Quantification of UV-B flux through time using UV-B-absorbing compounds contained in fossil Pinus sporopollenin.

UV-B radiation currently represents c. 1.5% of incoming solar radiation. However, significant changes are known to have occurred in the amount of incoming radiation both on recent and on geological timescales. Until now it has not been possible to reconstruct a detailed measure of UV-B radiation beyond c. 150 yr ago. Here, we studied the suitability of fossil Pinus spp. pollen to record variations in UV-B flux through time. In view of the large size of the grain and its long fossil history, we hypothesized that this grain could provide a good proxy for recording past variations in UV-B flux. Two key objectives were addressed: to determine whether there was, similar to other studied species, a clear relationship between UV-B-absorbing compounds in the sporopollenin of extant pollen and the magnitude of UV-B radiation to which it had been exposed; and to determine whether these compounds could be extracted from a small enough sample size of fossil pollen to make reconstruction of a continuous record through time a realistic prospect. Preliminary results indicate the excellent potential of this species for providing a quantitative record of UV-B through time. Using this technique, we present the first record of UV-B flux during the last 9500 yr from a site near Bergen, Norway.

UV-B absorbing compounds in present-day and fossil pollen, spores, cuticles, seed coats and wood: evaluation of a proxy for solar UV radiation.

UV-B absorbing compounds (UACs) in present-day and fossil pollen, spores, cuticles, seed coats and wood have been evaluated as a proxy for past UV. This proxy may not only provide information on variation of stratospheric ozone and solar UV in the period preceding and during the Antarctic ozone hole (1974-present day), but also on the development and variation of the stratospheric ozone layer and solar surface UV during the evolution of life on Earth. Sporopollenin and cutin are highly resistant biopolymers, preserving well in the geological record and contain the phenolic acids p-coumaric (pCA) and ferulic acid (FA). pCA and FA represent a good perspective for a plant-based proxy for past surface UV radiation since they are induced by solar UV-B via the phenylpropanoid pathway (PPP). UV-B absorption by these monomers in the wall of pollen and spores and in cuticles may prevent damage to the cellular metabolism. Increased pCA and FA in pollen of Vicia faba exposed to enhanced UV-B was found in greenhouse experiments. Further correlative evidence comes from UV-absorbing compounds in spores from 1960-2000 comparing exposure of land plants (Lycopodium species) to solar UV before and during ozone depletion and comparing plants from Antarctica (severe ozone depletion), Arctic, and other latitudes with less or negligible ozone depletion. Wood-derived compounds guaiacyl (G), syringyl (S), and p-hydroxyphenyl (P) are produced via the PPP. The proportions of P, G, and S in the lignin differ between various plant groups (e.g. dicotyledons/monocotyledons, gymnosperms/angiosperms). It is hypothesized that this lignin composition and derived physiological and physical properties of lignin (such as tree-ring wood density) has potential as a proxy for palaeo-UV climate. However validation by exposure of trees to enhanced UV is lacking. pCA and FA also form part of cutin polymers and are found in extant and fossil Ginkgo leaf cuticles as shown by thermally-assisted hydrolysis and methylation (THM)-pyrolysis-GC-MS. Potentially, the time scale for reconstruction of ozone column thickness and UV-B based on the UAC UV proxy may be decadal, centennial, millennial and possibly billenial. For further development of the UACs and pCA and FA-based UV proxy, it is necessary to obtain the UV dose-response (content of UACs, pCA and FA in sporopollenin and cutin) relationships for validation, based on outdoor UV radiation manipulations experiments with plants, and comparative analysis of stored plants (herbaria) or fossil material of the same or related plant species.

Flavonoid concentrations in three grass species and a sedge grown in the field and under controlled environment conditions in response to enhanced UV-B radiation.

An investigation was carried out to find whether enhanced levels of UV-B radiation induce increased concentrations of flavonoids in the leaves of the grass species Deschampsia antarctica, Deschampsia borealis and Calamagrostis epigeios and the sedge Carex arenaria. Whether the enhanced levels of UV-B influenced the proportions of the various flavonoids in the leaves was also studied. Increased flavonoid concentrations would improve the UV-B shielding of UV-B susceptible tissues. Using HPLC analysis the flavonoids orientin and luteolin were identified in D. antarctica, orientin in D. borealis and tricin in C. arenaria. Neither flavonoid concentrations nor the proportion of the various flavonoids in climate room grown D. antarctica and D. borealis plants differed between individuals grown under 0, ambient or elevated UV-B levels. After 12 weeks of growth biomass production and shoot-to-root ratios of D. antarctica were not affected by elevated UV-B radiation. Greenhouse grown C. epigeios plants contained higher concentrations and different proportions of flavonoids grown under elevated levels of UV-B than when grown under ambient or 0 UV-B. In C. epigeios plants grown in their natural habitat in the field under ambient or elevated levels of UV-B, flavonoid concentrations and proportions were the same in plants from both treatments. In the leaves of the sedge C. arenaria grown in a greenhouse flavonoid concentrations and proportions were not affected by UV-B radiation. Leaves were harvested four times during the growing season from C. arenaria plants grown in their natural habitat in the field under ambient or elevated levels of UV-B. Leaves harvested in January contained higher concentrations of flavonoids when grown under elevated UV-B than when grown under ambient UV-B radiation. In leaves harvested in May, September and December flavonoid concentrations were the same in plants grown under ambient or elevated UV-B. The proportion of the different flavonoids was the same for both treatments in all months. These results indicate that constitutive levels of flavonoids in these grass and sedge species are adequately high to protect them against ambient and elevated levels of solar UV-B radiation.

UV-B absorbance and UV-B absorbing compounds (para-coumaric acid) in pollen and sporopollenin: the perspective to track historic UV-B levels.

UV-B absorbance and UV-B absorbing compounds (UACs) of the pollen of Vicia faba, Betula pendula, Helleborus foetidus and Pinus sylvestris were studied. Sequential extraction demonstrated considerable UV-B absorbance both in the soluble (acid methanol) and insoluble sporopollenin (acetolysis resistant residue) fractions of UACs, while the wall-bound fraction of UACs was small. The UV-B absorbance of the soluble and sporopollenin fraction of pollen of Vicia faba plants exposed to enhanced UV-B (10 kJ m(-2) day(-1) UV-B(BE)) was higher than that of plants that received 0 kJ m(-2) day(-1) UV-B(BB). Pyrolysis gas chromatography-mass spectrometry (py-GC-MS) analysis of pollen demonstrated that p-coumaric acid and ferulic acid formed part of the sporopollenin fraction of the pollen. The amount of these aromatic monomers in the sporopollenin of Vicia faba appeared to increase in response to enhanced UV-B (10 kJ m(-2) day(-1) UV-B(BE)). The detection limit of pyGC-MS was sufficiently low to quantify these phenolic acids in ten pollen grains of Betula and Pinus. The experimental data presented provide evidence for the possibility that polyphenolic compounds in pollen of plants are indicators of solar UV-B and may be applied as a new proxy for the reconstruction of historic variation in solar UV-B levels.

Relation between heavy metal concentrations in salt marsh plants and soil.

The aim of the research reported here was to investigate the relation between heavy metal concentrations in salt marsh plants, extractability of the metals from soil and some soil characteristics. In April 1987, Spartina anglica and Aster tripolium plants and soil were collected from four salt marshes along the Dutch coast. The redox potential of the soil between the roots of the plants and at bare sites was measured. Soil samples were oven-dried and analyzed for chloride concentration, pH, fraction of soil particles smaller than 63 microm (f < 63 microm), loss on ignition (LOI) and ammonium acetate and hydrochloric acid extractable Cd, Cu and Zn concentrations. The roots and shoots of the plants were analyzed for Cd, Cu and Zn. Because drying of the soil prior to chemical analysis might have changed the chemical speciation of the metals, and therefore the outcome of the ammonium acetate extraction, a second survey was performed in October 1990. In this survey A. tripolium plants and soil were collected from two salt marshes. Fresh and matched oven-dried soil samples were analyzed for water, ammonium acetate and diethylene triaminepentaacetic acid (DTPA) extractable Cd, Cu and Zn concentrations. The soil samples were also analyzed for f < 63 microm, LOI and total (HNO(3)/HCl digestion) metal concentrations. Soil metal concentrations were correlated with LOI. Drying prior to analysis of the soil had a significant effect on the extractability of the metals with water, ammonium acetate or DTPA. Plant metal concentrations significantly correlated only with some extractable metal concentrations determined in dried soil samples. However, these correlations were not consistently better than with total metal concentrations in the soil. It was concluded that extractions of metals from soil with water, ammonium acetate or DTPA are not better predictors for metal concentrations in salt marsh plants than total metal concentrations, and that a major part of the variation in metal concentrations in the plants cannot be explained by variation in soil composition.

A survey of zinc, copper and cadmium concentrations in salt marsh plants along the Dutch coast.

In autumn 1986, plants and soil were collected from the lower and the higher salt marsh zones of salt marshes along the Dutch coast. The main purpose was to get an overview of Zn, Cu and Cd concentrations in six dominant species of salt marsh plants. The roots and shoots of the plants were analysed for Zn, Cu and Cd. The highest heavy metal concentrations were found in plants collected from salt marshes near harbour areas and/or that are known to receive contaminated fluvial sediment. Dicotyledonous plant species tended to have similar heavy metal concentrations in roots and shoots, whereas in monocotyledonous species the concentrations in the roots were two to three times higher than in the shoots. Differences in accumulation in the roots between elements and between plant species were found. Cd was accumulated more than Zn or Cu. Triglochin maritima shows a low Cd uptake by roots, whereas Spartina anglica and Scirpus maritimus tend to accumulate it. The fraction of soil particles smaller than 63 microm, loss on ignition and Zn, Cu and Cd concentrations were determined in soil samples. The highest Zn, Cu and Cd concentrations in the soil were found at salt marshes in the Western Scheldt area and were nine, five and 20 times higher than background levels, respectively.

Iron plaque on roots of Aster tripolium L.: interaction with zinc uptake.

The iron plaque on roots of Aster tripolium L. growing in waterlogged salt marsh soil adsorbed appreciable amounts of Zn and Cu, with maximum Zn/Fe and Cu/Fe ratios of 0.1 When concentrations of Zn or Cu adsorbed in the iron plaque are expressed as mg metal kg-1 FeOOH (assuming that iron plaque consists mainly of FeOOH), the Zn and Cu concentrations of the iron plaque was up to 680 and up to 2000 times higher than in the surrounding sediment, respectively. The Zn concentration in red roots (with iron plaque) was higher than in white roots (without iron plaque). Zn concentrations in field sampled roots were correlated with the amount of Zn on the roots and the Zn concentration in the soil, whereas Cu concentrations in the roots were only significantly correlated to the Cu concentration in the soil. In vitro experiments showed that red roots take up more Zn than white roots. Measurement of Zn uptake by excised roots showed that the uptake of Zn into the xylem fluid was significantly higher in roots with 500-2000 nmol Fe cm-2 on the root surface compared to roots with less than 500 or more than 2000 nmol Fe cm-2 on the root surface. The results indicate that iron plaque enhances uptake of Zn by the roots but may act as a barrier when large amounts of Fe are deposited on the root surface. The role of the iron plaque on roots of salt marsh plants growing in soil contaminated with heavy metals is discussed.

The cell wall of Chlamydomonas eugametos. Immunological aspects.

An antiserum was raised against the major cell wall glycoprotein of Chlamydomonas eugametos which after purification reacted specifically with all individual wall components but not with intact cell walls. The antigenic sites in intact walls appear to be cryptic but become exposed on partial enzymatic degradation or in situ during daughter-cell release when the insoluble component is digested. Using the antiserum as a specific label for cell walls in various stages of disintegration, cell wall digestion during asexual and sexual reproduction was studied. It is also shown that while cell wall material is associated with isolated flagella, it is not normally associated with the flagella of intact cells.

Sexual agglutination factor from Chlamydomonas eugametos.

Chlamydomonas eugametos gametes can sexually agglutinate via their flagellar surfaces whereas vegetative cells cannot. Therefore, flagellar glycoproteins, present in gamete cells but absent from vegetative cells, were investigated as prospective mt (-)agglutination factors. They were identified as periodic acid Schiff (PAS) stained bands separated in sodium dodecyl sulphate-polyacrylamide electrophoresis gels. Gamete-specific bands were determined by comparison with equivalent gels of vegetative flagella and by immunological techniques using antisera raised against isolated mt (-) gamete flagella. Four high molecular weight flagellar glycoproteins proved to be gamete specific (PAS-1.2, PAS-1.3, PAS-3 and PAS-4). They were extracted from flagella by 3 M guanidine thiocyanate, separated in a column of Sepharose 2B, and tested for in vitro agglutination activity on mt (+) gametes. A single peak of activity was found to be correlated with the presence of the PAS-1.2 band. It is shown that mt (-) agglutination activity is related to the concentration of this glycoprotein in flagellar membranes.