BVOC responses to realistic nitrogen fertilization and ozone exposure in silver birch.

Emission of BVOC (Biogenic Volatile Organic Compounds) from plant leaves in response to ozone exposure (O3) and nitrogen (N) fertilization is poorly understood. For the first time, BVOC emissions were explored in a forest tree species (silver birch, Betula pendula) exposed for two years to realistic levels of O3 (35, 48 and 69 ppb as daylight average) and N (10, 30 and 70 kg ha(-1) yr(-1), applied weekly to the soil as ammonium nitrate). The main BVOCs emitted were: α-pinene, ß-pinene, limonene, ocimene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and hexanal. Ozone exposure increased BVOC emission and reduced total leaf area. The effect on emission was stronger when a short-term O3 metric (concentrations at the time of sampling) rather than a long-term one (AOT40) was used. The effect of O3 on total leaf area was not able to compensate for the stimulation of emission, so that responses to O3 at leaf and whole-plant level were similar. Nitrogen fertilization increased total leaf area, decreased α-pinene and ß-pinene emission, and increased ocimene, hexanal and DMNT emission. The increase of leaf area changed the significance of the emission response to N fertilization for most compounds. Nitrogen fertilization mitigated the effects of O3 exposure on total leaf area, while the combined effects of O3 exposure and N fertilization on BVOC emission were additive and not synergistic. In conclusion, O3 exposure and N fertilization have the potential to affect global BVOC via direct effects on plant emission rates and changes in leaf area.

Interactions of water stress and solar irradiance on the physiology and biochemistry of Ligustrum vulgare.

We studied the interactive effects of water stress and solar irradiance on physiological and biochemical traits in Ligustrum vulgare L., with special emphasis on antioxidant enzymes and flavonoids. Water relations, photosynthetic performance, plant growth, activities of antioxidant enzymes and of phenylalanine-ammonia-lyase, and concentrations of nonstructural carbohydrates and phenylpropanoids were measured in plants growing in 12% (shade) or 100% (sun) sunlight and supplied with 100 or 40% of daily evapotranspiration-demand over a 4-week period. The mild water stress treatment caused leaf water potential and relative water content to decline on average by -0.22 MPa and 4.5%, respectively. In response to the water stress treatment, photosynthetic rates decreased more in sun plants than in shade plants, likely because of declines in photosystem II photochemistry, apparent maximum rate of carboxylation and apparent maximum electron transport rate coupled with significant reductions in stomatal conductance. Antioxidant enzymatic activities, which were much greater in sun leaves than in shade leaves under well-watered conditions, increased (particularly the enzymatic activities associated with hydrogen peroxide removal) in response to water stress only in shade leaves. Antioxidant phenylpropanoids, particularly quercetin and luteolin derivatives, markedly increased in response to full sunlight irrespective of water treatment; however, antioxidant phenylpropanoid concentrations increased in response to water stress only in shade leaves. We suggest that: (1) assimilated carbon in sun plants was used largely to support an effective antioxidant system capable of countering water-stress-induced oxidative damage--an example of cross tolerance; and (2) in shade plants, carbon was also diverted from growth to counter oxidative damage driven by the mild water-stress treatment. Both findings are consistent with the nearly exclusive distribution of L. vulgare in well-watered, partially shaded Mediterranean areas.

Morphology and biochemistry of non-glandular trichomes in Cistus salvifolius L. leaves growing in extreme habitats of the Mediterranean basin.

Here the functional roles of stellate and dendritic trichomes in Cistus salvifolius L leaves were studied by analysing i) both leaf surface and trichome morphology using scanning electron and light microscopy; and ii) the composition and localisation of polyphenols by coupling liquid chromatography with fluorescence spectroscopy and fluorescence microimaging. Red-coloured compounds were detected in the stalk cells and the channel in the trichome arm, and appeared to be released at the tip end of the trichome branch. We identified such metabolites as ellagitannins, namely punicalagin and two galloyl derivatives of punicalagin. These ellagitannins accounted for 4.3 % of leaf dry weight and their concentration in the leaf leachate averaged 289.4 mg L (-1). The trichome arms exhibited an appreciable orange-red autofluorescence (centred at 620 nm) when excited with UV light (at 365 nm) or emitted in the yellow waveband (peak centred at 566 nm) when stained with the Naturstoff reagent, and excited at 488 nm. The fluorescence signatures of the trichome arms were consistent with the presence of mono-hydroxy B-ring substituted flavonoids, which were identified as the mono- and di-coumaroyl derivative of a kaempferol 3-O-glycoside. Our data may provide some insights on the functional roles of stellate and dendritic trichomes in the response mechanisms of C. salvifolius to Mediterranean-type climate, based upon (i) the potential effect of released ellagitannins on the soil nitrogen dynamic and (ii) the ability of acylated kaempferol 3-O-glycosides to effectively absorb both the UV-B and UV-A wavelengths.

Identification and quantification of galloyl derivatives, flavonoid glycosides and anthocyanins in leaves of Pistacia lentiscus L.

Separation, identification and quantification of polyphenols was carried out on leaves of Pistacia lentiscus L., an evergreen member of the family Anacardiaceae, using semi-preparative HPLC, HPLC-photodiode array detection and HPLC-MS analysis, together with 1H- and 13C NMR. Three major classes of secondary metabolites were detected: (i) gallic acid and galloyl derivatives of both glucose and quinic acid; (ii) flavonol glycosides, i.e. myricetin and quercetin glycosides; and (iii) anthocyanins, namely delphinidin 3-O-glucoside and cyanidin 3-O-glucoside. Low amounts of catechin were also detected. The concentration of galloyl derivatives was extremely high, representing 5.3% of the leaf dry weight, and appreciable amounts of myricetin derivatives were also detected (1.5% on a dry weight basis). These findings may be useful in establishing a relationship between the chemical composition of the leaf extract and the previously reported biological activity of P. lentiscus, and may also assign a new potential role of P. lentiscus tissue extracts in human health care.

HPLC analysis of flavonoids and secoiridoids in leaves of Ligustrum vulgare L. (Oleaceae).

Identification and quantification of flavonol glycosides and secoiridoids was carried out on leaves of Ligustrum vulgare L. (Oleaceae) by means of HPLC-DAD and HPLC-MS analysis. In addition to previously reported secoiridoids (oleuropein, ligustaloside A, ligustaloside B, and ligstroside) four kaempferol glycosides (kaempferol 3-O-glucoside 7-O-rhamnoside, kaempferol 3, 7-O-dirhamnoside, kaempferol 3-O-rhamnoside, and kaempferol 3-O-glucoside) and two quercetin glycosides (quercetin 3-O-glucoside 7-O-rhamnoside and quercetin 3,7-O-dirhamnoside) were present in leaves of L. vulgare L. Although secoiridoids accounted for nearly the 76% of the total leaf polyphenols content (with ligustaloside A as the main component), kaempferol glycosides were also accumulated in the leaves of L. vulgare L. to a relatively high extent (23%). Contribution of quercetin derivatives was minor under our experimental conditions. Our findings suggest that flavonol glycosides may have a central role in both the ecology and the biology of L. vulgare L.

Flavonoids accumulate in leaves and glandular trichomes of Phillyrea latifolia exposed to excess solar radiation.

Experiments were conducted on Phillyrea latifolia plants grown under a dense overstorey of Pinus pinea (shade plants) or on seashore dunes (sun plants) in a coastal area of Tuscany (42° 46' N, 10° 53' E). Total integrated photon flux densities averaged 1.67 and 61.4 m mol m-2 d-1 for shade and sun sites, respectively. A leaf morphological-structural analysis, a qualitative and quantitative analysis of phenylpropanoids of leaf tissue and leaf surface, and a histochemical localization of flavonoids were conducted. The area of sun leaves reached 57% of that of shade leaves, whereas leaf angle (ß), sclerophylly index (ratio of leaf d. wt:leaf area), and trichome frequency (trichome number mm-2 ) were markedly greater in leaves exposed to full solar radiation than in leaves acclimated to shade. The total thickness of sun leaves was 78% higher than that of shade leaves, mostly owing to a greater development of both palisade parenchyma and spongy mesophyll. The concentration, but not the composition, of leaf tissue phenylpropanoids varied significantly between sun and shade leaves, with a marked increase in flavonoid glycosides in sun leaves. Flavonoids occurred almost exclusively in the upper epidermal cells of shade leaves. By contrast, flavonoids largely accumulated in the upper and lower epidermis, as well as in the mesophyll tissue of leaves that were acclimated to full sunlight. Flavonoid glycosides were found exclusively in the secretory products of glandular trichomes of P. latifolia leaves exposed to high levels of light; luteolin 7-O- glucoside and quercetin 3-O-rutinoside were the major constituents. By contrast, verbascoside and an unidentified caffeic acid derivative constituted 72% of total phenylpropanoids secreted by glandular trichomes of shade leaves, whereas they were not detected in glandular trichomes of sun leaves. These findings suggest that the light-induced synthesis of flavonoids in glandular trichomes of P. latifolia probably occurs in situ and concomitantly inactivates other branch pathways of the general phenylpropanoid metabolism. This is the first report of the key role of glandular trichomes and of flavonoid glycosides in the integrated mechanisms of acclimation of P. latifolia to excess light.

Analysis of leaf water relations in leaves of two olive (Olea europaea) cultivars differing in tolerance to salinity.

One-year-old rooted cuttings of olive (Olea europaea L. cvs. Frantoio and Leccino) were grown either hydroponically or in soil in a greenhouse. Plants were exposed to NaCl treatments (0, 100, and 200 mM) for 35 days, followed by 30 to 34 days of relief from salt stress to determine whether previously demonstrated genotypic differences in tolerance to salinity were related to water relations parameters. Exposure to high salt concentrations resulted in reductions in predawn water potential (Psi(w)), osmotic potential at full turgor (Psi(piFT)), osmotic potential at turgor loss point (Psi(piTLP)), and relative water content (RWC) in both cultivars, regardless of the growth substrate. Leaf Psi(w) and RWC returned to values similar to those of controls by the end of the relief period. The effect of salinity on Psi(pi) appeared earlier in Leccino than in Frantoio. Values for Psi(piFT) were -2.50, -2.87, and -3.16 MPa for the 0, 100, and 200 mM salt-treated Frantoio plants, respectively, and -2.23, -2.87, and -3.37 MPa for the corresponding Leccino plants. Recovery of Psi(pi) was complete for plants in the 100 mM salt treatment, but not for plants in the 200 mM salt treatment, which maintained an increased pressure potential (Psi(pi)) compared to control plants. Net solute accumulation was higher in Leccino, the salt-sensitive cultivar, than in Frantoio. In controls of both cultivars, cations contributed 39.9 to 42.0% of the total Psi(piFT), mannitol and glucose contributed 27.1 to 30.8%, and other soluble carbohydrates contributed 3.1 to 3.6%. The osmotic contribution of Na(+) increased from 0.1-2.1% for non-treated plants to 8.6-15.5% and 15.6-20.0% for the 100 mM and 200 mM salt-treated plants, respectively. The mannitol contribution to Psi(piFT) reached a maximum of 9.1% at the end of the salinization period. We conclude that differences between the two cultivars in leaf water relations reflect differences in the exclusion capacities for Na(+) and Cl(-) ions.