Cytological events induced by the inhibition of polyamine biosynthesis in thin cell layers of tobacco

Summary The proliferative growth of thin cell layers ofNicotiana tabacum cultured on a rhizogenic medium was markedly disturbed when polyamine biosynthesis was inhibited. Treatments with polyamine inhibitors led to cell expansion, accompanied by thinning of the cell wall and inhibition of cell division, and frequent cases of nucleolar extrusion, mainly in the parenchymal layer in contact with the medium. Nucleolar extrusion was not correlated with cell expansion. The highest incidence of nucleolar extrusion occurred when the pathways of putrescine biosynthesis were inhibited and when spermidine synthesis, via S-adenosylmethionine decarboxylase, was blocked. The duration of the growth phase with nuclear amitotic divisions was prolonged in the presence of the inhibitors and root meristem formation delayed. When polyamines were added with the inhibitors, all reactions proceeded as in the controls.Abbreviations CHA cyclohexylamine - DFMA DL--difluoromethyl-arginine - DFMO DL--difluoromethylornithine - LS longitudinal section - MGBG methylglyoxal-bis(guanylhydrazone) - PA polyamine - Pu putrescine - RLS radial longitudinal section - S.E. standard error - Spd spermidine     

Growth inhibition by methionine analog inhibitors of S-adenosylmethionine biosynthesis in the absence of polyamine depletion

Four methionine analog inhibitors of methionine adenosyltransferase, the enzyme which catalyzes S-adenosylmethionine biosynthesis, were tested in cultured L1210 cells for their effects on cell growth, leucine incorporation, S-adenosylmethionine (AdoMet) formation and polyamine biosynthesis. The IC50 values were as follows: selenomethionine, 0.13 mM; L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cis-AMB), 0.4 mM; cycloleucine, 5 mM and 2-aminobicyclo[2.1.1]hexane-2-carboxylic acid, 5 mM. At IC50 levels, the analogs significantly reduced AdoMet pools by approximately 50% while not similarly affecting leucine incorporation or polyamine biosynthesis. In combination with inhibitors of polyamine biosynthesis, growth inhibition was greatly increased with methylglyoxal bis(guanylhydrazone), an inhibitor of AdoMet decarboxylase, but only slightly increased with alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase. Overall, the data indicate that the methionine analogs, and particularly L-cis-AMB, seem to inhibit cell growth by interference with AdoMet biosynthesis. Since polyamine biosynthesis is not affected, the antiproliferative effect may be mediated through perturbations of certain transmethylation reactions.     

Regulation of polyamine biosynthesis in tobacco. Effects of inhibitors and exogenous polyamines on arginine decarboxylase, ornithine decarboxylase, and S-adenosylmethionine decarboxylase

Treatment of tobacco liquid suspension cultures with methylglyoxal bis(guanylhydrazone) (MGBG) an inhibitor of S-adenosylmethionine decarboxylase, resulted in a dramatic overproduction of a 35-kDa peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Malmberg, R.L., and McIndoo, J. (1983) Nature 305, 623-625). MGBG treatment also resulted in a 20-fold increase in the activity of S-adenosylmethionine decarboxylase. Purification of S-adenosylmethionine decarboxylase from MGBG-treated cultures revealed that the overproduced 35-kDa peptide and S-adenosylmethionine decarboxylase are identical. Precursor incorporation experiments using [3H] methionine and [35S]methionine revealed that MGBG does not induce any increased synthesis of S-adenosylmethionine decarboxylase but rather stabilizes the protein to proteolytic degradation. The half-life of the enzyme activity was increased when MGBG was present in the growth medium. In addition to stabilizing S-adenosylmethionine decarboxylase, MGBG also resulted in the rapid and specific loss of arginine decarboxylase activity with little effect ornithine decarboxylase. The kinetics of this effect suggest that arginine decarboxylase synthesis was rapidly inhibited by MGBG. Exogenously added polyamines had little effect on ornithine decarboxylase, whereas S-adenosylmethionine and arginine decarboxylase activities rapidly diminished with added spermidine or spermine. Finally, inhibition of ornithine decarboxylase was lethal to the cultures, whereas inhibition of arginine decarboxylase was only lethal during initiation of growth in suspension culture.     

The inhibition of capacitative calcium entry due to ATP depletion but not due to glucosamine is reversed by staurosporine.

The capacitative Ca2+ entry pathway in J774 macrophages is rapidly inhibited by the amino sugar glucosamine. This pathway is also inhibited by treatments such as 2-deoxy-D-glucose (2dGlc) or glucose deprivation that inhibit glycolysis and lead to significant decreases in cellular ATP and other trinucleotides. We sought to determine whether glucosamine's effect on capacitative Ca2+ entry was also due to ATP depletion, as has been suggested recently for its link to insulin resistance. In contrast to brief treatments with 2dGlc, there was no significant decrease in ATP following exposure to glucosamine. In addition, the 2dGlc-mediated inhibition of capacitative Ca2+ influx was reversed by staurosporine, a microbial alkaloid that inhibits a broad range of protein kinases. Staurosporine was also able to reverse the inhibition of capacitative Ca2+ entry seen following other treatments that decreased cellular ATP levels, including cytochalasin B and iodoacetic acid. Other inhibitors of protein kinase C, including bisindolylmaleimide, K252a, H-7, and calphostin C, were unable to mimic this effect of staurosporine. However, the inhibition of capacitative Ca2+ influx in the presence of glucosamine was not reversed by staurosporine. These data indicate that the inhibitory action on capacitative Ca2+ entry of glucosamine is distinct from that caused by ATP depletion.     

Enhanced flux of substrates into polyamine biosynthesis but not ethylene in tomato fruit engineered with yeast S-adenosylmethionine decarboxylase gene

S-adenosylmethionine (SAM), a major substrate in 1-C metabolism is a common precursor in the biosynthetic pathways of polyamines and ethylene, two important plant growth regulators, which exhibit opposing developmental effects, especially during fruit ripening. However, the flux of various substrates including SAM into the two competing pathways in plants has not yet been characterized. We used radiolabeled ¹⁴C-Arg, ¹⁴C-Orn, L-[U-¹⁴C]Met, ¹⁴C-SAM and ¹⁴C-Put to quantify flux through these pathways in tomato fruit and evaluate the effects of perturbing these pathways via transgenic expression of a yeast SAM decarboxylase (ySAMDC) gene using the fruit ripening-specific promoter E8. We show that polyamines in tomato fruit are synthesized both from Arg and Orn; however, the relative contribution of Orn pathway declines in the later stages of ripening. Expression of ySAMDC reversed the ripening associated decline in spermidine (Spd) and spermine (Spm) levels observed in the azygous control fruit. About 2- to 3-fold higher levels of labeled-Spd in transgenic fruit (556HO and 579HO lines) expressing ySAMDC confirmed the enzymatic function of the introduced gene. The incorporation of L-[U-¹⁴C]Met into Spd, Spm, ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC) was used to determine Met-flux into these metabolites. The incorporation of ¹⁴C-Met into Spd/Spm declined during ripening of the control azygous fruit but this was reversed in fruits expressing ySAMDC. However, incorporation of ¹⁴C-Met into ethylene or ACC during ripening was not altered by the expression of ySAMDC in the fruit. Taken together these results show that: (1) There is an inverse relationship between the production of higher polyamines and ethylene during fruit ripening, (2) the inverse relationship between higher polyamines and ethylene is modulated by ySAMDC expression in that the decline in Spd/Spm during fruit ripening can be reversed without significantly altering ethylene biosynthesis, and (3) cellular flux of SAM in plants is homeostatically regulated based on its demand for competing pathways.     

Adventitious rooting is enhanced by methyl jasmonate in tobacco thin cell layers

Adventitious roots (ARs) are induced by auxins. Jasmonic acid (JA) and methyl jasmonate (MeJA) are also plant growth regulators with many effects on development, but their role on ARs needs investigation. To this aim, we analyzed AR formation in tobacco thin cell layers (TCLs) cultured with 0.01–10 μM MeJA, either under root-inductive conditions, i.e., on medium containing 10 μM indole-3-butyric acid (IBA) and 0.1 μM kinetin, or without hormones. The explants were excised from the cultivars Samsun, Xanthii and Petite Havana, and from genotypes with altered AR-forming ability in response to auxin, namely the non-rooting rac mutant and the over-rooting Agrobacterium rhizogenes rolB transgenic line. Results show that NtRNR1 (G1/S) and Ntcyc29 (G2/M) gene activity, cell proliferation and meristemoid formation were stimulated in hormone-cultured TCLs by submicromolar MeJA concentrations. The meristemoids developed either into ARs and xylogenic nodules, or into xylogenic nodules only (rac TCLs). MeJA-induced meristemoid over-production characterized rolB TCLs. No rooting or xylogenesis occurred under hormone-free conditions, independently of MeJA and genotype. Endogenous JA progressively (days 1–4) increased in hormone-cultured TCLs in the absence of MeJA. JA levels were enhanced by 0.1 μM MeJA, on both days 1 and 4. Endogenous IBA was the only auxin detected, both in the free form and as IBA-glucose. Free IBA increased up to day 2, remaining constant thereafter (day 4). Its level was enhanced by 0.1 μM MeJA only on day 1, while IBA conjugation was not affected by MeJA. Taken together, these results show that an interplay between jasmonates and auxins regulates AR formation and xylogenesis in tobacco TCLs.     

Polyamine-mediated inhibition of in-vitro cell proliferation is not due to acrolein

The influence of acrolein or spermine on the viability and growth of phytohaemagglutinin-stimulated rat thymic lymphocytes in cultures supplemented with foetal calf serum have been investigated. Acrolein (greater than 20 microM) was cytotoxic; spermine had little effect on viability, but inhibited [3H]TdR incorporation at low concentrations (approximately 10 microM). Cells treated with greater than 8 microM acrolein 3 hr before stimulation exhibited irreversible inhibition of protein synthesis, whereas 50 microM spermine had no effect, even when cells were treated for 24 hr before stimulation. However, addition of 25 microM spermine after stimulation did inhibit both [3H]-uridine incorporation and protein synthesis: this was reversible if cells were freed of polyamine within 4 hr, but not if washed after 24 hr. These results show that, contrary to several previous reports, in-vitro inhibition of cell proliferation by spermine is not due to the formation and action of acrolein.     

The role of polyamine depletion and accumulation of decarboxylated S-adenosylmethionine in the inhibition of growth of SV-3T3 cells treated with alpha-difluoromethylornithine.

The effects of alpha-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, on cell growth rate, polyamine content and the content of decarboxylated S-adenosylmethionine in SV-3T3 transformed mouse fibroblasts were studied. DL-alpha-Difluoromethylornithine at 1 mM or higher concentrations decreased the growth rate by over 90% after 2 or more days of exposure, but the cells remained viable, although quiescent for at least 9 days. Addition of 10 microM-spermidine or -spermine or 50 microM-putrescine at any time throughout this period completely reversed the inhibition of growth. Treatment with alpha-difluoromethylornithine decreased putrescine and spermidine contents by more than 98% and that of spermine by 60%, but cells exposed to exogenous polyamines did not require complete replenishment of the polyamine pools to resume growth. In fact, a virtually normal growth rate was obtained in cells lacking putrescine, having 2% of normal spermidine content and 156% of normal spermine. These results suggest that the well-known increase in putrescine and spermidine in cells stimulated for growth is not essential for this to occur and that mammalian cells can utilize spermine as their only polyamine. A substantial reversal of the growth-inhibitory effect of alpha-difluoromethylornithine was produced by a number of polyamines not normally found in mammalian cells, including the spermidine analogues aminopropylcadaverine and sym-homospermidine, which were partially converted into their respective spermine analogues by addition of an aminopropyl group within the cell. The spermine analogue sym-norspermine was also effective, but the maximal growth rate produced by these unphysiological polyamines was only 60-70% of that produced by the normal polyamines. These results indicate that spermidine and spermine have the optimal length for activation of the cellular processes critically dependent on polyamines and should help in identifying these processes. Exposure to alpha-difluoromethylornithine leads to an enormous rise in the concentration of decarboxylated S-adenosylmethionine, which reached a peak at 530-fold after 3 days of exposure and steadily declined to 140-fold after 11 days. This increase was abolished by addition of exogenous polyamines, which rapidly decreased the activity of S-adenosylmethionine decarboxylase. The increase in decarboxylated S-adenosylmethionine is unlikely to be solely responsible for the decrease to the same extent by spermine, sym-norspermidine and sym-homospermidine, which produce 97%, 16% and 60% of the control growth rate, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of pyridoxal phosphate in mammalian polyamine biosynthesis. Lack of requirement for mammalian S-adenosylmethionine decarboxylase activity.

1. Polyamine concentrations were decreased in rats fed on a diet deficient in vitamin B-6. 2. Ornithine decarboxylase activity was decreased by vitamin B-6 deficiency when assayed in tissue extracts without addition of pyridoxal phosphate, but was greater than in control extracts when pyridoxal phosphate was present in saturating amounts. 3. In contrast, the activity of S-adenosylmethionine decarboxylase was not enhanced by pyridoxal phosphate addition even when dialysed extracts were prepared from tissues of young rats suckled by mothers fed on the vitamin B-6-deficient diet. 4. S-Adenosylmethionine decarboxylase activities were increased by administration of methylglyoxal bis(guanylhydrazone) (1,1'-[(methylethanediylidine)dinitrilo]diguanidine) to similar extents in both control and vitamin B-6-deficient animals. 5. The spectrum of highly purified liver S-adenosylmethionine decarboxylase did not indicate the presence of pyridoxal phosphate. After inactivation of the enzyme by reaction with NaB3H4, radioactivity was incorporated into the enzyme, but was not present as a reduced derivative of pyridoxal phosphate. 6. It is concluded that the decreased concentrations of polyamines in rats fed on a diet containing vitamin B-6 may be due to decreased activity or ornithine decarboxylase or may be caused by an unknown mechanism responding to growth retardation produced by the vitamin deficiency. In either case, measurements of S-adenosylmethionine decarboxylase and ornithine decarboxylase activity under optimum conditions in vitro do not correlate with the polyamine concentrations in vivo.     

Effect of aldehydes on polyamine metabolism. II. Methylglyoxal-induced changes in S-adenosylmethionine decarboxylase (SAMD) activity and RNA synthesis and degradation in isolated hepatocytes

In isolated rat liver cells methylglyoxal (MeG) inhibits S-adenosylmethionine decarboxylase activity and RNA and protein synthesis; MeG also stimulated RNA degradation. The changes induced by MeG in RNA metabolism are partially prevented (in the case of RNA synthesis inhibition) or totally abolished (in the case of RNA degradation stimulation) by exogenous spermidine addition. This suggests the effects of MeG on RNA metabolism are dependent, at least in part, on SAMD inhibition.     

The functional intronless S-adenosylmethionine decarboxylase gene of the mouse (Amd-2) is linked to the ornithine decarboxylase gene (Odc) on Chromosome 12 and is present in distantly related species of the genus Mus

S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the biosynthesis of polyamines. We have previously identified a mouse AdoMetDC gene that exhibits the hallmarks of a retroposon; that is, it has no introns, is flanked by direct repeats, and has a poly(dA) tract at its 3′-end. This gene, termed Amd-2, is not a processed pseudogene; rather, it is transcribed in a variety of mouse tissues and encodes a functional enzyme. In the current report, we present the sequence of a 6.7-kb genomic segment of the Amd-2 locus. Several sequences of interest, including an intercisternal A particle (IAP) element, a transposon-related sequence, and several expressed sequence tags (ESTs), were found within or near Amd-2. We also show, through analysis of an interspecific backcross, that Amd-2 is located on Chr 12, tightly linked to the gene (Odc) that encodes ornithine decarboxylase, another key enzyme in polyamine synthesis. Finally, we show that Amd-2 is present among several divergent species of the genus Mus. Thus, the integration event that generated Amd-2 may have occurred early during Mus evolution. Received: 1 December 1998 / Accepted: 31 March 1999     

Effects of Ri TL-DNA from Agrobacterium rhizogenes and the inhibitors of polyamine synthesis on growth, floral development, sexual organogenesis and polyamine metabolism in tobacco

Our recent work associated the phenotypic alterations caused by the Ri TL-DNA (root-inducing, left-hand, transferred DNA) from Agrobacterium rhizogenes with a general decrease in the accumulation of polyamines and their derivatives and showed that irreversible and specific inhibition of putrescine biosynthesis causes a phenotype similar to that attributed to the Ri TL-DNA. In this review we report recent data supporting a correlation between the degree of expression of the transformed phenotype due to the root-inducing, left-hand, transferred DNA and inhibition of polyamine accumulation, strongly suggesting that genes carried by the root-inducing, transferred DNA may act through interference with polyamine production via the ornithine pathway.     

Suppression of S-adenosylmethionine decarboxylase activity is a major cause for high-temperature inhibition of pollen germination and tube growth in tomato (Lycopersicon esculentum Mill.)

Possible involvement of impaired polyamine biosynthesis in the poor performance of tomato pollen (Lycopersicon esculentum Mill.) at high temperatures was investigated. Incubation of pollen at 38 degrees C suppressed the increase of S-adenosylmethionine decarboxylase (SAMDC) activity in germinating pollen with little influence on arginine decarboxylase activity. Consequently, spermidine and spermine content in the pollen did not increase at 38 degrees C, while putrescine content increased at both 25 degrees C and 38 degrees C. High-temperature inhibition of pollen germination was alleviated by the addition of spermidine or spermine but not of putrescine to the germination medium. Cycloheximide inhibited SAMDC activity in parallel with pollen germination at 25 degrees C, whereas actinomycin D had no effect on either of them, indicating that enhanced SAMDC activity is associated with de novo protein synthesis. Incubation of crude enzyme extracts at 40 degrees C for 1 h did not affect SAMDC. In addition, high temperatures did not enhance protease activity in germinating pollen. These results indicate that low activity of SAMDC, probably due to impaired protein synthesis or functional enzyme formation, is a major cause for the poor performance of tomato pollen at high temperatures.     

Intracellular depletion of insulin by oleate is due to an inhibited synthesis and not to an increased secretion

In the polyclonal rat pancreatic beta-cell line INS-1, immunoreactive insulin (IRI, insulin and its precursors) and C-peptide (surrogate marker for mature insulin) were quantified after a 1-h incubation at 16.7 mM glucose with or without oleate. Oleate caused a 20% decrease (P 相似文献   

Oxalacetate decarboxylase activity in muscle is due to pyruvate kinase.

The enzyme from cod fish muscle that catalyzes the irreversible decarboxylation of oxalacetate and is homogeneous by several criteria contains very significant pyruvate kinase activity. For every unit of decarboxylase activity (0.90 unit/mg) there are 235 units of pyruvate kinase activity (212 units/mg). The inability to separate the two activities by a variety of physical techniques indicates that both are due to a single enzyme protein. Improtantly, the two activities appear to take place at the same or overlapping sites on the enzyme. Phosphoenolpyruvate and 4-ethyloxalacetate are strong linear competitive inhibitors of the decarboxylase activity with respect to oxalacetate having dissociation constants of 3.2 and 10.2 muM, respectively, while 4-ethyloxalacetate is a linear competitive inhibitor of the pyruvate kinase activity with respect to phosphoenolpyruvate, Ki - 13.5 muM. In addition, both activities exhibit sigmoidal kinetics for substrates. The differential influence of effectors on substrate cooperativity for the two reactions indicates that the decarboxylase reaction may be an important tool for studying allosteric mechanisms in this enzyme.     

Enhanced susceptibility of photosynthesis to low-temperature photoinhibition due to interruption of chill-induced increase of S-adenosylmethionine decarboxylase activity in leaves of spinach (Spinacia oleracea L.)

The possible involvement of polyamines in the chilling tolerance of spinach (Spinacia oleracea L.) was investigated focusing on photosynthesis. During chilling at 8/5C (day/night) for 6 d, S-adenosylmethionine decarboxylase (SAMDC) activity increased significantly in leaves in parallel with the increase in putrescine and spermidine (Spd) content in leaves and chloroplasts. Treatment of leaves with methylglyoxal-bis(guanylhydrazone) (MGBG), an SAMDC inhibitor, resulted in the deterioration of plant growth and photosynthesis under chilling conditions, which was reversed by the concomitant treatment with Spd through the roots. Plants treated with MGBG showed lower photochemical efficiency of PSII than either the control or plants treated with MGBG plus Spd during chilling and even after transfer to warm conditions, suggesting an increase of photoinhibition due to low Spd in chloroplasts. Indeed, MGBG-treated plants had much lower activities of thylakoid electron transport and enzymes in carbon metabolism as well as higher degrees of lipid peroxidation of thylakoid membranes compared to the control. These results indicate that the enhanced activity of SAMDC with a consequential rise of Spd in chloroplasts is crucial for the cold acclimation of the photosynthetic apparatus in spinach leaves.