Effects of the Suicide Inhibitors of Arginine and Ornithine Decarboxylase Activities on Organogenesis, Growth, Free Polyamine and Hydroxycinnamoyl Putrescine Levels in Leaf Explants of Nicotiana Xanthi n.c. Cultivated in Vitro in a Medium Producing Callus Formation

Abstract

We studied the effects of DL-a-difluoromethylarginine (DFMA) and DL-a-difluoromethylomithine (DFMO), specific, irreversible inhibitors of arginine decarboxylase (ADC) and omithine decarboxylase (ODC), respectively, on organogenesis growth and titers of free polyamines and conjugated putrescines (hydroxycinnamoyl putrescines) in tobacco (Nicotiana tabacum cv Xanthi n.c.)calli. These results suggest that ADC and ODC regulate putrescine biosynthesis during early and later stages of tobacco callus development, respectively. ADC appears active in biosynthesis of large levels of free amines (agmatine and putrescine) while ODC appears active only in biosynthesis of large levels of putrescine conjugates (hydroxycinnamoyl putrescines). DFMA inhibits the fresh and dry weight increases of tobacco calli, whereas DFMO even promoted the fresh and dry weight increases, thus supporting the view that ADC is important for cell division and callus induction. Inhibition of ODC activity by DFMO resulting in an amide deficiency after 4 weeks of culture facilates the expression of differentiated cell functions. Formation of buds is associated with a significant decrease of hydroxycinnamoyl putrescines.Polyamines and their biosynthetic enzymes may play an important role in many aspects of plant development including growth, differentiation, senescence, and response to stress (1, 10, 26). Increased polyamine synthesis appears necessary for cell proliferation to occur (3). Amides formed between hydroxycinnamic acid and amines are widely distributed in the plant kingdom (17, 18). Accumulation of hydroxycinnamic acid amides correlates with leaf emergence and flowering in several plants (5,17,19). Tobacco stem apices, callus tissues, and cell cultures often contain polyamines conjugated with cinnamic acids (4,5,11,20). Some functional roles of these compounds have been suggested. Caffeoyl putrescine and other amides such as caffeoyl spermidine have been found in large concentrations in reproductive organs oftobacco, and it has been proposed that their formation is functionally linked with reproduction (5). In addition, since tobacco plants infected with tobacco mosaic virus react by increasing levels of polyamine conjugates, and virus multiplication is retarded in their presence, a role for these compounds in virus resistance has been suggested (15, 19). Recent results indicate that hydroxycinnamoyl putrescines, when included in the medium, interfere in vitro with hormones in the control of cell multiplication and differentiation of tobacco leaf explants (16).For in vitro cultures, it appears that the intracellular levels of putrescine and hydroxycinnamoyl putrescines are markedly enhanced when the cultures are induced to proliferate (14). The differentiation of buds is always accompanied by significant changes in putrescine and hydroxycinnamoyl putrescine levels (14). Increasing levels were found during the first week in culture when cell multiplication is rapid. Then levels declined sharply after 3 weeks of culture as the rate of ...

Polyamines, Hydroxycinnamoylputrescines, and Root Formation in Leaf Explants of Tobacco Cultivated in Vitro

“…There is an arginase-mediated hydrolysis of DFMA in tobacco ovary tissues (29) resulting in the accumulation ofDFMO and significant inhibition ofODC activity in these tissues. Similar results are obtained when DFMA is added in tobacco callus cultures cultivated with a cytokinin + 2,4-D (8). In these cultures the inhibition of Put biosynthesis by DFMA is explained by the conversion of DFMA to DFMO after the first 2 weeks of culture.…”

“…These results report that a change in morphology or growth is accompanied by changes in Put levels and the largest changes occur in conjugated Put specifically. The (8).…”

“…20 and 31). Recent results (8,22) report that a change in morphology or growth of tobacco leaf explants in vitro is accompanied by changes in putrescine levels, and the largest changes occur in conjugated putrescine content specifically.Are polyamines and conjugated polyamines esssential for root formation, or are they simply secondary by-products of a growth response? One way to approach this question is by utilizing specific inhibitors of polyamine synthesis and then examining the physiological properties of tissues.…”

“…20 and 31). Recent results (8,22) report that a change in morphology or growth of tobacco leaf explants in vitro is accompanied by changes in putrescine levels, and the largest changes occur in conjugated putrescine content specifically.…”

Métabolisme et rôle des polyamines dans le développement de la plante

“…Les deux principaux inhibiteurs synthetises sont Ia 2-difluoromethylarginine (DFMA), inhibiteur specifique et irreversible de l'ADC (Kallio et al, 1981 ;Burtin et al, 1989), et Ja 2-difluoromethylornithine (DFMO), inhibiteur specifique et irreversible de l'ODC (Metcalf et al, 1978 ;Slocum et Gals ton, 1987;Burtin et al, 1991). Ces inhibiteurs sont des pseudo-substrats, chimiquemcnt inertes pour l'enzyme, qui se fixent sur le site actif, et sont transformes en un compose qui inactive )'enzyme {Bey et al, 1987).…”