Arginine Decarboxylase Is Localized in Chloroplasts.

Plants, unlike animals, can use either ornithine decarboxylase or arginine decarboxylase (ADC) to produce the polyamine precursor putrescine. Lack of knowledge of the exact cellular and subcellular location of these enzymes has been one of the main obstacles to our understanding of the biological role of polyamines in plants. We have generated polyclonal antibodies to oat (Avena sativa L.) ADC to study the spatial distribution and subcellular localization of ADC protein in different oat tissues. By immunoblotting and immunocytochemistry, we show that ADC is organ specific. By cell fractionation and immunoblotting, we show that ADC is localized in chloroplasts associated with the thylakoid membrane. The results also show that increased levels of ADC protein are correlated with high levels of ADC activity and putrescine in osmotically stressed oat leaves. A model of compartmentalization for the arginine pathway and putrescine biosynthesis in active photosynthetic tissues has been proposed. In the context of endosymbiote-driven metabolic evolution in plants, the location of ADC in the chloroplast compartment may have major evolutionary significance, since it explains (a) why plants can use two alternative pathways for putrescine biosynthesis and (b) why animals do not possess ADC.

Epithelial cell hyperproliferation after biliopancreatic reflux into the esophagus of rats.

BACKGROUND: Chronic reflux of duodenal contents into the esophagus of rats produces severe esophagitis and exerts a co-carcinogenic effect on the proliferating cells by enhancing the formation of nitrosamine-induced esophageal carcinomas. We investigated the effect of the different components of the duodenal reflux on the epithelial cell proliferation of the lower esophagus. METHODS: Sprague-Dawley rats underwent three surgical reflux models (biliopancreatic, pancreatic, and biliary) and a sham operation. Animals were sacrificed at 72 hours, 6 weeks, and 9 weeks after the operation. Histology and cell proliferation, determined by ornithine decarboxylase activity, polyamine (putrescine, spermidine, spermine) levels, and proliferating cell nuclear antigen labeling index of the basal and suprabasal layers, were studied in the distal esophagus. RESULTS: Both biliopancreatic and pancreatic reflux induced severe esophagitis starting on week 6. Suprabasal proliferating cell nuclear antigen labeling index significantly increased throughout the 9 weeks of the study in the biliopancreatic and pancreatic reflux groups, although this increase was earlier in the former group. Ornithine decarboxylase activity and polyamine levels were significantly increased in the biliopancreatic and pancreatic groups on week 6, decreasing on week 9. CONCLUSIONS: Increased esophageal cell proliferation after both biliopancreatic and pancreatic reflux into the lower esophagus may therefore be one mechanism by which duodenal-content reflux stimulates esophageal carcinogenesis in experimental animals.

Arginine decarboxylase as the source of putrescine for tobacco alkaloids.

The putrescine which forms a part of nicotine and other pyrrolidine alkaloids is generally assumed to arise through the action of ornithine decarboxylase (ODC). However, we have previously noted that changes in the activity of arginine decarboxylase (ADC), an alternate source of putrescine, parallel changes in tissue alkaloids, while changes in ODC activity do not. This led us to undertake experiments to permit discrimination between ADC and ODC as enzymatic sources of putrescine destined for alkaloids. Two kinds of evidence presented here support a major role for ADC in the generation of putrescine going into alkaloids: (a) A specific 'suicide inhibitor' of ADC effectively inhibits the biosynthesis of nicotine and nornicotine in tobacco callus, while the analogous inhibitor of ODC is less effective, and (b) the flow of 14C from uniformly labelled arginine into nicotine is much more efficient than that from ornithine.

Modified alkaloid pattern in developing tobacco callus.

Developing Nicotiana tabacum L. cv. Wisconsin-38 callus grown on modified Murashige-Skoog (MS) medium with Kao organic acids (pyruvic, citric, malic and fumaric acids) contains abnormally high levels of nornicotine and total alkaloids when compared with the leaves of the donor plant. Nornicotine/nicotine ratios observed during callus development suggest that nicotine is converted into nornicotine in the callus, with subsequent movement of alkaloids into roots formed on the callus and into the agar medium. Addition of Kao organic acids to the medium increases alkaloid levels, but cannot account for the abnormal increase in nicotine demethylation. This study thus reports two new findings: (a) that the total alkaloid content of tobacco callus can be greatly enhanced to 3.75% on a dry weight basis by exogenous organic acids, and (b) that endogenous nornicotine can accumulate in tobacco tissue cultures.

Polyamine metabolism and osmotic stress. II. Improvement of oat protoplasts by an inhibitor of arginine decarboxylase.

We have attempted to improve the viability of cereal mesophyll protoplasts by pretreatment of leaves with DL-alpha-difluoromethylarginine (DFMA), a specific 'suicide' inhibitor of the enzyme (arginine decarboxylase) responsible for their osmotically induced putrescine accumulation. Leaf pretreatment with DFMA before a 6 hour osmotic shock caused a 45% decrease of putrescine and a 2-fold increase of spermine titer. After 136 hours of osmotic stress, putrescine titer in DFMA-pretreated leaves increased by only 50%, but spermidine and spermine titers increased dramatically by 3.2- and 6-fold, respectively. These increases in higher polyamines could account for the reduced chlorophyll loss and enhanced ability of pretreated leaves to incorporate tritiated thymidine, uridine, and leucine into macromolecules. Pretreatment with DFMA significantly improved the overall viability of the protoplasts isolated from these leaves. The results support the view that the osmotically induced rise in putrescine and blockage of its conversion to higher polyamines may contribute to the lack of sustained cell division in cereal mesophyll protoplasts, although other undefined factors must also play a major role.

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-alpha-diffluromethylarginine (DFMA) and D-arginine (D-Arg), both inhibitors of ADC activity, were more effective than DL-alpha-difluromethylorinithine (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.

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.

Rapid high-performance liquid chromatographic method for the quantitation of polyamines as their dansyl derivatives: application to plant and animal tissues.

A rapid high-performance liquid chromatographic method for the separation of polyamines as their dansyl derivative has been developed. The chromatographic system used consisted of a reversed-phase column and a mobile phase of acetonitrile and water. The separation of 1,3-diaminopropane, putrescine, cadaverine, spermidine and spermine takes only 9 min. This method provides a good resolution between 1,3-diaminopropane and putrescine. It has been applied to quantify polyamines from seeds of wheat, petals of Phalaenopsis hybrids and various rat tissues.

Correlation between polyamines and pyrrolidine alkaloids in developing tobacco callus.

Since the diamine putrescine can be metabolized into the pyrrolidine ring of tobacco alkaloids as well as into the higher polyamines, we have investigated the quantitative relationship between putrescine and these metabolites in tobacco callus cultured in vitro. We measured levels of free and conjugated putrescine and spermidine, and pyrrolidine alkaloids, as well as activities of the putrescine-biosynthetic enzymes arginine and ornithine decarboxylase. In callus grown on high (11.5 micromolar) alpha-naphthalene acetic acid, suboptimal for alkaloid biosynthesis, putrescine and spermidine conjugates were the main putrescine derivatives, while in callus grown on low (1.5 micromolar) alpha-naphthalene acetic acid, optimal for alkaloid formation, nornicotine and nicotine were the main putrescine derivatives. During callus development, a significant negative correlation was found between levels of perchloric acid-soluble putrescine conjugates and pyrrolidine alkaloids. The results suggest that bound putrescine can act as a pool for pyrrolidine alkaloid formation in systems where alkaloid biosynthesis is active. In addition, changes in arginine decarboxylase activity corresponding to increased alkaloid levels suggest a role for this enzyme in the overall biosynthesis of pyrrolidine alkaloids.

Polyamine metabolism and osmotic stress. I. Relation to protoplast viability.

Cereal leaves subjected to the osmotica routinely used for protoplast isolation show a rapid increase in arginine decarboxylase activity, a massive accumulation of putrescine, and slow conversion of putrescine to the higher polyamines, spermidine and spermine (HE Flores, AW Galston 1984 Plant Physiol 75: 102). Mesophyll protoplasts from these leaves, which have a high putrescine:polyamine ratio, do not undergo sustained division. By contrast, in Nicotiana, Capsicum, Datura, Trigonella, and Vigna, dicot genera that readily regenerate plants from mesophyll protoplasts, the response of leaves to osmotic stress is opposite to that in cereals. Putrescine titer as well as arginine and ornithine decarboxylase activities decline in these osmotically stressed dicot leaves, while spermidine and spermine titers increase. Thus, the putrescine:polyamine ratio in Vigna protoplasts, which divide readily, is 4-fold lower than in oat protoplasts, which divide poorly. We suggest that this differing response of polyamine metabolism to osmotic stress may account in part for the failure of cereal mesophyll protoplasts to develop readily in vitro.

Endogenous polyamine concentrations in juvenile, adult and in vitro reinvigorated hazel.

We investigated endogenous polyamine concentrations in leaves from juvenile and mature hazel (Corylus avellana L.) shoots, as well as leaves from shoots obtained by both forced outgrowth and micropropagation of adult tissues. To determine if the observed in vitro reinvigoration was associated with polyamine metabolism, we tested the effect of serial subcultures on polyamine concentrations. Polyamines, mostly free putrescine, were higher in juvenile tissues. Adult tissues micropropagated for 14 subcultures had polyamine concentrations characteristic of juvenile tissues. However, with additional subcultures, total polyamine concentrations decreased. The putrescine to spermidine plus spermine ratio was higher in juvenile and micropropagated tissues than in adult tissues, but decreased in micropropagated tissues after 20 subcultures. This ratio may reflect a balance between vegetative growth and reproductive processes. Thus, an analysis of polyamine concentrations may provide a simple assay for determining the juvenility of plant tissues and, hence, their suitability for micropropagation.

Inducible overexpression of oat arginine decarboxylase in transgenic tobacco plants.

To test the possible interaction of polyamines in plant growth responses, transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase (ADC) gene under the control of a tetracycline-inducible promoter were generated. Inducible overexpression of oat ADC in transgenic tobacco led to an accumulation of ADC mRNA, increased ADC activity and changes in polyamine levels. Transgenic lines, induced during vegetative stage, displayed different degrees of an altered phenotype, the severity of which was correlated with putrescine content. These phenotypic changes were characterized by short internodes, thin stems and leaves, leaf chlorosis and necrosis, as well as reduced root growth. This is the first report to show altered phenotypes as a consequence of polyamine changes under tetracycline-induction in in vivo conditions. Interestingly, overexpression of oat ADC in tobacco resulted in similar detrimental effects to those observed by ADC activation induced by osmotic stress in the homologous oat leaf system. In the context of the role of specific polyamines in plant growth and development, the present results indicate that activation of the ADC pathway leading to high levels of endogenous putrescine (or its catabolytes) is toxic for the vegetative growth of the plant. In contrast, no visible phenotypic effects were observed in flowering plants following tetracycline induction. Further characterization of the different transgenic lines may shed light on the action of specific polyamines in different plant developmental processes.

Manipulation of S-adenosylmethionine decarboxylase activity in potato tubers. An increase in activity leads to an increase in tuber number and a change in tuber size distribution.

S-Adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) is a key enzyme in the biosynthesis of the polyamines spermidine and spermine from putrescine and its activity is rate limiting in this pathway. Transgenic potato (Solanum tuberosum L. cv. Desiree) plants containing both sense and antisense SAMDC constructs driven by the tuber-specific patatin promoter have been generated and analysed. In sense transformants, developing tubers expressed higher steady-state levels of the SAMDC-specific transcript, had higher levels of SAMDC activity and contained significantly higher levels of spermidine than vector-transformed controls. Additionally, there was a significant shift in tuber size distribution with larger numbers of smaller tubers but no overall change in tuber yield. In developing tubers from the antisense transformed lines, there was a decrease in SAMDC transcript level, SAMDC activity and total polyamine levels. However, no obvious phenotypic effect was detected in the tuberisation physiology of the antisense lines.