Methyl jasmonate differentially affects tocopherol content and tyrosine amino transferase activity in cultured cells of Amaranthus caudatus and Chenopodium quinoa.

Tocopherols are lipid-soluble compounds synthesised exclusively by photosynthetic organisms. In this study, in vitro callus cultures were established from two plants that are naturally rich in tocopherols, Amaranthus caudatus and Chenopodium quinoa, in order to examine whether callus cultures were able to produce these compounds at levels comparable to those observed in planta. In both species, cotyledon explants produced the best callus induction and, once established, callus cultures were grown under two different hormonal treatments to check for effects of growth and to induce chloroplast differentiation in the cells. A rapid differentiation of chloroplasts occurred only in C. quinoa cell aggregates grown in the presence of benzyladenine, leading to the production of a homogeneous green callus. In both species, only alpha-tocopherol was produced by callus cultures, although levels were much lower than in planta, and the production was not influenced by the hormonal conditions. Interestingly, cell cultures of the two species responded in different ways to methyl jasmonate (MJ). In A. caudatus cultures, treatment with 100 mum MJ increased the production of alpha-tocopherol up to fivefold, and the inductive effect was influenced by the hormonal composition of the medium. This increase in alpha-tocopherol was associated with a proportional increase in tyrosine aminotransferase (TAT) activity, one of the key enzymes involved in tocopherol biosynthesis. By contrast, in C. quinoa cultures, elicitation with MJ did not have any effect, neither on tocopherol production, nor on TAT activity. These results are discussed in relation to chloroplast differentiation and the interplay between jasmonates and phytohormones.

Bis(guanylhydrazones) negatively affect in vitro germination of kiwifruit pollen and alter the endogenous polyamine pool.

Bis(guanylhydrazones) are a class of compounds known to interfere with the metabolism of polyamines (PAs). Among them, the methylglyoxal derivative (MGBG) has been studied most thoroughly. Because PAs and their biosynthetic enzymes are strongly involved in pollen tube organization, emergence and elongation, a number of these inhibitors have been studied in the present work for their effects on the in vitro performance of kiwifruit (Actinidia deliciosa) pollen. Increasing concentrations of several bis(guanylhydrazones) in the range 0.05-1 mM were checked for their effect on pollen germination. Most of the compounds tested showed a dose-dependent inhibitory effect on tube emergence, which was established very early during incubation. At 0.5 mM, the methylpropylglyoxal derivative (MPGBG) had a stronger inhibitory effect than MGBG. To verify whether the inhibitors reached their metabolic target, PA levels and S-adenosylmethionine decarboxylase (SAMDC) activity were determined in pollen germinated in the presence or absence (controls) of 0.5 mM bis(guanylhydrazones). Spermidine (Spd) content was significantly reduced in the treated pollen, and this effect was more pronounced after treatment with MGBG than with MPGBG. An early and strong reduction in SAMDC activity was observed after exposure to either inhibitor. Inhibition of pollen germination by MGBG or MPGBG could not be reversed by the addition of exogenous Spd, which per se was inhibitory. Taken together, our results suggest that bis(guanylhydrazones) alter PA metabolism and negatively affect kiwifruit pollen germination, even though a strict cause-effect relationship could not be established, and other mechanisms, unrelated to PA activity, must be involved.

Genetic and phytochemical difference between some Indian and Italian plants of Withania somnifera (L.) Dunal.

The geographical distribution of Withania somnifera (L.) Dunal is quite wide. However, in Italy, this species is very rare and grows spontaneously only in Sicily and in Sardinia. The PCR-RAPD technique has been utilized in this work to determine the genetic relationship among Sicilian, Sardinian and Indian samples and the HPLC analysis of whitaferin A was used as a marker to evaluate the phytochemical differences. The genetic difference between Indian and Sicilian plants of W. somnifera turned out to be smaller than that between Indian and Sardinian plants of this species. The phytochemical analysis as well showed that the Sardinian specimen strongly differed from the Indian and Sicilian ones in its contents of withaferin A. Our results seem to confirm the hypothesis that the Italian populations of this species may not be indigenous but naturalised. Due to the high withaferin A content of the Sardinian samples, these plants could be used as a source for pharmaceutical purposes.

Vitamin C content and antioxidant activity of the fruit and of the Ayurvedic preparation of Emblica officinalis Gaertn.

Emblica officinalis Gaertn. is one of the most important plants of Ayurved, the traditional Indian medicine. In this ancient medicine, the fruit of Emblica officinalis is processed according to a method named "Svaras Bhavana", whereby the therapeutic potential of the plant is enhanced by treating the main herb with its own juice. For many years, the activity of the fruits was attributed to the high content of ascorbic acid; however, this has recently been questioned. The aim of the paper is to clarify this matter. A reliable and feasible HPLC method with diode array detection has been developed for the determination of ascorbic acid in Emblica fruit and particularly in Emblica fruit processed according to the Ayurvedic method. The antioxidant effects have also been evaluated in comparison to the real levels of Vitamin C by different antioxidant tests. The data obtained show that the Emblica fruit contains ascorbic acid (0.4%, w/w), and that the Ayurvedic method of processing increases the healthy characteristics of the fruit thanks to a higher antioxidant activity and a higher content of ascorbic acid (1.28%, w/w). It has also been found that Vitamin C accounts for approximately 45-70% of the antioxidant activity.

Effect of Bis(guanylhydrazones) on Growth and Polyamine Uptake in Plant Cells.

In the present work the effect of several bis(guanylhydrazones) on the growth of Helianthus tuberosus tuber explants was studied. Different aliphatic congeners of glyoxal bis(guanylhydrazone) were tested. Most of the compounds displayed an inhibitory effect on growth, and a correlation between the structure of the molecule and the inhibitory activity was observed. Experiments carried out with glyoxal bis(guanylhydrazone) and its congeners methyl-, ethylmethyl-, and methylpropylglyoxal bis(guanylhydrazones) show that as the total number of side chain carbon atoms in the molecule increases, the inhibitory potency also increases. A depletion of spermidine levels was also found in the explants treated with ethylmethylglyoxal bis(guanylhydrazone), which turned out to be one of the most potent growth inhibitors. The addition of spermidine caused a significant reversion of the antiproliferative action of glyoxal bis(guanylhydrazone). The effect of these compounds on spermidine uptake in protoplasts isolated from carrot phloem parenchyma was also investigated. Only a slight competition was found when antagonists were present at concentrations 20 times higher than the polyamine, thus suggesting that bis(guanylhydrazones) do not share, at least at low concentrations, the polyamine transport system in plant cells.Key Words. Bis(guanylhydrazones)-Carrot protoplasts-Growth-Helianthus tuberosus-Polyamines-Uptakehttp://link.springer-ny.com/link/service/journals/00344/bibs/18n1p39.html

Transcriptional inhibition of the operon for the spermidine uptake system by the substrate-binding protein PotD.

Inhibition of spermidine uptake in Escherichia coli, which occurs in the presence of accumulated polyamines, has been studied using the spermidine uptake operon consisting of the potA, -B, -C, and -D genes. Transcription of the potABCD operon was inhibited by PotD, a spermidine-binding protein usually found in the periplasm, and the inhibitory effect of PotD was increased by spermidine. Transcription was not affected by bovine serum albumin, PotA, or PotF, suggesting that the effects of PotD are specific to the PotD protein. In the presence of 8 mM spermidine, a 50% inhibition of transcription was observed with a molar ratio of approximately 1:500 of template DNA:PotD. It was found that PotD bound to regions -258 to -209 nucleotides upstream and +66 to +135 nucleotides downstream of the ATG initiation codon of the potA gene. Binding of PotD to the downstream site was stimulated by spermidine. Overexpression of PotD in Escherichia coli DH5alpha inhibited the uptake of spermidine, the synthesis of PotABCD mRNA, and expression of a lacZ reporter gene fused downstream of a potA gene containing the PotD binding sites. In cells overexpressing PotD, a large amount of PotD existed as PotD precursor in spheroplasts. Our results indicate that PotD precursor can also inhibit spermidine transport. The amino acid residues in PotD that are involved in its interaction with the potABCD operon were determined using mutated PotD proteins. Thr-35 and Ser-85 of PotD were found to be important for this interaction. These results suggest that transcription of the spermidine transport (potABCD) operon is inhibited in vivo by PotD precursor rather than PotD through its binding to two regions close to the transcriptional initiation site of the operon.

Characterization of spermidine binding to solubilized plasma membrane proteins from zucchini hypocotyls

In this work [14C]spermidine binding to total proteins solubilized from plasma membrane purified from zucchini (Cucurbita pepo L.) hypocotyls was investigated. Proteins were solubilized using octyl glucoside as a detergent. Specific polyamine binding was thermolabile, reversible, pH dependent with an optimum at pH 8.0, and had a Kd value of 5 &mgr;M, as determined by glass-fiber-filter assays. Sephadex G-25 M gel-filtration assays confirmed the presence of a spermidine-protein(s) complex with a specific binding activity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native polyacrylamide gel electrophoresis of collected fractions having the highest specific spermidine-binding activity, several protein bands (113, 75, 66, and 44 kD) were identified. The specificity of spermidine binding was examined by gel-filtration competition experiments performed using other polyamines and compounds structurally related to spermidine. Partial purification on Sephadex G-200 led to the identification of 66- and 44-kD protein bands, which may represent the putative spermidine-binding protein(s) on the plasmalemma.

Polyamine Binding to Plasma Membrane Vesicles Isolated from Zucchini Hypocotyls.

The general features of [14C]spermidine binding to plasmalemma vesicles isolated from zucchini (Cucurbita pepo L.) etiolated hypocotyls are reported in the present paper. The specific interaction of the polyamine with the plasma membranes was reversible and thermolabile, since it decreased by about 50% in the assay performed at 40[deg]C compared to that carried out on ice. On the contrary, nonspecific binding was unaffected by temperature. Specific spermidine binding showed a pH dependence with a maximum at pH 8.0 and it reached saturation between 0.75 and 1 mM external spermidine concentration. The value of the dissociation constant calculated from Scatchard analysis was 4.4 x 10-5 M. Specific spermidine interaction appeared to be sensitive to detergents and was markedly reduced by the presence of divalent cations, such as Mg2+ and Ca2+, whereas it was stimulated by monovalent cations. Polyamine binding sites were highly sensitive to pronase treatment. Competition experiments, performed using a series of compounds structurally related to spermidine, may provide some indication of the characteristics of spermidine binding sites. The results presented here suggest that specific spermidine binding occurs mainly with the protein component of the plasma membrane.

Kinetics and calcium-specificity of polyamine uptake in carrot protoplasts.

The kinetics of putrescine and spermidine uptake and the influence of calcium on the kinetic parameters of the transport process were investigated in protoplasts isolated from carrot phloem parenchyma. Spermidine uptake dependence on external concentration was biphasic, both in the absence and in the presence of 1 mM CaCl2. In the first case, saturation was reached at 0.1 to 0.25 mM and the Km value was 43µM. When calcium was added, the Km and Vmax increased. A similar pattern was found with regard to putrescine uptake. Moreover, in order to clarify the mode of action of calcium on polyamine uptake, lanthanides (lanthanum and gadolinium) were utilised as Ca(+2)-channel antagonists. When protoplasts were preincubated with these lanthanides, the stimulatory effect exerted by Ca(+2) on polyamine uptake was almost totally abolished. On the other hand, if lanthanum was supplied instead of calcium, it gave rise to a small enhancement of polyamine transport. These results induce us to suggest that calcium acts on polyamine uptake both by binding to external sites on the plasmalemma and by penetrating into the cell.

Effects of Polyamines on the Oxidation of Exogenous NADH by Jerusalem Artichoke (Helianthus tuberosus) Mitochondria.

The effect of polyamines (putrescine, spermine, and spermidine) on the oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L. cv. OB1) mitochondria, have been studied. Addition of spermine and/or spermidine to a suspension of mitochondria in a low-cation medium (2 millimolar-K(+)) caused a decrease in the apparent K(m) and an increase in the apparent V(max) for the oxidation of exogenous NADH. These polycations released by screening effect the mitochondrially induced quenching of 9-aminoacridine fluorescence, their efficiency being dependent on the valency of the cation (C(4+) > C(3+)). Conversely, putrescine only slightly affected both kinetic parameters of exogenous NADH oxidation and the number of fixed charges on the membranes. Spermine and spermidine, but not putrescine, decreased the apparent K(m) for Ca(2+) from about 1 to about 0.2 micromolar, required to activate external NADH oxidation in a high-cation medium, containing physiological concentrations of Pi, Mg(2+) and K(+). The results are interpreted as evidence for a role of spermine and spermidine in the modulation of exogenous NADH oxidation by plant mitochondria in vivo.

Spermidine Uptake by Mitochondria of Helianthus tuberosus.

In the present work evidence is provided that spermidine, a polyamine largely present in plant tissues, may be transported, at physiological concentrations, into the matrix space of mitochondria isolated from tubers of Helianthus tuberosus L. cv OB1 (Jerusalem artichoke). It is concluded that the movement of spermidine strictly depends on membrane potential, since it is drastically blocked by valinomycin and only slightly sensitive to nigericin. Mg(2+) and K(+) inhibit the transport of spermidine in line with the general concept that these cations compete for the same binding sites on the mitochondrial membrane. In contrast to previous data on mammalian mitochondria, spermidine uptake by plant mitochondria does not depend on the presence of inorganic phosphate. This latter result, along with evidence that Ca(2+) does not affect accumulation of spermidine, indicates that the control of the polyamine uptake mechanism in plant mitochondria is distinct from that of mammalian systems.