Josef Eder

Melatonin : Occurrence and daily rhythm in Chenopodium rubrum

Abstract The occurrence of melatonin (5-methoxy-N-acetyltryptamine), a common animal hormone, in extracts of the above-ground parts of 15-day-old plants of Chenopodium rubrum was confirmed by liquid chromatography/tandem mass spectrometry. Using both this method and radioimmunoassay, changes in melatonin content during a 12 hr light/12 hr dark cycle were demonstrated. The melatonin concentration remained low or undetectable during the light period and increased during the darkness reaching a maximum at hours 4–6 of the dark period before rapidly decreasing. Both the nocturnal increase and the range of concentration are similar to those known in animals.

Abscisic acid, polyamines and phenolic acids in sessile oak somatic embryos in relation to their conversion potential

Two types of sessile oak (Quercus petraea (Matt) Liebl.) somatic embryos with well-developed ivory or green-coloured cotyledons and root apices differing in their development, i.e. in the conversion into plantlets, were examined for the endogenous contents of abscisic acid (ABA), polyamines (PAs), aromatic monoamines (AMs), phenolic acids (PhAs), lignin and peroxidase activity. The conversion did not occur in embryos with deep green cotyledons (NCE, nonconverting embryos) while almost 56 % of embryos with ivory or light green cotyledons converted into plantlets (CE, converting embryos). High conversion capacity is associated with (a) lower content of ABA, (b) lower levels of free putrescine and its soluble conjugates, and higher content of spermidine soluble conjugates, (c) markedly increased level of phenylethylamine and its soluble conjugates, and (d) significantly lower content of total PhAs represented by the sum of free, soluble ester- and glycoside-, and insoluble cell wall-bound PhAs as compared to the contents in NCE. Higher levels of all determined free PhAs, and ten and three times higher contents of sinapic acid esters and glycosides, were found in NCE. On the contrary, three times higher ferulic acid content was found in the cell walls of CE. Higher content of lignin in NCE positively correlated with the amount of soluble phenolics and ionically bound peroxidase activity (EC 1.11.1.7). The results indicate that the alterations in phenylpropanoid metabolism in sessile oak somatic embryos are closely related to their developmental capability.

Peroxidase activities and contents of phenolic acids in embryogenic and nonembryogenic alfalfa cell suspension cultures

Contents of phenolic acids, peroxidase activities and growth curves showed significant differences in embryogenic (EC) and nonembryogenic (NEC) suspension cultures ofMedicago sativa L. NEC gave a typical growth curve while in EC the distinct phases were absent. The total content of phenolic acids was higher in NEC (related to EC), changed during the growth cycle and most of the acids occurred in ester-bound methanol soluble form. The level of phenolic acids in EC was significantly lower and did not change during 12-d cultivation. The major fraction was formed by phenolic acids ester-bound to the cell wall. The cytoplasmic peroxidase activity in NEC increased continuously during the growth and reached the maximum value at the end of exponential phase. In EC the extremely low cytoplasmic peroxidase activity did not change during cultivation. Ionically bound peroxidases in NEC represented 14 to 30% of the total extracted activity in dependence on the growth phase while in EC formed about 50% of the total activity and did not change during studied period. A possible participation of ionic peroxidase in the incorporation of phenolics into the cell wall is discussed.

Effect of inhibition of biosynthesis of phenylpropanoids on sessile oak somatic embryogenesis

Abstract The inhibition of phenylpropanoid biosynthesis by 2-aminoindan-2-phosphonic acid (AIP) in embryogenic culture of sessile oak (Quercus petraea (Matt.) Liebl.) was associated with a strong reduction of the amount of cinnamic acid derivatives, decline in lignin content and changes in the proportion of different types of somatic embryos. The decrease in the level of phenylpropanoid lignin precursors after AIP application decreased the amount of embryos with deep green cotyledons that are in control cultures characterized by high content of lignin. Their proportion in the total yield represented 30% and 10% in the control and AIP media, respectively. On the contrary, the reduced endogenous pool of cinnamic acids stimulated the cell division activity and the formation of early stages of somatic embryos. Globular, heart- and torpedo-shaped stages of somatic embryos represented in AIP-treated culture about 25% from the total yield of embryos, while only 10% in the control culture. Higher amounts of free putrescine (Put), spermidine and spermine and significantly lower levels of their conjugated forms were characteristic for embryos cultured for 7 weeks on AIP medium (related to controls). Significant increase in Put contents in cotyledonary embryos after 7-week culture with AIP might reflect the stress caused by lowered levels of phenylpropanoids in treated cells.

Changes in the levels of free IAA and cytokinins in potato tubers during dormancy and sprouting

Changes in the levels of free indol-3-ylacetic acid (IAA) and free cytokinins were determined in the course of dormancy and sprouting period in potato tubers(Solanum tuberosum L., cv. Nevskii) stored at 4 °C. The same analyses were performed in potato tubers after Ethrel application, which prolongs dormancy. No significant changes were found in free IAA level during dormancy followed by a rapid decrease during sprouting. After Ethrel application a significant lower IAA level was found 3 weeks after application, but further on no changes in free IAA level between treated and non-treated tubers were detected. Cytokinin level was relatively low and constant till sprouting and increases then by about 55 %, mainly due to an increase in the level of zeatin riboside and isopentenyladenosine. Ethrel application decreased cytokinin level during dormancy slightly, but postpones the increase coupled with sprouting by about 1 month. Thus, IAA does not seem to have a significant effect on tuber dormancy, while cytokinins are probably necessary for sprouting initiation.

Role of phytohormones in organogenic ability of elm multiplicated shoots

The study presents the comparative analyses of endogenous contents of auxin (IAA), cytokinins (CKs), polyamines (PAs), and phenolic acids (PhAs) in apical and basal parts of elm multiplicated shoots with regard to the organogenic potential. The shoot-forming capacity was higher in the apical part than in the basal part. However, the timing of root formation was in the apical type of explant significantly delayed (compared with the organogenic potential of basal part). Significantly higher contents of free bases, ribosides and ribotides of isopentenyl adenine, zeatin and dihydrozeatin that were found in the apical segments, might be considered as the most important factor affecting in vitro shoot formation. The content of endogenous free IAA was approximately three times higher in the basal shoot parts than in the apical parts. The amounts of putrescine and spermidine were higher in the apical part which generally contains less differentiated tissues than the basal part of shoot. The predominant PhA in both types of explants was caffeic acid, and concentrations of other PhAs decreased in the following order: p-coumaric, ferulic, sinapic, vanillic, chlorogenic, p-hydroxybenzoic and gallic acids. The contents of all determined PhAs in their free forms and higher contents of glycoside-bound p-coumaric, ferulic and sinapic acids, precursors for lignin biosynthesis, were found in the basal parts.

Morphogenesis of potato plants in vitro. II. Endogenous levels, distribution, and metabolism of IAA and cytokinins

The levels of endogenous IAA and cytokinins (zeatin, zeatin riboside, isopentenyladenine, and isopentenyladenosine) were determined in potato plants cultured in vitro under red light (R) and blue light (B) on medium with or without hormones. On medium without hormones in B, plants contained much higher cytokinin levels, particularly in leaves and roots, and also slightly elevated IAA levels. Kinetin in the medium in B changed the distribution of cytokinins and significantly increased IAA level in roots. In R, the presence of kinetin led to an increased cytokinin level in the whole plant, while the IAA level was slightly lower. IAA in the medium in B decreased cytokinin level in all plant parts, while the IAA level did not change significantly. In R, the presence of IAA in the medium led to a moderate increase of CK level and to a significant increase in IAA level, especially in roots. Uptake of 1-14C-IAA and of 3H-zeatin was generally higher in B than in R. Higher percentage of IAA taken up in B was converted to conjugates in the roots. Metabolism of 3H-zeatin was similar in R and B with only slight differences in metabolite amounts.Thus, in all experimental situations in which tuber formation was stimulated, IAA level in roots and stolons rose significantly, stressing the importance of an IAA gradient for tuber formation.

Evaluation of biological activity of new synthetic brassinolide analogs

The responses of plants to exogenous treatment with new synthetic brassinosteroids (BRs) were assessed and compared with the activity of natural 24-epibrassinolide (24-EPI). Morphological experiments on plants of pea and flax showed that the boundary between stimulatory and inhibitory concentrations of individual BRs and 24-EPI used is very narrow and differs also with the plant species. Moreover brassinosteroids can exhibit effects similar to various other plant hormones. This was proven also in our experiments, where auxin, anti-auxin and cytokinin like effects were achieved by BRs application. One of the explanations of the different morphological effects could be the influence of brassinosteroid application on the level of endogenous hormones. There are changes in the levels of indole-3-acetic acid, 6-benzylaminopurine, trans-zeatin and dihydrozeatin in rape and wheat plants caused by BR 4 and 24-EPI application, but there is no general trend explaining unequivocally their influence. The fact that all tested BRs significantly increased the dry weight accumulation in comparison with non-treated reference rape plants can be accounted for the known BRs characteristics to avoid biotic stresses.

Induced changes in phenolic acids and stilbenes in embryogenic cell cultures of Norway spruce by culture filtrate of Ascocalyx abietina

The aim of this work was to identify whether the Ascocalyx abietina culture filtrate has the ability to induce changes in the contents of phenolic substances that might be indicative for the disease response of Norway spruce (Picea abies). We focused on the accumulation of soluble and cell wall-bound phenolics, stilbenes as well as extracellular peroxidase activities elicited in the embryogenic cell cultures of Norway spruce by A. abietina culture filtrate. Treatment of spruce cells with fungal culture filtrate (5 and 20% v/v concentrations) evoked an increase in the total contents of phenolic acids (represented by the sum of free, methanol soluble ester- and glycoside-bound phenolics and methanol insoluble cell-wall bound phenolic esters). The challenge with filtrate was in particular manifested in the increase (compared with the control) in the contents of cell wall-bound phenolic acids (by about 100 and 130% in 5 and 20% filtrate, respectively) and soluble phenolic glycosides (by 37 and 65% in 5 and 20% filtrate, respectively) already 6 h after filtrate addition. Significantly decreased concentrations of stilbene glycosides, isorhapontin, astringin and piceid, were determined in filtrate-treated spruce cell cultures 6 and12 h after filtrate addition. The culture filtrate did not influence significantly the extracellular peroxidase activity 12 h after filtrate addition. Reduced extracellular peroxidase activity at 24 and 48 h in treated cells and decline in the amounts of soluble phenolics coincided with the rate of browning of filtrate-treated cells.ZusammenfassungDas Ziel dieser Arbeit bestand in der Überprüfung, ob das Kulturfiltrat des Ascomyceten Ascocalyx abietina die Gehalte phenolischer Abwehrsubstanzen der Gemeinen Fichte (Picea abies) beeinflussen kann. Unser Augenmerk richtete sich auf die Anreicherung löslicher und zellwandgebundener Phenole, Stilbene, als auch extrazellulärer Peroxidaseaktivitäten, die in embryogenen Zellkulturen der Gemeinen Fichte durch das Kulturfiltrat des Pilzes induziert wurden. Die Behandlung der Fichtenzellen mit dem Filtrat (Konzentrationen von 5 und 20% v/v) bewirkte einen Anstieg des Gesamtgehalts der Phenolsäuren (repräsentiert durch die Summe der freien, methanollöslichen ester- und glycosidgebundenen Phenole sowie der methanolunlöslichen zellwandgebundenen Phenolester). Die Wirkung der pilzlichen Kulturfiltrate manifestierte sich insbesondere in einem Anstieg der Gehalte zellwandge-bundener Phenolsäuren (um etwa 100 bzw. 130% in den 5-und 20%-Filtraten) und löslicher Phenolglycoside (um 37 bzw. 65% in den 5- und 20%-Filtraten) bereits 6 h nach Zugabe des Filtrats. Signifikante Konzentrationsabnahmen der Stilbenglycoside Isorhapontin, Astringin und Piceid wurden in den filtratbehandelten Fichtenzellkulturen 6 und 12 h nach Zugabe festgestellt. Das Kulturfiltrat beeinflusste die extrazelluläre Peroxidaseaktivität 12 h nach Filtratzugabe nicht. Eine verminderte Aktivität extrazellulärer Peroxidasen 24 und 48 h nach Filtratzugabe und die Konzentrations-abnahme löslicher Phenole korrelierten mit dem Verbräunungsgrad der filtratbehandelten Fichtenzellen.

Effect of 2-aminoindan-2-phosphonic acid on cell cycle progression in synchronous meristematic cells of Vicia faba roots

Abstract 2-Aminoindan-2-phosphonic acid (AIP), a potent inhibitor of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), was applied to highly synchronous meristematic cells of Vicia faba roots containing cells in G1/S and G2/M cell cycle checkpoints. The inhibition resulted in a slight decline of the content of hydroxycinnamic derivatives 3.5 h after the AIP application. Flow cytometric analyses and BrdU immunolabelling of replicated cells showed that AIP applied in the G1-phase of the cell cycle delayed the progression through S-phase. The transient delay in DNA replication in treated cells was accompanied by accumulation of free spermidine and perchloric acid (PCA)-soluble spermidine conjugates. The cell cycle checkpoint G2/M appeared to be less sensitive to the inhibitor than G1/S. Mitotic activity of asynchronous root meristems was decreased only after the highest tested AIP concentration was applied (100 μM) for 24 h. Significant increase in free putrescine and perchloric acid soluble-putrescine conjugates in cells after AIP-treatment might represent stress response in cells after AIP application. Increased PA conjugation suggests that it has an important role in the control of free PA contents in cells.