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This proceedings volume contains the invited and a selection of the contributed papers of the 8th International Workshop on Sulfur Metabolism in Higher Plants, which was held at Department of Forest and Ecosystem Science, University of Melbourne, Water Street, Creswick, Victoria 3363, Australia, from November 22-27, 2010. The content of the volume shows that the understanding of sulfur metabolism in plants and the interaction of the environment are rapidly progressing. This volume covers various aspects of the regulation of sulfate uptake and assimilation in plants, from a cellular to a whole plant level, and additionally emphasizes interactions with other minerals. Moreover the significance of sulfur metabolism in biotic and abiotic stress responses, in food security and quality, and in relation to interactions with global change factors is discussed in detail.

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The level of glutathione (GSH) in plants is important in defence reactions against biotic and abiotic stresses and can place considerable demand of the sulphur assimilation pathway. Enzymes involved in sulphur assimilation and GSH metabolism are not evenly distributed between different subcellular compartments or between different cell types in leaves or roots; however, there is little information on the effect that such asymmetries have on the actual GSH concentration in each compartment or cell type. In the present study in situ labelling with monochlorobimane (MCB) in combination with confocal laser scanning microscopy was used to quantify GSH in each of the main cell types of poplar leaves from fluorescence of the GSB conjugate formed. Comparison of results from the in situ approach with total GSH levels measured in vitro by high-performance liquid chromatography suggested that only the cytosolic GSH pool was labelled using this approach. This suggests that an appropriate GST was not present within the chloroplasts to catalyse the conjugation reaction and that chloroplastic GSH does not rapidly exchange with the cytoplasmic pool under the conditions of the assay. Cytosolic GSH levels were between 0.2 and 0.3 mm for both photosynthetic and non-photosynthetic (epidermal) cell types in wild-type poplar leaves. Cytosolic levels increased by around two-fold in transgenic poplars over-expressing bacterial gamma-glutamylsynthetase (gamma-ECS) in the cytosol of all cell types, but there was no concomitant increase in the chloroplastic GSH pool.

This study with poplar (Populus tremula x Populus alba) cuttings was aimed to test the hypothesis that sulfate uptake is regulated by demand-driven control and that this regulation is mediated by phloem-transported glutathione as a shoot-to-root signal. Therefore, sulfur nutrition was investigated at (a) enhanced sulfate demand in transgenic poplar over-expressing gamma-glutamylcysteine (gamma-EC) synthetase in the cytosol and (b) reduced sulfate demand during short-term exposure to H2S. H2S taken up by the leaves increased cysteine, gamma-EC, and glutathione concentrations in leaves, xylem sap, phloem exudate, and roots, both in wild-type and transgenic poplar. The observed reduced xylem loading of sulfate after H2S exposure of wild-type poplar could well be explained by a higher glutathione concentration in the phloem. In transgenic poplar increased concentrations of glutathione and gamma-EC were found not only in leaves, xylem sap, and roots but also in phloem exudate irrespective of H2S exposure. Despite enhanced phloem allocation of glutathione and its accumulation in the roots, sulfate uptake was strongly enhanced. This finding is contradictory to the hypothesis that glutathione allocated in the phloem reduces sulfate uptake and its transport to the shoot. Correlation analysis provided circumstantial evidence that the sulfate to glutathione ratio in the phloem may control sulfate uptake and loading into the xylem, both when the sulfate demand of the shoot is increased and when it is reduced.

The dynamic-chamber technique was used to investigate the correlation between NH3 and NO, fluxes and different climatic and physiological parameters: air temperature; relative air humidity; photosynthetic photon fluence rate; NH3 and NO2 concentrations; transpiration rate; leaf conductance for water vapour; and photosynthetic activity. The experiments were performed with twigs from the sun crown of mature beech trees (Fagus sylvatica) at a field site (Höglwald, Germany), and with 12-wk-old beech seedlings under controlled conditions. Both sets of experiments showed that NO2 and NH3 fluxes depended linearly on NO2 and NH3 concentration, respectively, in the concentration ranges representative for the field site studied, and on watervapour conductance as a measure for stomatal aperture. The NO2 compensation point determined in the field studies (the atmospheric NO2 concentration with no net NO2 flux) was 1.8-1.9 nmol mol high -1, The NH3 compensation point varied between 3.3 and 3.5 nmol mol high -1, in the field experiments, and was 3.0 nmol mol -1 in the experiments under controlled conditions. The climatic factors T and PPFR were found to influence both NO2 and NH3 fluxes indirectly, by changing stomatal conductance. Whilst NO2 flux showed a response to changing relative humidity that could be explained by altered stomatal conductance, increased NH3 flux with increasing relative humidity (> 50 %) depended on other factors. The exchange of NO2 between above-ground parts of beech trees and the atmosphere could be explained exclusively by uptake or emission of NO2 through the stomata, as indicated by the quotient between measured and predicted NO2 conductance of approx. 1 under all environmental conditions examined. Neither internal mesophyll resistances nor additional sinks could be observed for adult trees or for beech seedlings. By contrast, the patterns of NH3 flux could not be explained by an exclusive exchange of NH3 through the stomata. Deposition into additional sinks on the leaf surface, as indicated by an increase in the quotient between measured and predicted NH3 conductance, gained importance in high air humidity, when the stomata were closed or nearly closed and/or when atmospheric NH3 concentrations were high. Although patterns of NH3 gas exchange did not differ between different months or years at high NH3 concentrations (c. 140 nmol mol high -1), it must be assumed that emission or deposition fluxes at low ambient NH3 concentration (0.8 and 4.5 nmol mol high -1) might vary significantly with time because of variation in the NH3 compensation point.

Aerenchyma, developed in both root and aboveground parts of rice plants, is predominantly responsible for plant-mediated transfer of methane (CH4) form the soil to the atmosphere. To clarify the pathways of CH4 transport through the rice plant and find differences that may determine the large variation in the patterns of methane transport capacity (MTC) of rice cultivars, we examined the appearance, the distribution pattern, and the density of aerenchyma in different parts of rice plants of three widely varying rice cultivars during panicle initiation, flowering, and maturity stages. The data on the amount and density of small (> 1 x 10 high 3 - 10 high 3 µm high 2), medium (> 5 x 10 high 3 - 20 x 10 high 3 µm high 2), and large aerenchyma lacunae (> 20 x 10 high 3 µm high 2) were collected using a computer assisted image-analyzing system. The brightfield optical microscopy of roots of all tested rice plants demonstrated the continuity of aerenchyma channels in the roots that function as conduits for bi-directional transport of gases. The aerenchyma channels of primary roots showed direct connection with those of culms. Intercalary meristems were found at the transition zone of rootculm aerenchyma connections. Well-developed aerenchyma lacunae present in the internodal region of the culm base were uniformly distributed in the peripheral contical zone. The nodal region had relatively fewer and smaller aerenchyma lacunae that showed a non-uniform distribution pattern. As a result, few aerenchyma channels continued from the internodal region through to the nodal region. The aerenchyma in the cortex zone of the culm expanded along with the growing secondary tiller, development continuity between the culm and the secondary tiller. The micrographs of longitudinal sections of different specimens of culm-leaf sheath intersection showed the continuity of aerenchyma channels from the culm to the leaf. The amount of medium and large aerenchyma lacunae in the leaf sheath was respectively 2 and 33 times greater as compared to those of the tiller. The proportion of the large lacunae in the total amount of aerenchyma in leaf sheath was 75 % as compared to only 8 % in the tiller, revealing higher number and larger size of aerenchyma in the former. There were significant differences in amount and density of aerenchyma between individual cultivars at a given growth stage, as well as in the development patterns. While the amount and density of medium and small aerenchyma lacunae in the internodal region of the culm base did not show any relationship with MTC of rice cultivars, large aerenchyma lacunae exhibited highly significant correlations with MTC of different cultivars, suggesting that the wide variation in MTC of rice plants during different growth stages are related to these structural features.

The Interregional Research Program on Methane Emissions from Rice Fields established a network of eight measuring stations in five Asian countries. These stations covered different environments and encompassed varying practices in crop management. All stations were equipped with a closed chamber system designed for frequent sampling and long-term measurements of emission rates. Even under identical treatment - e. g., continuous flooding and no organic fertilizers - average emission rates varied from 15 to 200 kg CH sub 4 ha high -1 season high -1. Low temperatures limited CH sub 4 emissions in temperate and subtropical stations such as northern China and northern India. Differences observed under given climates, (e. g., within the tropics) indicated the importance of soil properties in regulating the CH sub 4 emission potential. However, local variations in crop management superseded the impact o soil- and climate-related factors. This resulted in uniformly high emission rates of about 300 kg CH sub 4 ha high -1 season high -1 for the irrigated rice stations in the Philippines (Maligaya) and China (Beijing and Hangzhou). The station in northern India (Delhi) was characterized by exceptionally low emission rates of less than 20 kg CH sub 4 ha high -1 season high -1 under local practice. These findings also suggest opportunities for reducing CH sub 4 emission through a deliberate modification of cultural practice for most irrigated rice fields.

Three-year-old seedlings of black alder (Alnus glutinosa (L) Gaertn.), a common European wetland tree species, were grown in native soil taken from an alder swamp. Fluxes of methane (CH4) and nitrous oxide (N2O) between the tree stem and the atmosphere were determined under controlled conditions. Both CH4 and N2O were emitted through the bark of the stem into the atmosphere when the root zone exhibited 'higher-than-ambient' CH4 and N2O gas mixing ratios. Flooding of the soil caused a decreased N2O emission but an increased CH4 efflux from the stem. Immediately after flooding of the soil, N2O was emitted from the seedlings' bark at a rate of 350 my mol N2O m(-2) h(-1) whereas CH4 flux could not be detected. After more than 40 days of flooding CH4 fluxes up to 3750 my mol CH4 m(-2) h(-1) from the stem were measured, while N2O emission had decreased below the limit of detection. Gas efflux decreased with increasing stem height and correlated with gas mixing ratios in the soil, indicating diffusion through the aerenchyma as the major path of gas transport. From these results it is assumed that woody species with aerenchyma can serve as conduits for soil-derived trace gases into the atmosphere, to date only shown for herbaceous plants. This, yet unidentified, 'woody plant pathway' contributes to the total greenhouse gas source strength of wetlands.

In order to compare the effects of excess pedospheric and atmospheric nitrogen supply on nitrate reductase activity (NR, EC 1.6.6.1) excised spruce branches were exposed to nitrate solutions or were fumigated with NO2. Immersion of spruce branches in 6 mM nitrate caused an increase in NR activity by a factor of 14 or 19 in current-year and in one-year-old needles, respectively, as compared to controls incubated in tap water. Exposure to 65 nl l(-1) NO2 increased NR activity by a factor of 1.5 in current-year needles and by a factor of 2.5 in one-year-old needles as compared to non-fumigated controls. Addition of cycloheximide (0.17 my M) or puromycin (200 my M) to the incubation solution prevented the induction of NR activity from both nitrate and NO2 exposure. This finding indicates that induction of NR activity by both atmospheric NO2 or increased nitrate supply of the needles is both caused by denovo synthesis of NR protein. The increase in NR activity in needles of branches still a ttached to the tree as a consequence of exposure to 65 nl l(-1) NO2 was found to be a transient phenomenon. The increase persisted for several days only and was no longer observed after one week of sustained NO2 exposure. An interruption of phloem transport by girdling, applied subsequent to the induction of NR activity by atmospheric NO2, prevented the decrease in NR activity. Apparently, export out of the exposed needles and phloem transport within the stem are involved in the regulation of NR activity upon NO2 exposure.

Methan ist ein atmosphärisches Spurengas und für den Treibhauseffekt mitverantwortlich. Geeignete Düngungs- und Bewässerungsmaßnahmen, Reisvarietäten mit geringen Methan transportkapazitäten und Biogasanlagen bieten erfolgversprechende Möglichkeiten zur Reduktion der Methanemission aus Reisfeldern.

The dynamic chamber technique was applied to investigate NO2 influx into Picea abies (L.) Karsten branches, and its effects on net photosynthesis and transpiration, as well as its dependency on irradiance, temperature and relative humidity. The study aimed to quantify effects of climate on atmospheric NO2 fluxes to spruce. Experiments were performed with 3- to 4-yr-old branches of 8- to 9-yr-old potted trees under controlled environmental conditions. With ambient NO2 concentrations increasing from 3.5 to 50 nl l(-1) a linear increase in the NO2 influx of up to c. 6 x 8 my mol m(-2) s(-1) was observed. From this increase a compensation point of 1.64 nl l(-1) NO2 was calculated by linear regression analysis. In the range of the NO2 concentrations studied, net photosynthesis of spruce was not affected. The responsiveness of the stomata to changes in irradiance and relative humidity was reduced at 45 nl l(-1) NO2 compared with 25 nl l(-1) NO2. With increasing irradiance up to 1000 my mol m (-2) s(-1) PAR, increasing NO2 flux to spruce branches was observed, which was attributed to a light-dependent increase in stomatal aperture. Variation of the temperature between 14 and 35 deg C did not affect the NO2 flux in light or in darkness. Higher temperatures, up to 45 deg C, resulted in an increase in NO 2 influx in the light; in darkness, changes in NO2 flux were not observed under these conditions. An increase in relative humidity from 5 to 60 per cent in the light caused an increase in NO2 influx, whereas in darkness NO2 influx was not affected by changes in relative humidity. The increase in NO2 flux in response to r.h. observed in the light could not be explained by changes in stomatal aperture. A solution of NO2 in ultra-thin water films covering the needle surface might explain this phenomenon.

Spinach plants (Spinacea oleracea L. cv. Estivato) were grown an nutrient solutions under deficient, normal and excess sulfate supply. In both young and mature plants net uptake of sulfate and its transport to the shoot increased with increasing sulfate supply, but bath processes proceeded at a higher rate in young as compared to mature plants. The relative sulfate transport, i.e. the relative amount of the sulfate taken up that is transported to the shoot, decreased with increasing sulfate supply. Apparently, net uptake of sulfate is not strictly controlled by the sulfur demand of the shoot, but xylem loading appears to counteract excess transport of sulfate to the shoot. Fumigation with H2S or SO2 reduced net uptake of sulfate by the roots in sulfur-deficient plants and absolute as well as relative sulfate transport to the shoot independent of the three sulfate levels supplied to the plant. At the same time thiol contents of the shoot and the root were enhanced by fumigation with H2S and SO2. These findings are consistent with the idea that thiols produced in the leaves can mediate demand-driven control of sulfate uptake by the roots and its transport to the shoot.

Sulfur nutrition of herbaceous plants is characterized by sulfate uptake and xylem loading of sulfate in the roots, assimilatory sulfate reduction in the leaves, and a cycling pool of both, sulfate and glutathione, the main long distance transport form of reduced sulfur. Also in spruce and beech sulfate aquisition by the roots, sulfate transport to, and assmiliatory sulfate reduction in the leaves are important processes required to fulfil the tree's needs for sulfur in growth and development. However, sulfate reduction for protein synthesis may take place in roots of beech as well as in roots and stems of spruce trees. In addition, storage of reduced sulfur and its mobilization is required in spruce and beech to support the early growth of the new flush. In spruce reduced sulfur in the form of glutathione is stored in the older needles during winter and is transported in phloem and xylem in acropetal direction to the new generation in spring. In beech glutathione exported from the leaves is removed from the phloem during basipetal transport; its sulfur seems to be stored as protein in the stem and apparently undergoes mobilization in spring during bud break. As a consequence, high amounts of cysteine are transported to the developing leaves in the xylem.

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Considerable amounts of low M(r) thiols were found in leaves, flowers and stems of the European mistletoe Viscum album (L). Leaf thiol contents were the highest so far reported for green tissue. Thiol composition was similar in all mistletoe organs analysed. Glutathione was the predominant low M(r) thiol of V. album independent of xylem sap thiol composition of the host. From the present findings it may be concluded that either cysteine removed from the xylem sap of the host is rapidly incorporated into glutathione in the endophytic system, or glutathione is selectively removed from the xylem sap of the host. The significance of de novo reduction of sulphate taken from the xylem sap of the host for the mistletoe's thiol composition and content remains to be elucidated.

Xylem sap of poplar trees was collected by vacuum extraction. Samples were taken year round and concentrations of thiols as well as amino acids were determined. Throughout the season glutathione was the predominant thiol in the xylem sap of poplar trees with concentrations ranging from 0. 1 to 13 myM Cysteine and gamma-glutamylcysteine concentrations were 0. 1 -4 and 0.01 -3 myM respectively. The glutathione content was subject to strong seasonal fluctuations. Highest amounts of glutathione (13 myM) were observed in March, immediately before flowering of the catkins. Like gamma-glutamylcysteine and cysteine, glutathione values reached further maxima at the onset of budbreak and leaf expansion in April and May. Also the amino acids investigated (glutamine, asparagine, glutamic acid, methionine, arginine) showed strong seasonal fluctuations in the xylem sap of poplar, with glutamine being the most abundant organic nitrogen compound (max. 13 mM). Highest amounts of amino acids were detect ed during April and May, the period of budbreak and leaf expansion, presumably owing to an increased degradation of storage protein. The sulfur-containing amino acid methionine was present in high amounts during the early growing season. It appeared to be the dominant reduced sulfur compound translocated in the xylem sap of poplar trees in April and May, whereas glutathione was the predominant organic sulfur compound in the xylem sap in March. The significance of storage and mobilization of reduced sulfur and organic nitrogen compounds for development of reproductive (catkins) and vegetative tissue (leaves and sprouts) in poplar trees is discussed.