Effect of feeding precursors on phenylalanine ammonia-lyase activity and coumarin accumulation in leaves of <Emphasis Type="Italic">Matricaria chamomilla</Emphasis> L.

Four-week-old chamomile (Matricaria chamomilla) plants were exposed for 72 h to 0.01, 0.1 and 1 mM phenylalanine (Phe) or tyrosine (Tyr). Phe at all concentrations significantly increased phenylalanine ammonia-lyase (PAL) activity (by 30, 76 and 90%, respectively) as well as accumulation of coumarin-related compounds (herniarin and its precursors (Z)- and (E)-2-β-D-glucopyranosyloxy-4-methoxycinnamic acids). Free Phe content increased significantly at the highest dose tested. Lower Tyr concentrations (0.01 and 0.1 mM) significantly increased PAL activity and increased free Tyr content, however free Phe content decreased. This indicated that Tyr-mediated stimulation of PAL is coupled to Phe consumption. Notwithstanding, Tyr had no effect on coumarin accumulation. Therefore we speculate that in chamomile a regulation/signalling mechanism could be operating in the pathway leading to coumarin synthesis. The malondialdehyde accumulation, an usual marker of stress in plants, was not significantly changed by amino acid supplements, suggesting that membrane damage is not the signal causing coumarin accumulation. In parallel experiment we observed that neither lower (0.25 × full strength), nor higher (3 × full strength) nitrogen concentration of nutrient solution compared to normal (1 × full strength, 205 mg N l^-1) solution used for Phe/Tyr supply affected herniarin and GMCAs accumulation. This indicates that Phe had stimulatory effect on PAL activity and coumarin metabolism.     

Circadian rhythm of (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxy cinnamic acids and herniarin in leaves of Matricaria chamomilla

Chamomile (Matricaria chamomilla) in the above-ground organs synthesizes and accumulates (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxy cinnamic acids (GMCA), the precursors of phytoanticipin herniarin (7-methoxycoumarin). The diurnal rhythmicity of the sum of GMCA (maximum before daybreak) and herniarin (acrophase at 10 h 21 min of circadian time) was observed under artificial lighting conditions LD 12:12. The acrophase is the time point of the maximum of the sinusoidal curve fitted to the experimental data. In continuous light, the circadian rhythms of both compounds were first described with similar acrophases of endogenous rhythms; a significantly different result from that in synchronized conditions. The rhythms’ mesor (the mean value of the sinusoidal curve fitted to the experimental data) under free-running conditions was not influenced. Abiotic stress under synchronized conditions decreased the average content of GMCA to half of the original level and eliminated the rhythmicity. In contrast, the rhythm of herniarin continued, though its content significantly increased. Nitrogen deficiency resulted in a significant increase in GMCA content, which did not manifest any rhythmicity while the rhythm of herniarin continued. Circadian control of herniarin could be considered as a component of the plant’s specialized defence mechanisms.     

Salicylic acid-induced changes to growth and phenolic metabolism in <Emphasis Type="Italic">Matricaria chamomilla</Emphasis> plants

The influence of salicylic acid (SA) doses of 50 and 250 μM, for a period of up to 7 days, on selected physiological aspects and the phenolic metabolism of Matricaria chamomilla plants was studied. SA exhibited both growth-promoting (50 μM) and growth-inhibiting (250 μM) properties, the latter being correlated with decrease of chlorophylls, water content and soluble proteins. In terms of phenolic metabolism, it seems that the higher SA dose has a toxic effect, based on the sharp increase in phenylalanine ammonia-lyase (PAL) activity (24 h after application), which is followed by an increase in total soluble phenolics, lignin accumulation and the majority of the 11 detected phenolic acids. Guaiacol-peroxidase activity was elevated throughout the experiment in 250 μM SA-treated plants. In turn, some responses can be explained by mechanisms associated with oxidative stress tolerance; these mitigate acute SA stress (which is indicated by an increase in malondialdehyde content). However, PAL activity decreased with prolonged exposure to SA, indicating its inhibition. Accumulation of coumarin-related compounds (umbelliferone and herniarin) was not affected by SA treatments, while (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxycinnamic acids increased in the 250 μM SA-treated rosettes. Free SA content in the rosettes increased significantly only in the 250 μM SA treatment, with levels tending to decrease towards the end of the experiment and the opposite trend was observed in the roots.     

Matricaria chamomilla is not a hyperaccumulator, but tolerant to cadmium stress

The influence of low (3 μM) and high (60 and 120 μM) cadmium (Cd) concentrations were studied on selected aspects of metabolism in 4-week-old chamomile (Matricaria chamomilla L.) plants. After 10 days’ exposure, dry mass accumulation and nitrogen content were not significantly altered under any of the levels of Cd. However, there was a significant decline in chlorophyll and water content in the leaves. Among coumarin-related compounds, herniarin was not affected by Cd, while its precursors (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxycinnamic acids (GMCAs) increased significantly at all the levels of Cd tested. Cd did not have any effect on umbelliferone, a stress metabolite of chamomile. Lipid peroxidation was also not affected by even 120 μM Cd. Cd accumulation was approximately seven- (60 μM Cd treatment) to eleven- (120 μM Cd treatment) fold higher in the roots than that in the leaves. At high concentrations, it stimulated potassium leakage from the roots, while at the lowest concentration it could stimulate potassium uptake. The results supported the hypothesis that metabolism was altered only slightly under high Cd stress, indicating that chamomile is tolerant to this metal. Preferential Cd accumulation in the roots indicated that chamomile could not be classified as a hyperaccumulator and, therefore, it is unsuitable for phytoremediation.     

Salicylic acid regulates secondary metabolites content in leaves of Matricaria chamomilla

The influence of increasing doses of salicylic acid (SA) on selected physiological parameters and the content of coumarin-related compounds of diploid and tetraploid cultivars of Matricaria chamomilla plants were studied. Forty-eight hours after treatment SA showed growth-promoting effect with decrease in tissue water content, chlorophylls and soluble proteins. High doses of SA led to an increase of reactive oxygen species (hydrogen peroxide and superoxide radical) resulted in membrane damage (measured by accumulation of malondialdehyde). Changes in secondary metabolites accumulation in leaves were also observed. The pattern of quantitative changes of studied compounds was similar in tetraploid and diploid plants. The content of herniarin and its precursor (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxycinnamic acid increased simultaneously. A considerable increase of umbelliferone and decrease in chlorogenic acid was registered. The rise of ene-yne-dicycloether in treated plant was also detected.     

Phenolic compounds composition and physiological attributes of Matricaria chamomilla grown in copper excess

Four-week old plants of chamomile (Matricaria chamomilla) cultivated in nutrient solution were exposed to copper (3, 60 and 120 μM) for 10 days. At 120 μM, Cu decreased dry mass production, water, chlorophyll and nitrogen content in both the leaf rosettes and roots. Five phenolic acids were detected in methanol extracts of the leaf rosettes (protocatechuic, p-hydroxybenzoic, vanillic, chlorogenic and salicylic acid) and six additional compounds (gentisic, syringic, caffeic, sinapic and o-/p-coumaric acid) were released after acid hydrolysis. Most of the 11 phenolic acids detected increased in 60 μM Cu but in the 120 μM treatment their contents were lower or not significantly different from the control. Among coumarin-related compounds, (Z)- and (E)-2-ß-d-glucopyranosyloxy-4-methoxycinnamic acids increased in 60 and 120 μM Cu while herniarin rose in the 3 and 60 μM Cu by the end of the experiment. The amounts of umbelliferone were not affected by any of the doses tested. These facts in relation to antioxidative properties of phenolic metabolites are also discussed. The malondialdehyde content of the leaf rosettes was not affected by exposure of plants to 120 μM Cu in a time-course experiment but in the roots a sharp increase was observed after 24 and 48 h of treatment. At 120 μM, Cu stimulated a 9-fold higher K⁺ loss than the 60 μM treatment while at the lowest concentration it stimulated potassium uptake. Cu accumulation in the roots was 3-, 49- and 71-fold higher than that in the leaf rosettes in the 3, 60, and 120 μM Cu treatments, respectively. Results suggest that 120 μM Cu dose is limiting for chamomile growth under the conditions of present research.     

Quantitative changes of secondary metabolites of Matricaria chamomilla by abiotic stress

The responses of young plants of diploid and tetraploid Matricaria chamomilla cultivars to abiotic stress were studied. The course of quantitative changes of main leaf secondary metabolites was evaluated within an interval from 6 h before to 54 h after spraying the leaf rosettes with aqueous CuCl2 solution. The content of herniarin in the treated plants rose approximately 3 times, simultaneously with a decline of its precursor (Z)- and (E)-2-beta-D-glucopyranosyloxy-4-methoxycinnamic acid. The highest amounts of umbelliferone in stressed plants exceeded 9 times and 20 times those observed in control plants of the tetraploid and diploid cultivar, respectively. Due to stress the concentration of ene-yne-dicycloether in leaves decreased by more than 40%. The pattern of quantity changes of the examined compounds in tetraploid and diploid plants was similar.     

The effect of copper toxicity on the contents of nitrogen compounds in Silene vulgaris (Moench) Garcke

The effect of copper on the uptake of nitrogen and the tissue contents of inorganic nitrogen, amino acids and proteins were studied in cooper-sensitive Silene vulgaris (Moench) Garcke, grown at different nitrogen sources (NH₄ ⁺ or NO₃ ^-). All the toxic copper levels tested, i.e. 4, 8, 16 M Cu²⁺, strongly inhibited the uptake of nitrogen, especially of NO₃ ^-, and decreased the content of NO₃ ^-, amino acids and proteins. Especially at 4 and 8 M Cu²⁺, NH₄ ⁺ accumulated in the plants, suggesting that the conversion of NH₄ ^- into amino acids was inhibited.     

Changes of phenolic metabolism and oxidative status in nitrogen-deficient Matricaria chamomilla plants

The effects of nitrogen deficiency on selected physiological attributes, phenylalanine ammonia-lyase (PAL, EC. 4.3.1.5) activity, phenolic contents, peroxidase (EC. 1.11.1.7) and catalase (EC. 1.11.1.6) activities, lipid peroxidation status and H₂O₂ accumulation were studied in N-deficient Matricaria chamomilla (L.) over 12 days. N deficiency enhanced root growth and inhibited shoot growth. Chlorophyll composition and F _v/F _m were not affected by N stress, but nitrogen and soluble proteins decreased in both the rosettes and the roots. PAL activity, expressed per mg protein, was enhanced in N-deficient rosettes and tended to decrease by the end of the experiment, while in the roots PAL activity was maintained. Total phenolic contents increased in both rosettes and roots. Peroxidase and catalase activities in N-deficient rosettes tended to decrease by the end of the experiment, while in the roots they increased on the 12th day of deficiency. Furthermore, lipid peroxidation status increased in N-deficient roots on the 12th day, indicating that antioxidative protection was insufficient to scavenge reactive oxygen species being generated. Surprisingly, H₂O₂ content was even lower in N-deficient roots by the end of the experiment, while in the leaves increased. This observation in correlation to lipid peroxidation and H₂O₂ degradation is discussed. The importance of PAL activity and phenolic metabolites in combination with antioxidative enzymes for plant protection against oxidative stress and the significance of PAL activity for the mobilization of N availability in N-deficient tissue are also discussed in view of existing information.     

High nitrogen supply affects the metabolism of Matricaria chamomilla leaves

N-status of the two Matricaria chamomilla cultivars grown in the presence of high potassium nitrate concentration was evaluated and compared with ammonium nitrate supply. After 5 days of potassium nitrate treatment the visible increase of dry mass together with total chlorophyll accumulation were observed. In both cultivars, ammonium nitrate application led to increased accumulation of N-containing compounds in chamomile leaves. NH₄NO₃ nitrogen supply influenced activity of nitrate reductase positively. In vivo nitrate reductase activity reached maximum in lower nitrate supply and decreased in higher nitrate availability significantly. Among the most abundant leaf secondary metabolites, the high nitrate availability both KNO₃ and NH₄NO₃ significantly increased umbelliferone level. The highest potassium nitrate dose (60 mmol per plant) caused an osmotic stress accompanied with lower tissue water content and turgor loss. In such condition the decrease in (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxycinnamic acid, herniarin and dicycloethers, as well as PAL activity was observed. On the other hand, strong increase of umbelliferone is likely a stress response and is related to its antioxidant activity.     

Effects of low and high levels of magnesium on the response of sunflower plants grown with ammonium and nitrate

The effect of the nitrogen source (ammonium and nitrate) and its interaction with magnesium on various physiological processes was studied in sunflower plants (Helianthus annuusL.). Plants were grown in hydroponic culture with nitrate (5 mM) or ammonium (5 mM) and four concentrations of magnesium (0.1, 0.8, 5 and 10 mM). After 2 weeks, growth, gas exchange and fluorescence parameters, soluble carbohydrates, free amino acids, soluble protein and mineral elements were determined. Ammonium nutrition resulted in a reduction of dry matter accumulation, as well as in a decrease in the CO₂ assimilation. Moreover, ammonium-fed plants showed a greater content of free amino acids, soluble protein, Rubisco and anions, and a lower cation content, mostly Mg²⁺. The presence of high levels of Mg²⁺ in the nutrient solution containing NH₄ ⁺ resulted in a stimulation of growth and CO₂ assimilation to the levels observed in nitrate-fed plants. The lower photosynthetic rate of ammonium-fed plants grown with low level of magnesium does not seem to be due to a lower photosynthetic pigment content, or a deficiency in Photosystem II activity, or to lower Rubisco content. Hence, Rubisco activity or other enzymes involved in CO₂ fixation could have been affected in ammonium-fed plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cell wall polymers of Actinomadura carminata INA 4281

The cell walls of Actinomadura carminata INA 4281 were found to contain peptidoglycan, teichoic acid, and nonpeptidoglycan amino acids. The peptidoglycan was of the A1 type and contained a small amount of ll-DAP in addition to m-DAP. The teichoic acid was an 1,3-poly(glycerol phosphate) chain composed of about eight glycerophosphate units, two of which had a 2-acetamido-2-deoxy--d-galactopyranosyl substituent and one, a 3-O-methyl--d-galactopyranosyl-(1 3)-2-acetamido-2-deoxy--d-galactopyranosyl residue at C2 of glycerol. The structure of the polymer was identified by chemical analysis and ¹³C-NMR spectroscopy. The teichoic acid contained 3-O-methyl-d-galactose (madurose) — the first ever finding of this compound within a teichoic acid. The nonpeptidoglycan amino acids made up some 30% of the cell wall's dry weight, about a quarter of the amino acids being removable with sodium dodecyl sulfate. Further treatment of the cell walls with LiCl and guanidine hydrochloride caused only a small loss of the amino acids and slight changes in their molar ratio.Abbreviations Gro glycerol - GroP monophosphate glycerol - GroP₂ diphosphate glycerol - Gro2P -monophosphate glycerol - P_TA phosphorus of teichoic acids - P_NA phosphorus of nucleic acids - TA teichoic acid     

Agrobacterium-mediated transformation of white clover using direct shoot organogenesis

Summary White clover (Trifolium repens L.) plants from the cultivars Grasslands Huia and Grasslands Tahora have been transformed using Agrobacterium-mediated T-DNA transfer. Transgenic plants regenerated directly from cells of the cotyledonary axil. To transform white clover, shoot tips from 3 day old seedlings were co-cultivated with A. tumefaciens strain LBA4404 carrying the plasmid vector pPE64. This vector contains the neomycin phosphotransferase II gene (nptII) and -glucuronidase reporter gene (gus) both under the control of the CaMV 35S promoter. Kanamycin-resistant plants regenerated within 42 days after transfer onto selective media. Integration of the nptII and gus genes into the white clover genome was confirmed using Southern blotting, and histochemical analysis indicated that the gus gene was expressed in a variety of tissues. In reciprocal crosses between a primary transformant and a non-transformed plant the introduced gus gene segregated as a single dominant Mendelian trait.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - MS Murashige and Skoog - GUS -glucuronidase - X-GLUc 5-bromo-4-chloro-3-indolyl--D-glucuronide - MUG methylumbelliferyl--D-glucuronide - CaMV Cauliflower Mosaic Virus - NPTII neomycin phosphotransferase II - OCS octopine synthase - 4-MU 4-methyl umbelliferone     

Amino acids as determinants of host preference for the xylem feeding leafhopper,Homalodisca coagulata (Homoptera: Cicadellidae)

Summary Homalodisca coagulata is a highly polyphagous xylem feeder with distinct seasonal patterns in it's selection of host plants. These patterns were examined in relation to the amino acid content of the xylem for four common host species; Lagerstroemia indica, Baccharis halimifolia, Prunus persica, and Prunus salicina. Xylem fluid was collected from each host species at times when numbers of feeding leafhoppers were both low and high. In each case, concentrations of amino acids were greatest when numbers were high. Similarly, comparisons between host species at given times showed that concentrations of amino acids were positively correlated with host selection. In a second study, amino acids of xylem were manipulated by budding scions of a non-preferred host (P. persica) on rootstocks of preferred (P. salinica) and non-preferred (P. persica) hosts. Morphology and phenology of the budded trees were similar to that of the scion species yet the xylem composition of amino acids was primarily dependent on the rootstock. Concentrations of amino acids and the preference of leafhoppers were roughly two-fold greater for scions of the preferred than the non-preferred rootstock. In both studies, amides (glutamine plus asparagine) were the amino acids most highly correlated with host selection. These compounds are the predominant amino acids in xylem fluid, have high nitrogen to carbon ratios, and account for a high percentage of the caloric value in xylem fluid. Many of the less abundant amino acids were positively correlated with host preference, but the correlations were less consistent and correlation coefficients were generally lower.Florida Agricultural Experiment Station Journal Series No. 9672     

Systemic induction of volatile release in cotton: How specific is the signal to herbivory?

Plants attacked by herbivorous insects release chemical signals that attract natural enemies of the herbivores to the damaged plants. Feeding of Spodoptera exigua larvae on the lower leaves of cotton (Gossypium hirsutum L.) for multiple feeding periods of 9–12 h with a 12 h, interval in between when the caterpillars are removed overnight, will induce a systemic release of volatile compounds that is comparable to the volatiles released in response to continuous feeding damage on the lower leaves for several days. The systemic volatile release in response to herbivory can be mimicked by mechanically damaging the lower leaves and applying caterpillar oral secretion to the injured leaves over 4 days. Cotton plants that are only mechanically damaged systemically release significantly less -pinene, myrcene, (Z)-3-hexenyl acetate, (E)--farnesene and (E,E)--farnesene after 4 days compared to plants damaged mechanically with application of caterpillar regurgitant. However, multiple 9–12 h mechanical damage alone induces a significantly higher systemic release of (Z)-3-hexenyl acetate, myrcene, (E)--ocimene, and (E)--farnesene after 4 days compared to undamaged control plants. This indicates that multiple mechanical damage alone cannot mimic completely the response induced by mechanically injuring the leaves and applying caterpillar regurgitant. A specific elicitor in the regurgitant of the caterpillar enhances the amount of several systemically released volatiles. Thus, the systemic release of volatile compounds by herbivore-damaged cotton plants appears to be regulated by at least two different mechanisms.     

A β-ketoacyl-acyl carrier protein synthase III gene (fabH) is encoded on the chloroplast genome of the red alga Porphyra umbilicalis

DNA sequencing of a region of the chloroplast genome of the red alga Porphyra umbilicalis revealed an open reading frame of 326 amino acids. Databank searches indicated that this ORF is 34% identical to an E. coli gene (fabH) encoding -ketoacyl-carrier protein synthase III. In addition, a leucine tRNA gene (trnL(GAG)) was detected just downstream. Neither of these genes are encoded on the chloroplast genomes of land plants.     

New Triterpene Glycosides from an Ulosa sp. Sponge

New triterpene glycosides, ulososides C, (20S,22S,23R,24S)-3,22,23-trihydroxy-3-O-(-D-glucopyranosyl)-32-nor-24-methyllanost-8(9)-ene-30-oic acid, D, (20S,22S,23R,24S)-3,22,23-trihydroxy-3-O-(-D-N-acetylglucosaminopyranosyl)-32-nor-24-methyllanost-8(9)-ene-30-oic acid, and E, (20S,22S,23R,24S)-3,22,23-trihydroxy-3-O-(-D-glucuronopyranosyl-(1 2)--D-arabinopyranosyl-32-nor-24-methyllanost-8(9)-ene-30-oic acid, were isolated from an Ulosa sp. sponge. Their structures were determined by spectral methods and chemical transformations. Specific features of their structures are discussed.     

Effects of aluminium and pH on growth and potassium uptake by three ectomycorrhizal fungi in liquid culture

Clonal plants of white clover (Trifolium repens L.) were grown in a controlled environment with either low or high rates of applied nitrate-N (providing, notionally, insufficient or sufficient N for unrestricted growth), or in the absence of applied N. Plants receiving no nitrate-N were inoculated with Rhizobia and fixed their own N₂. All plants were maintained with a maximum of three fully unfolded leaves per apex (lenient defoliation) until day 68 when half of the plants were severely defoliated. The export and translocation of carbohydrates from the first fully unfolded main stolon leaf was measured three days later using ¹⁴C.Reduced carbon translocation to stolon tissue and roots, and increased translocation to young branches, occurred following severe defoliation in all three nitrogen treatments. However, N-deficient plants showed large reductions in total export of carbohydrates (44 vs. 17% of ¹⁴C assimilated for lenient vs. severe defoliation) whereas N-sufficient plants (either receiving nitrate-N or fixing their own N₂) showed small increases in total export (means of 54% vs. 62% in the respective defoliation treatments). Furthermore, carbohydrate translocation to old branches ceased altogether in severely defoliated, N-deficient plants, but increased in severely defoliated, N-sufficient plants, illustrating that plant responses to multiple-factor stresses may differ greatly from those seen as the result of single-factor stresses. Interactions between nitrogen nutrition and defoliation in total carbohydrate export, and in carbohydrate supply to old branches, could have serious negative effects on the short-term C economy and physiological integration, and hence on the adaptability, of clonal plants growing with a mineral deficiency in the presence of grazing animals.     

Feeding by emerald ash borer larvae induces systemic changes in black ash foliar chemistry

The exotic wood-boring pest, emerald ash borer (EAB), Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), has been threatening North American ash (Fraxinus spp.) resources, this being recognized since its first detection in Michigan, USA and Ontario, Canada in 2002. Ash trees are killed by larval feeding in the cambial region, which results in disruption of photosynthate and nutrient translocation. In this study, changes in volatile and non-volatile foliar phytochemicals of potted 2-yr-old black ash, Fraxinus nigra Marshall, seedlings were observed in response to EAB larval feeding in the main stem. EAB larval feeding affected levels of six compounds [hexanal, (E)-2-hexenal, (Z)-3-hexenyl acetate, (E)-β-ocimene, methyl salicylate, and (Z,E)-α-farnesene] with patterns of interaction depending upon compounds of interest and time of observation. Increased methyl salicylate emission suggests similarity in responses induced by EAB larval feeding and other phloem-feeding herbivores. Overall, EAB larval feeding suppressed (Z)-3-hexenyl acetate emission, elevated (E)-β-ocimene emission in the first 30 days, but emissions leveled off thereafter, and generally increased the emission of (Z,E)-α-farnesene. Levels of carbohydrates and phenolics increased overall, while levels of proteins and most amino acids decreased in response to larval feeding. Twenty-three amino acids were consistently detected in the foliage of black ash. The three most abundant amino acids were aspartic acid, glutamic acid, glutamine, while the four least abundant were α-aminobutyric acid, β-aminoisobutyric acid, methionine, and sarcosine. Most (16) foliar free amino acids and 6 of the 9 detected essential amino acids decreased with EAB larval feeding. The ecological consequences of these dynamic phytochemical changes on herbivores harbored by ash trees and potential natural enemies of these herbivores are discussed.