenesis. Furthermore, OGs are known to affect mitotic activity leading to specific morphogenic events, and PAs are known to affect mitotic activity leading to xylogenesis. Thus, it may be reasonable to assume that OGs and PAs affect mitotic activity in the same cell types, and in the same hormone-induced morphogenic processes, e.g., xylogenesis. To gain further insight into this aspect, the effects of OGs, and of putrescine (Put) and spermidine (Spd), on auxin (indoleacetic acid, IAA) plus benzyladenine (BA)-induced morphogenesis in tobacco leaf explants were investigated histologically. The effect of PA biosynthetic inhibitors in the culture medium was also monitored, as well as the combined application of the inhibitor with the corresponding PA. Results show that vascular mitoses consistently occurred in the control (IAA+BA-treated) explants, leading exclusively to xylogenic nodule formation. The application of OGs resulted in an inhibition of vascular mitoses, and into a strong reduction of vascular nodule formation. By contrast, Spd enhanced both vascular mitoses and nodule formation, and Put was less effective than Spd on both events. Taken together, the results reveal a new biological activity of OGs and Spd in morphogenesis, obtained under the same hormonal conditions, and in the same tissue (i.e., the vascular parenchyma), namely the inhibition of xylogenesis by OGs, and its promotion by Spd. The fact that the effects of Spd and OGs on this morphogenic event may involve a different relationship with auxin is discussed.

The Plant Oncogene Rolb Enhances Meristem Formation in Tobacco thin Cell Layers

The expression and the effects of Agrobacterium rhizogenes rolB gene during in vitro flower, root and vegetative organogenesis were investigated. Thin cell layers were excised from floral pedicels, inflorescence rachis and stems of tobacco plants containing either rolB and its promoter fused to the GUS reporter gene or only this latter construct. Organogenesis and GUS activity were monitored in pedicel and stem expiants cultured under conditions for floral, root, and vegetative neoformation. Flowering, rhizogenesis and caulogenesis were enhanced in rolB-transformed expiants as compared to untransformed controls: the neoformed meristems were much more numerous and appeared sooner. rolB promoter was specifically active in the initial cells of all the meristem types. Rachis expiants from rolB-plants produced, upon culture under flowering conditions, also an exceeding number of vegetative buds. Thus, rolB seems to stimulate the formation of meristems regardless of their subsequent differentiation and of the hormone controlling the morphogenic event.