Lichtabhängigkeit der Phytolakkumulation

Summary Phytol is identified by gas chromatography and mass spectrometry and its concentration determined (range 0.005–3 g) in darkgrown and irradiated plants. Seeds of oats (Avena sativa L.), wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) contain bound phytol (2–5 g/g). The phytol content decreases during germination in the dark. Phytol synthesis in dark-grown seedlings starts in the light and stops in the dark again. The degradation of phytol in the dark is much slower than that of chlorophyll. The action spectra of phytol and chlorophyll accumulation are identical. The phytol/chlorophyll ratio increases at higher intensities of the monochromatic light, independent of the wavelength.     

Auxin level and regeneration of Begonia leaves

Summary As previously found, both the level of ether-extractable auxin (presumably indole-3-acetic acid) and the root-forming ability of B.xcheimantha leaves are increased under long-day conditions by high temperature, whereas the capacity for adventitious bud formation is reduced. However, this relation is present under relatively high light intensity only. Under the low light intensities in late fall neither auxin level nor regeneration ability were significantly affected by temperature.Dark treatment of detached leaves for 2 to 16 days greatly counteracted the inhibitory effect of high temperature on bud formation and reduced both the auxin level and the root-forming ability of the leaves.The great seasonal changes in the regeneration ability of Begonia leaves seem to be the result of a complex interaction of temperature, day-length, and daily light energy on the level of endogenous auxin and other growth regulators.     

The Pool of Chlorophyllous Pigments in Barley Seedlings of Different Ages under Heat Shock and Water Deficit

The effects of a high temperature (3 h, 40°C) and water deficit (45 h on 3% PEG 6000) on the pool of chlorophyllous pigments in the leaves of 4-, 7-, and 11-day-old barley (Hordeum vulgare L.) seedlings were studied. Heating resulted in a decrease in the total content of chlorophylls (Chl) (a + b) in 4-day-old plants but not in the older leaves. Water deficit induced an increase in the pigment content in young seedlings but reduced it in the leaves of 11-day-old plants. In young seedlings, hyperthermia and dehydration affected similarly Chl (a + b) degradation, leading to a marked inhibition of the chlorophyllase (Chlase) activity hydrolyzing Chl to chlorophyllides and phytol. In old leaves, an activation of this enzyme was observed. The stress factors under study affected different stages of pigment biosynthesis. High temperature inhibited the activity of dark and light stages of Chl(a + b) biosynthesis. Dehydration did not change markedly the resynthesis of protochlorophyllide, while the enzymes of the light stage of Chl biosynthesis were activated in young but inhibited in old barley leaves. The results thus obtained allowed us to conclude that heat treatment and dehydration specifically affected the Chl biosynthesis. At the same time, the Chlase response was nonspecific.     

Adventitious shoot formation in decapitated dicotyledonous seedlings starts with regeneration of abnormal leaves from cells not located in a shoot apical meristem

Regeneration of new shoots in plant tissue culture is often associated with appearance of abnormally shaped leaves. We used the adventitious shoot regeneration response induced by decapitation (removal of all preformed shoot apical meristems, leaving a single cotyledon) of greenhouse-grown cotyledon-stage seedlings to test the hypothesis that such abnormal leaf formation is a normal regeneration progression following wounding and is not conditioned by tissue culture. To understand why shoot regeneration starts with defective organogenesis, the regeneration response was characterized by morphology and scanning electron and light microscopy in decapitated cotyledon-stage Cucurbita pepo seedlings. Several leaf primordia were observed to regenerate prior to differentiation of a de novo shoot apical meristem from dividing cells on the wound surface. Early regenerating primordia have a greatly distorted structure with dramatically altered dorsoventrality. Aberrant leaf morphogenesis in C. pepo gradually disappears as leaves eventually originate from a de novo adventitious shoot apical meristem, recovering normal phyllotaxis. Similarly, following comparable decapitation of seedlings from a number of families (Chenopodiaceae, Compositae, Convolvulaceae, Cucurbitaceae, Cruciferae, Fabaceae, Malvaceae, Papaveraceae, and Solanaceae) of several dicotyledonous clades (Ranunculales, Caryophyllales, Asterids, and Rosids), stems are regenerated bearing abnormal leaves; the normal leaf shape is gradually recovered. Some of the transient leaf developmental defects observed are similar to responses to mutations in leaf shape or shoot apical meristem function. Many species temporarily express this leaf development pathway, which is manifest in exceptional circumstances such as during recovery from excision of all preformed shoot meristems of a seedling.     

Correlation between chlorophyllide esterification, Shibata shift and regeneration of protochlorophyllide650 in flash-irradiated etiolated barley leaves

The pigments of etiolated leaves of barley ( Hordeum vulgare L.) were analysed during dark periods after flash illumination, and the results were compared with in vivo spectroscopy of the leaves. Pretreatment of the leaves with kinetin slightly stimulated and pretreatment with NaF and anaerobiosis inhibited the esterification of chlorophyllide a (Chlide) at 10–40 min after the flash, whereas the rapid esterification within 30 s after the flash remained unchanged. Irrespective of pretreatment, the amount of esterified pigment was, at any time, identical with the amount of pigment that had shifted its absorption from 684 to 672 nm (Shibata shift). Cycloheximide (CHI) had only a small inhibitory effect on esterification, but drastically inhibited the hydrogenation of geranylgeraniol to phytol, bound to Chlide. The regeneration of long-wavelength protochlorophyllide a (Pchlide650) was stimulated by kinetin and inhibited by CHI and NaF. During the rapid phase (0–30 s after the flash), the esterification was faster than the regeneration of Pchlide650, and this, in turn, was faster than the formation of photoactive Pchlide. The kinetics changed after pretreatment with 5-aminolaevulinic acid: regeneration of Pchlide650 was the fastest reaction and the Shibata shift preceded the esterification of Chlide. The results are discussed as pigment exchange reactions at NADPH:protochlorophyllide oxidoreductase (POR; EC 1.6.99.1).     

Induction of Adventitious Buds on Undetached Leaves, Excised Leaves and Leaf Fragments of Heloniopsis orientalis

Adventitious buds were induced on intact, undetached leaves, isolated leaves, and both green and etiolated leaf fragments excised from young plants of Heloniopsis orientalis (Thunb.) C. Tanaka (Liliaceae) in darkness. Morphactin promoted bud initiation on undetached leaves. The regeneration loci on excised leaves were different in darkness and in light, and they were also modified by etiolation and by morphactin or benzyladenine. Experiments with pre-incubation in darkness, with successive treatments by sorbitol and sucrose, and with DCMU-treatment in light, led to the conclusion that bud formation on isolated leaves and leaf fragments is controlled by a photosynthetic system as well as the hormonal level.     

Zur Dynamik des Phytolgehaltes während des Chlorophyllauf- und - abbaues in Primärblättern von Weizenkeimpflanzen (Triticum aestivum L.)

The amounts of total phytol and its chlorophyll-bound and free portions have been determined quantitatively at different developmental stages of primary leaves of wheat using thin-layer chromatography combined with a potassium permanganate reaction. During the greening of etiolated leaves a close correlation exists between chlorophyll and phytol contents. Phytol is always present in excess and only in a bound state. During induced leaf senescence phytol is essentially degraded more slowly than chlorophyll. The liberated phytol does not influence the hydrolytic activity of chlorophyllase.     

A salvage pathway for phytol metabolism in Arabidopsis

Chlorophyll is the most abundant photosynthetic pigment in higher plants. During senescence, chlorophyll is hydrolyzed, resulting in the release of free phytol and chlorophyllide. Although the degradation of chlorophyllide has been studied in depth, the metabolic fate of phytol in plants is less clear. Here, we provide evidence that phytol can be incorporated into chlorophyll, tocopherol, and lipid esters by Arabidopsis seedlings. Phytol is phosphorylated to phytyl-phosphate and phytyl-diphosphate by two successive kinase activities associated with chloroplast envelope membranes of Arabidopsis. Although phytol kinase is CTP-dependent, the second kinase reaction, phytyl-phosphate kinase, shows broader specificity for CTP, GTP, UTP, and ATP. Therefore, in addition to de novo synthesis from geranylgeranyl-diphosphate, phosphorylation of free phytol represents an alternative route for phytyl-diphosphate production as the precursor for chloroplast prenyl lipid synthesis. Lipid esters are produced after feeding phytol to Arabidopsis seedlings, and they also accumulate in large amounts in leaves during senescence. The predominant phytyl ester that accumulates during senescence is hexadecatrienoic acid phytyl ester. Fatty acid phytyl ester synthesis by protein extracts of Arabidopsis is stimulated in the presence of phytol- and acyl-CoA esters. Thus, Arabidopsis contains a distinct enzymatic machinery for redirecting free phytol released from chlorophyll degradation into chloroplast lipid metabolism.     

PIGMENT TURNOVER IN THE MARINE DIATOM THALASSIOSIRA WEISSFLOGII. I. THE 14CO2-LABELING KINETICS OF CHLOROPHYLL a1

The turnover of chlorophyll a (chl a) was investigated in the diatom Thalassiosira weissflogii (Grunow) Fryxell and Hasle using a new method based on the incorporation of ¹⁴C into chl a. The alga was maintained in its exponential growth phase under continuous light; ¹⁴C was supplied as bicarbonate. The time course of label accumulation into the tetrapyrrole ring and the phytol side chain was determined for time periods equivalent to 1–2 cell doublings. The labeling kinetics of the tetrapyrrole ring and the phytol side chain were described satisfactorily by a simple precursor-pigment model with two free parameters, the precursor turnover rate and the pigment turnover rate, both having dimensions of time^?1. The model was fit to the experimental data to determine the values of these two free parameters. The turnover rates of the tetrapyrrole ring and the phytol side chain were not significantly different, ranging from 0.01 to 0.1 per day. These rates are equivalent to turnover times ranging from days to weeks. Growth rate-normalized turnover rates did not vary with irradiance (7.5–825 μE · m^?2· s^?1). The precursor turnover rates of the tetrapyrrole ring and the phytol side chain differed by an order of magnitude. These results indicate that chl a is not degraded significantly in cultures of T. weissflogii grown under continuous light. Neither irradiance nor growth rate affected growth rate-normalized chlorophyll turnover rates. Our results are inconsistent with the hypothesis that steady-state cellular concentrations of chl a are maintained by a dynamic equilibrium between rates of synthesis and degradation.     

Changes in the Content of Phytyl and Geranylgeranyl Esters of Germinating Polytrichum commune Spores

Polytrichum commune spores contained esterified phytol and geranylgeraniol, 706 and 114 μg, respectively, per 100 mg dry weight of freshly collected spores. After storage for 9 months the level of esterified phytol of the spores was decreased by c. 600 μg, whereas the level of esterified geranylgeraniol was more or less unchanged. The changes in the level of esterified prenols during germination follow the same pattern in freshly collected and in 9 month-old spore material. An immediate steep decrease between 0 and 3 h was followed by an increase in the level of esterified phytol between 3 and 12 h and by a constant value for esterified geranylgeraniol during the same period. Between 12 and 48 h the level of both types of esterified prenols decreased. In the freshly collected spores the amount of esterified prenols increased after 48 h of germination, in the older spores after 72 h. Free phytol was found in trace amounts in dry and germinating spores and in the protonema.     

Plant regeneration fromin vitro leaf culture of severalGerbera species

Efficient bud regeneration was obtained from a clone ofGerbera hybrida Bol. L. leaf explants cultured on modified Murashige and Skoog medium supplemented with 10 µM benzyladenine and 2.5 µM naphthalenacetic acid. The morphogenic potential varied with the developmental stage of the leaves. Up to 90% of excised developing leaves formed 3 to 5 shoots per explant. Bud regeneration was not obtained on the same medium with fully expanded leaves. Addition of 0.05 µM or 0.5 µM thidiazuron to the above medium significantly promoted regeneration from mature leaves but was ineffective for similar explants of a recalcitrant clone. The two wild speciesG. viridifolia Schultz Bip. andG. piloselloides L. Cass. also displayed specific multiplication habits and regeneration abilities. Bud regeneration occurred from callus. Chromosome counts and DNA flow cytometry indicated that all the regenerants were typically diploid, as were the various tissues of the mother plants. Afterin vitro rooting and acclimatization, no phenotypic variations were detected among the regenerants during both their vegetative and reproductive phases.     

Compositional heterogeneity of protochlorophyllide ester in etiolated leaves of higher plants

The formation and degradation of protochlorophyllide esters, i.e., protochlorophylls, were studied in etiolated leaves of kidney bean in relation to their aging. By the sensitive analysis of the pigments using high-performance liquid chromatography, the presence of four protochlorophylls esterified with phytol, tetrahydrogeranylgeraniol (THGG), dihydrogeranylgeraniol (DHGG), and geranylgeraniol (GG) was detected in kidney bean grown in the dark. Similar components were also observed in the etiolated seedlings of cucumber, sunflower, and corn. The content of each protochlorophyll species changed with the plant species and age of plants. In the case of kidney bean, the content of protochlorophyll phytol reached a maximal level at 9 days, then decreased rapidly during the subsequent development, in spite of the total protochlorophyll content remaining unchanged. In contrast to the degradation of protochlorophyll phytol, the other three protochlorophylls esterified with THGG, DHGG, and GG accumulated. These results may indicate that (i) protochlorophyll phytol is formed from the first esterified protochlorophyll GG through the next three hydrogenation steps as in the case of chlorophyll a phytol formation; (ii) the esterification reaction stops at 9 days and then reaction proceeds in sequence in the reverse direction, leading to the dehydrogenation of the alcohol moiety of protochlorophyll phytol to protochlorophylls THGG, DHGG, and GG.     

Photoinduced formation of pheophytin/chlorophyll-containing complexes during the greening of plant leaves

Illumination of etiolated maize leaves with low-intensity light produces a chlorophyll/pheophytin-containing complex. The complex contains two native chlorophyll forms Chl 671/668 and Chl 675/668 as well as pheophytin Pheo 679/675 (with chlorophyll/pheophytin ratio of 2/1). The complex is formed in the course of two successive reactions: reaction of protochlorophyllide Pchlde 655/650 photoreduction resulted in chlorophyllide Chlde 684/676 formation, and the subsequent dark reaction of Chlde 684/676 involving Mg substitution by H₂ in pigment chromophore and pigment esterification by phytol. Out data show that the reaction leading to chlorophyll/pheophytin-containing complex formation is not destructive. The reaction is in fact biosynthetic, and is competitive with the known reactions of biosynthesis of the bulk of chlorophyll molecules. The relationship between chlorophyll and pheophytin biosynthesis reactions is controlled by temperature, light intensity and exposure duration.The native complex containing pheophytin a and chlorophyll a is supposed to be a direct precursor of the PS II reaction centre in plant leaves.Abbreviations Chl chlorophyll - Chlde chlorophyllide - Pchl protochlorophyll - Pchlde protochloropyllide - Pheo pheophytin - PS II RC Photosystem II reaction centres. Abbreviations for native pigment forms: the first number after pigment symbol corresponds to the maximum position of low-temperature fluorescence band (nm); the second number corresponds to the maximum position of long wave absorption band     

The Effect of Phytohormones on Chlorophyll(ide), Protochlorophyll(ide) and Carotenoid Formation in Greening Dark Grown Wheat Leaves

The influence of phytohormones on chlorophyll and carotenoid formation during the greening of irradiated dark grown wheat leaves (Triticum aestivum L. cv. Starke II Weibull) was studied. Leaves were floated on solutions of abscisic acid, gibberellic acid and kinetin for 24 h. The chlorophyll and carotenoid contents were determined during a subsequent period of 48 h of continuous irradiation. Leaves treated with abscisic acid showed a longer lag phase and a lower rate of accumulation of chlorophyll as compared to the control than did leaves treated with gibberellic acid and kinetin. The carotenoid content was low both in leaves treated with abscisic acid and in those treated with gibberellic acid. Treatment with abscisic acid lowered the protochlorophyllide regeneration after a saturating light flash while gibberellic acid as well as kinetin had no effect. The influence of ABA was partly dependent on an increase of the wounded part of the cut leaf segments. The accumulation of protochlorophyllide in leaves treated with δ-aminolevulinic acid was not affected by the different hormonal treatments. These results suggest that the main effect of abscisic acid is probably outside the chloroplast, i.e. on the formation or transport of δ-aminolevulinic acid.     

Modelling photosynthesis in fluctuating light with inclusion of stomatal conductance, biochemical activation and pools of key photosynthetic intermediates

Photosynthetic carbon gain in rapidly fluctuating light is controlled by stomatal conductance, activation of ribulose-1,5-bisphosphate carboxylase-oxygenase, a fast induction step in the regeneration of ribulose-1,5-bisphosphate, and the build-up of pools of photosynthetic intermediates that allow post-illumination CO₂ fixation. Experimental work over recent years has identified and characterised these factors. A physiologically-based dynamic model is described here that incorporates these factors and allows the simulation of carbon gain in response to any arbitrary sequence of light levels. The model output is found to conform well to previously reported plant responses of Alocasia macrorrhiza (L.) G. Don. observed under widely differing conditions. The model shows (i) responses of net assimilation rate and stomatal conductance to constant light levels and different CO₂ concentrations that are consistent with experimental observations and predictions of a steady-state model; (ii) carbon gain to continue after the end of lightflecks, especially in uninduced leaves; (iii) carbon gain to be only marginally reduced during low-light periods of up to 2 s; (iv) a fast-inducing component in the regeneration of ribulose-1,5-bisphosphate to be limiting for up to 60 s after an increase in light in uninduced leaves: the duration of this limitation lengthens with increasing CO₂ concentration and is absent at low CO₂ concentration; (v) oxygen evolution to exceed CO₂ fixation during the first few seconds of a lightfleck, but CO₂ fixation to continue after the end of the lightfleck whereas oxygen evolution decreases to low-light rates immediately. The model is thus able to reproduce published responses of leaves to a variety of perturbations. This provides good evidence that the present formulation of the model includes the essential rate-determining factors of photosynthesis under fluctuating light conditions. Received: 27 January 1997 / Accepted: 15 April 1997     

In vitro shoot regeneration of Centranthus ruber DC from hairy roots induced by Agrobacterium rhizogenes,and determination of valepotriate content

ABSTRACT

Hairy roots were induced by infection with Agrobacterium rhizogenes strain LBA 9402 containing the plasmid 1855 from the valepotriate-producing medicinal plant Centranthus ruber. Plants were regenerated from callus derived from the hairy roots. The induction of shoot domes was obtained when hairy root calli, after a period of 3 months of dark incubation in MS (Murashige & Skoog, 1962) medium without growth regulators, were transferred for one week on the same medium under a 16-h light / 8-h dark photoperiod. Shoot regeneration increased with benzyladenine alone applied monthly at 2.5 µM concentration after the 3 months period of dark incubation. No bud formation was observed in untransformed tissues grown under the same light and hormonal conditions. Callus cultures of non-transformed plants was obtained by using leaves and roots as source material in the presence of 10.7 µM NAA and 1 µM kinetin as growth regulators, and 9 µM BA and 2.6 µM NAA for the next phase of shoot regeneration. BA and NAA together were not used for plant regeneration from transformed tissue. The morphological characteristics of the transgenic plants were analysed during two years of ex vitro growth. In the greenhouse, the transgenic plants showed pale pink flowers, heterostyly, leaves smaller than those of wild type plants, and a larger amount of roots. Roots of transgenic plants continued to produce valepotriates.     

Application of in vitro techniques in mutation breeding of chrysanthemum

Rooted cuttings of Chrysanthemum morifolium cv. Maghi, a small flowered, late blooming cultivar, were treated with different doses of gamma rays. Somatic mutations in flower colour (light mauve, white, light yellow and dark yellow) and chlorophyll variegation in leaves were detected as chimeras in treated populations. Attempts were made to standardize a microtechnique for plant regeneration from mutated tissues of stem node, stem internode, shoot tip and ray floret. All these explants were cultured on Murashige and Skoog's medium with 3% sucrose, 0.8% agar and different concentrations and combinations of growth regulators. Plant regeneration was successful from all of the mutated tissues. Plants with chlorophyll variegation in leaves and two new flower colours (light mauve and white) were isolated in pure form with 64% and 100% efficiency of mutant recovery, respectively. Attempts are being made to use this technique to establish new varieties from chimeric tissues to meet the increasing demand of the floriculture trade.     

Phytol has antibacterial property by inducing oxidative stress response in Pseudomonas aeruginosa

Phytol, isolated from Aster yomena, is widely distributed as a constituent of chlorophyll. In the present study, we confirmed the antibacterial activity of phytol and its mechanism inducing oxidative cell death in Pseudomonas aeruginosa. In phytol-treated cells, elevated level of intracellular reactive oxygen species (ROS) and transient NADH depletion were observed. These results demonstrated that phytol induced ROS accumulation and that the electron transport chain was involved in increase of ROS. Due to this ROS generation, the imbalance developed between intracellular ROS and the antioxidant defense system, leading to decrease of reduced glutathione (GSH). Moreover, severe DNA damage was shown after treatment with phytol. DNA electrophoresis and a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were conducted with pretreatment with the antioxidant N-acetylcysteine (NAC) to evaluate the cause of DNA damage. In NAC-pretreated cells, alleviated damage was confirmed and it supports that phytol induces oxidative stress-mediated DNA damage. In conclusion, phytol exerts the antibacterial property via inducing oxidative stress response in P. aeruginosa.     

Metabolic profiling reveals altered pattern of central metabolism in navel orange plants as a result of boron deficiency

We focused on the changes of metabolite profiles in navel orange plants under long‐term boron (B) deficiency using a gas chromatography–mass spectrometry (GC–MS) approach. Curling of the leaves and leaf chlorosis were observed only in the upper leaves (present before start of the treatment) of B‐deficient plants, while the lower leaves (grown during treatment) did not show any visible symptoms. The metabolites with up‐accumulation in B‐deficient leaves were mainly proline, l ‐ornithine, lysine, glucoheptonic acid, fucose, fumarate, oxalate, quinate, myo‐inositol and allo‐inositol, while the metabolites with down‐accumulation in B‐deficient leaves were mainly serine, asparagine, saccharic acid, citrate, succinate, shikimate and phytol. The levels of glucose and fructose were increased only in the upper leaves by B deficiency, while starch content was increased in all the leaves and in roots. The increased levels of malate, ribitol, gluconic acid and glyceric acid occurred only in the lower leaves of B‐deficient plants. The increased levels of phenols only in the upper leaves indicated that the effects of B on phenol metabolism in citrus plants may be a consequence of disruptions in leaf structure. Metabolites with opposite reactions in upper and lower leaves were mainly glutamine, glycine and pyrrole‐2‐carboxylic acid. To our knowledge, the phenomena of allo‐inositol even higher than myo‐inositol occurred characterized for the first time in this species. These results suggested that the altered pattern of central metabolism may be either specific or adaptive responses of navel orange plants to B deficiency.     

Remobilization of Phytol from Chlorophyll Degradation Is Essential for Tocopherol Synthesis and Growth of Arabidopsis

Phytol from chlorophyll degradation can be phosphorylated to phytyl-phosphate and phytyl-diphosphate, the substrate for tocopherol (vitamin E) synthesis. A candidate for the phytyl-phosphate kinase from Arabidopsis thaliana (At1g78620) was identified via a phylogeny-based approach. This gene was designated VITAMIN E DEFICIENT6 (VTE6) because the leaves of the Arabidopsis vte6 mutants are tocopherol deficient. The vte6 mutant plants are incapable of photoautotrophic growth. Phytol and phytyl-phosphate accumulate, and the phytyl-diphosphate content is strongly decreased in vte6 leaves. Phytol feeding and enzyme assays with Arabidopsis and recombinant Escherichia coli cells demonstrated that VTE6 has phytyl-P kinase activity. Overexpression of VTE6 resulted in increased phytyl-diphosphate and tocopherol contents in seeds, indicating that VTE6 encodes phytyl-phosphate kinase. The severe growth retardation of vte6 mutants was partially rescued by introducing the phytol kinase mutation vte5. Double mutant plants (vte5 vte6) are tocopherol deficient and contain more chlorophyll, but reduced amounts of phytol and phytyl-phosphate compared with vte6 mutants, suggesting that phytol or phytyl-phosphate are detrimental to plant growth. Therefore, VTE6 represents the missing phytyl-phosphate kinase, linking phytol release from chlorophyll with tocopherol synthesis. Moreover, tocopherol synthesis in leaves depends on phytol derived from chlorophyll, not on de novo synthesis of phytyl-diphosphate from geranylgeranyl-diphosphate.