Ravindar Kaur-Sawhney

The physiology and biochemistry of polyamines in plants

Our entry into the polyamine field was accidental. As plant physiologists interested in the regeneration of entire plants from isolated single cells and protoplasts, we had turned our attention to the two most important groups of food plants, the cereals and the legumes (1, 2). When we began this work about a decade ago, there had been no successful protoplast regeneration of any plants in these groups, although numerous researchers had been successful with various members of the Solanaceae (tobacco, petunia, potato, tomato), as well as a few species in other families (3). Today, several of the Leguminosae have been successfully regen erated from protoplasts (4-6), but only one report of cereal regeneration from protoplasts, in pearl millet, exists in the literature (7). Why should cereal leaf protoplasts behave so diflerently from protoplasts of other species? We had been working with oat, and our attention was drawn to a report that the rapid senescence of detached leaves of this plant could be delayed substantially by the application of arginine (8). It is well known to plant physiologists that leaves detached from the plant initiate a rapid and massive hydrolysis of proteins and nucleic acids, then senesce, yellow, and die. These events can be delayed, and sometimes prevented, by cytokinins, exemplified by N-isopentenyl adenosine or zeatin (9). We had, of course, applied cytokinins to our isolated oat leaf protoplasts, but without positive

Polyamines as Endogenous Growth Regulators

It is probable that all cells contain the diamine putrescine (Put; 1,4-diaminobutane) and the triamine spermidine (Spd), while eukaryotic cells contain the tetraamine spermine (Spm) as well (6, 13). In both prokaryotes and eukaryotes (53), including higher plants (30), mutants lacking the ability to biosynthesize polyamines (PAs) are unable to grow and develop normally (53). Since the addition of PAs to these mutants generally restores normal growth and development, it is reasonable to conclude that PAs are essential to all cells. This conclusion is reinforced by the demonstrable effects of “suicide inhibitors” of the main PA-bio- synthetic enzymes, ornithine decarboxylase (ODC) and arginine decarboxylase (ADC: Fig. 1). These compounds, a-difluoromethyl- ornithine (DFMO) and a-difluoromethylarginine (DFMA), specifically and irreversibly bind to and inhibit ODC and ADC, respectively. The ensuing decline in cellular PA titers is accompanied by a diminution or cessation of growth and development, which are restored upon the addition of the relevant PA.

Spermidine and flower-bud differentiation in thin-layer explants of tobacco.

Three lines of evidence indicate a connection between high spermidine levels and floral initiation in thin-layer tissue cultures of Wisconsin-38 tobacco (Nicotiana tabacum L.). (1) Spermidine levels are much higher in floral buds than in vegetative buds. (2) Inhibition of spermidine synthesis by cyclohexylamine prevents the rise in spermidine titer, inhibits floral initiation and promotes the formation of vegetative buds instead. (3) Application of exogenous spermidine causes floral initiation in cultures which would otherwise form vegetative buds.

Polyamines, ribonuclease and the improvement of oat leaf protoplasts

Abstract L-arginine and L-lysine delay spontaneous lysis of oat leaf protoplasts, preserve uniformity of chloroplast distribution in protoplasts and prevent protoplast aggregation and adhesion to the substratum. Osmotic shock, exogenous RNAase and cell-free centrifugal supernatant fractions of mechanically-lysed protoplasts all induce protoplast lysis; arginine and lysine protect against these stresses as well. Arginine and lysine also produce effects at the metabolic level that may be related to their morphological stabilization of protoplasts. For example, the RNAase activity of freshly extracted oat leaf protoplasts is low, but rises progressively as the protoplasts are incubated in vitro; the same rise occurs during incubation of excised leaves prior to protoplast extraction. The RNAase increase in both systems can be prevented by 10–50 mM L-arginine or L-lysine. Concomitantly, these compounds support higher levels of incorporation of [ 3 H] uridine into RNA. All the above effects can also be produced by polyamines (putrescine, cadaverine and spermidine) which are metabolically related to lysine and arginine; spermine is less effective at the concentrations tested.

Inhibition of protease activity by polyamines: Relevance for control of leaf senescence

The polyamines spermidine and spermine and the related diamines putrescine and cadaverine are polycations which are synthesized in most living cells [l-3] and have been implicated as essential growth factors for plants [4,5]. Polyamines applied exogenously are potent inhibitors of senescence of oat leaf protoplasts [6,7] and of leaves and storage tissue from several plants [8,9]. We have also reported that polyamine-biosynthetic enzyme activities and titer decrease in senescing attached and detached oat leaves incubated in the dark [lo], and in potato tubers during dormancy [ll]. When growth of pea buds and internodes is stimulated or inhibited by phytochrome conversion [ 12,131 or hormonal treatment [ 141, polyamine synthesis and titer show rapid and parallel changes. These observations, together with the demonstrated roles of polyamines in cell proliferation suggest that they are involved in processes which control plant growth and senescence. One of the early events in leaf senescence is the well-documented rise in protease activity [ 151. However, little is known about the regulation of proteolysis itself during leaf senescence. Considerable evidence has accumulated which shows that involvement of polyamines in cellular activity may

Chromosomal localization of osmotic and salt stress-induced differential alterations in polyamine content in wheat

Abstract Osmotic and salinity-induced polyamine accumulation were compared in callus cultures of drought and salt tolerant wheat (Triticus aestivum L) cultivars and in disomic substitution lines. Putrescine, spermidine and spermine occurred in all cultures. Mannitol-induced osmotic stress increased putrescine in all, and cadaverine in two varieties, while salt stress increased spermidine titer, the accumulation rate being higher in sensitive than in tolerant varieties. Specific chromosome (5A and 7A) involvement in osmotic stress induced spermidine accumulation revealed that mannitol was the most effective stress agent and only spermidine titer of Chinese Spring was significantly changed as a consequence of chromosome substitution. The A genome of Cappelle Desprez (donor) substituted into Chinese Spring (recipient) appears to carry genes involved in the control of osmotic stress induced spermidine accumulation, and the genes controlling cadaverine biosynthesis may be localized in chromosome 5B.

Effect of polyamine biosynthetic inhibitors on alkaloids and organogenesis in tobacco callus cultures

We studied the effects of inhibitors of ornithine decarboxylase (ODC), arginine decarboxylase (ADC) and spermidine synthase (Spd synthase) on organogenesis and the titers of polyamines (PA) and alkaloids in tobacco calli. DL-α-difluoromethylarginine (DFMA) and D-arginine (D-Arg), both inhibitors of ADC activity, were more effective than DL-α-difluoromethylornithine (DFMO), an inhibitor of ODC, in reducing titers of PA and the putrescine (Put)-derived alkaloids (nornicotine and nicotine). Dicyclohexylammonium sulfate (DCHA), an inhibitor of Spd synthase, was also more efficient than DFMO in reducing PA and alkaloid levels. Root organogenesis is inversely related to the titers of Put and alkaloids. Thus, DFMA and D-Arg, which strongly inhibit Put and alkaloid biosynthesis, markedly promote root organogenesis, while control callus with high Put and alkaloid content showed poor root organization. These results suggest that morphological differentiation is not required for activation of secondary metabolic pathways and support the view that ADC has a major role in the generation of Put going to the pyrrolidine ring of tobacco alkaloids.

Effect of cycloheximide and kinetin on yield, integrity and metabolic activity of oat leaf protoplasts

Abstract The yield of protoplasts from cellulase-treated peeled oat leaf segments is increased if the leaves have previously been floated on cycloheximide (CH), rather than on water. The optimal CH level is 0.5 to 1.0 μg/ml. No effects are produced by pre-incubation times less than 6 h, and the optimal pretreatment time is about 18 h. Protoplasts from CH-pretreated leaves are more resistant to spontaneous lysis, and are more active in incorporating labeled l -leucine, uridine and thymidine into trichloroacetic acid (TCA)-insoluble materials than are protoplasts from leaves floated on water. Kinetin (Kn) acts like CH in almost all respects.

The cytokinin-like action of methyl-2-benzimidazolecarbamate on oat leaves and protoplasts☆

Abstract Chlorophyll breakdown in excised oat leaves is retarded by methyl-2-benzimidazolecarbamate, benzimidazole, and kinetin. Protoplasts derived from treated leaves showed lower nuclease activity, increased leucine incorporation, and decreased uridine and thymidine incorporation into trichloroacetic acid-insoluble materials relative to water-treated controls. Methyl-2-benzimidazolecarbamate is more active than benzimidazole in affecting macromolecular synthesis, and both compounds resemble kinetin in their effects on host-plant physiology. Protoplasts can be useful tools in the analysis of the action of pesticides.

Further experiments on spermidine-mediated floral-bud formation in thin-layer explants of Wisconsin 38 tobacco

We studied the effects of various polyamines on bud regeneration in thin-layer tissue explants of vegetative and floweringNicotiana tabacum L. cv. Wisconsin 38, in which application of exogenous spermidine (Spd) to vegetative cultures causes the initiation and development of some flower buds (Kaur-Sawhney et al. 1988 Planta173, 282). We now show that this effect is dependent on the time and duration of application, Spd being required from the start of the cultures for about three weeks. Neither putrescine nor spermine is effective in the concentration range tested. Spermidine cannot replace kinetin (N6-furfurylaminopurine) in cultures at the time of floral bud formation, but once the buds are initiated in the presence of kinetin, addition of Spd to the medium greatly increases the number of floral buds that develop into normal flowers. Addition of Spd to similar cultures derived from young, non-flowering plants did not cause the appearance of floral buds but rather induced a profusion of vegetative buds. These results indicate a morphogenetic role of Spd in bud differentiation.