Monitoring of anatabine release by methyl jasmonate elicited BY-2 cells using surface-enhanced Raman scattering.

A new application of surface-enhanced Raman scattering (SERS) in the field of plant material analysis is proposed in this study. The aim was to monitor the release of anatabine by methyl jasmonate (MeJa) elicited Bright Yellow-2 (BY-2) cells. Gold nanoparticles (AuNps) were used as SERS substrate. The first step was to study the SERS activity of anatabine in a complex matrix comprising the culture medium and BY-2 cells. The second step was the calibration. This one was successfully performed directly in the culture medium in order to take into account the matrix effect, by spiking the medium with different concentrations of anatabine, leading to solutions ranging from 250 to 5000µgL(-1). A univariate analysis was performed, the intensity of a band situated at 1028cm(-1), related to anatabine, was plotted against the anatabine concentration. A linear relationship was observed with a R(2) of 0.9951. During the monitoring study, after the MeJa elicitation, samples were collected from the culture medium containing BY-2 cells at 0, 24h, 48h, 72h and 96h and were analysed using SERS. Finally, the amount of anatabine released in the culture medium was determined using the response function, reaching a plateau after 72h of 82µg of anatabine released/g of fresh weight (FW) MeJa elicited BY-2 cells.

Changes in polyamines, auxins and peroxidase activity during in vitro rooting of Fraxinus angustifolia shoots: an auxin-independent rooting model.

Among shoots of Fraxinus angustifolia Vahl raised in vitro, 76% rooted after culture on root induction medium for 5 days in darkness followed by culture on root expression medium for 15 days in light. The addition of 20.7 microM indole-butyric acid (IBA) to the root induction medium did not significantly increase the rooting percentage (88%). Putrescine, spermidine, cyclohexylamine (CHA) and aminoguanidine (AG) enhanced rooting up to 100% (98.66% for AG), when applied during root induction in the absence of IBA, otherwise these compounds inhibited rooting, as did spermine and difluoromethylornithine (DFMO) + difluoromethylarginine (DFMA). The root induction phase was characterized by a temporary increase in endogenous free indole-acetic acid (IAA) and putrescine concentrations during root induction, whereas the root expression phase was characterized by increased peroxidase activity and low concentrations of polyamines. These changes were specifically associated with the rooting process and did not depend on the presence of exogenous IBA, because application of exogenous IBA enhanced the amount of IAA in the cuttings but did not affect rooting or the pattern of changes in polyamines and peroxidase. The effects of CHA, AG and DFMO + DFMA on endogenous concentrations of auxins and polyamines highlight the close relationship between the effects of IAA and putrescine in root induction and suggest that polyamine catabolism has an important role in root formation and elongation.

Are hyperhydric shoots of Prunus avium L. energy deficient?

The content of oxidized and reduced pyridine nucleotides and some enzymatic activities of the oxidative pentose phosphate and glycolytic pathways were compared in normal (NS, growing on agar) and hyperhydric (HS, growing on gelrite) shoots of Prunus avium L. after 4 weeks of in vitro culture. The chlorophyll fluorescence from leaves and the redox capacity of the plasma membrane (reduction of exogenously added ferricyanide) of both types of shoots were recorded. The pool of oxidized and reduced pyridine nucleotides was lower in HS than in NS. These results suggested a reduced metabolism of HS in comparison to normal ones. This hypothesis was also supported by other observations. First, chlorophyll fluorescence measurements showed a lower chlorophyll content and a slight reduction of the photosynthetic capacity in HS. Second, the low activity of some enzymes of oxidative pentose phosphate pathway (OPP) and glycolysis indicated a decline of these biochemical pathways in HS with the consequence of a reduced production of chemical energy in the form of NAD(P)H and ATP. Finally, the lower reduction of ferricyanide by HS suggested a lower rate of redox reactions at the level of the plasma membrane of these shoots in comparison to NS.

Involvement of indole-3-acetic acid in the circadian growth of the first internode of Arabidopsis.

The extension rate of the first inflorescence node of Arabidopsis was measured during light/dark or continuous light exposure and was found to exhibit oscillations which showed a circadian rhythmicity. Decapitation induced a strong inhibition of stem extension. Subsequent application of IAA restored growth and the associated extension-rate oscillations. In addition, IAA treatments, after decapitation, re-established the circadian rhythmicity visible in the intact plants during free run. This indicates that the upper zone of the inflorescence has a major influence on the extension rate of floral stems and implies a role for auxin. Application of N-(1-naphthyl)phthalamic acid, an IAA transport inhibitor, to an intact floral stem inhibited growth and the rhythmicity in the extension rate oscillations, indicating that IAA polar transport may play a role in the dynamics of stem elongation. Furthermore, IAA-aspartate application, after decapitation, did not restore growth and rhythmicity. Nevertheless, biochemical analysis of IAA and IAA-aspartate demonstrated circadian fluctuations of the endogenous levels of both compounds. These observations suggest that IAA metabolism is an essential factor in the regulation of the circadian growth rhythm of Arabidopsis floral stems.

Shemin pathway and peroxidase deficiency in a fully habituated and fully heterotrophic non-organogenic sugarbeet callus: an adaptative strategy or the consequence of modified hormonal balances and sensitivities in these cancerous cells? A review and reassessment.

There are many arguments for considering a specific fully habituated (auxin and cytokinin-independent) and fully heterotrophic non-organogenic (HNO) sugarbeet callus cell line as terminating a neoplastic progression, and thus to be made of cancerous cells. The similarities with animal tumour and cancer cells are recalled. All types of habituated tissues examined in the literature share at least three common biochemical characteristics: low apparent peroxidase activity, high content of polyamines (PAs) and low production of ethylene. However, results concerning their auxin and cytokinin levels are not consistent. Peroxidase synthesis in the achlorophyllous HNO callus appears to arise from aminolevulinic acid (ALA) synthesis through the Shemin pathway, commonly used by animals and fungi. This pathway is limited by disturbed nitrogen metabolism that diverts glutamate (directly used for ALA synthesis in green higher plants) from the Kreb's cycle into PA synthesis. There is no argument to suggest that the low ethylene production is caused by a competition with PAs for their common precursor, S-adenosylmethionine. The results we report here indicate modified anabolic and catabolic pathways of auxins and cytokinins but also the possibilities of unusual compounds playing similar roles (dehydrodiconiferyl alcohol glucosides, for instance). A higher turnover of PAs is shown in the HNO callus, which could suggest a role for H2O2 and gamma-aminobutyric acid, products or intermediates in the PA catabolic pathway, as secondary messengers. The habituated cells retain some sensitivity towards exogenous auxins and cytokinins. Their increased sensitivity to PAs and ethylene suggests modified hormonal balances for the control of these actively dividing cells.

Changes in the concentrations of auxins and polyamines during rooting of in-vitro-propagated walnut shoots.

Rooting was induced in in-vitro-propagated walnut (Juglans regia L.) shoots by subculturing the shoots on rooting medium containing agar and 3 mg l(-1) indole-3-butyric acid (IBA) for 7 days in darkness. Changes in the concentrations of endogenous free indole-3-acetic acid (IAA), indole-3-acetylaspartic acid (IAAsp) and free polyamines were determined during culture on root-inducing medium. In extracts of whole shoots, the concentration of free IAA showed a transient peak at 60 h (around 48 h in extracts from basal shoot portions) and then remained at a relatively low concentration for the remainder of the 7-day culture period. The concentration of IAAsp in extracts of whole shoots peaked at about the same time as the concentration of free IAA, whereas the IAAsp concentration in extracts from basal shoot portions peaked earlier, at around 12 h. The concentrations of free polyamines in extracts of whole shoots increased soon after the shoots were transferred to root-inducing medium. The concentrations of IAA and IAAsp remained stable when the rooted shoots were transferred to a vermiculite/gelrite mixture (without auxin) and grown in light.

Masking in plants.

Circadian rhythms in plants are liable to masking, i.e. alterations by environmental influencing agents. Experiments have been reported for both positive and negative masking, attributed to a Zeitgeber which may either increase or decrease the amplitude of a circadian rhythm (CR). In some instances, the CR may even be unexpressed. This inhibition, however, may be alleviated by synchronizing agents. Reports are also available for changes in the shape or pattern of an oscillation. The latter may be prevented, at least in Acetabularia in certain conditions, by a phytohormone antagonist. Masking may also be brought about by water stress, relative humidity, bacterial infection and alteration in the relative direction of the gravitational force. Finally, subjecting plants to constant conditions, particularly continuous light, alters the physiological state of the organism.

Auxin-polyamine interaction with the calcium-controlled secretion of peroxidase by sugarbeet cells.

Spermidine and ornithine given to normal auxin-requiring cell suspensions of sugarbeet inhibited peroxidase secretion in the absence of Ca(2+). Habituated (organogenic or not) cells did not respond. Both compounds counteracted the Ca(2+) - promoted enzyme secretion by three cell lines. Auxins (2,4-D and BSAA) did not modify the extracellular level of peroxidase activity in the absence of Ca(2+) When Ca(2+) was added, auxins increased its effect in normal cells and had practically no effect in habituated cells. The inhibitory effect of spermidine and ornithine was somewhat reduced by auxins in normal cells and increased in habituated cells. It was hypothesized that the effect of auxins did not involve the mediation of polyamines and that both types of compounds directly interacted with Ca(2+) at the membrane level.