Experimental evidence for diel δ15N-patterns in different tissues, xylem and phloem saps of castor bean (Ricinus communis L.).

Nitrogen isotope signatures in plants might give insights in the metabolism and allocation of nitrogen. To obtain a deeper understanding of the modifications of the nitrogen isotope signatures, we determined δ(15)N in transport saps and in different fractions of leaves, axes and roots during a diel course along the plant axis. The most significant diel variations were observed in xylem and phloem saps where δ(15)N was significantly higher during the day compared with during the night. However in xylem saps, this was observed only in the canopy, but not at the hypocotyl positions. In the canopy, δ(15)N was correlated fairly well between phloem and xylem saps. These variations in δ(15)N in transport saps can be attributed to nitrate reduction in leaves during the photoperiod as well as to (15)N-enriched glutamine acting as transport form of N. δ(15)N of the water soluble fraction of roots and leaves partially affected δ(15)N of phloem and xylems saps. δ(15)N patterns are likely the result of a complex set of interactions and N-fluxes between plant organs. Furthermore, the natural nitrogen isotope abundance in plant tissue is not constant during the diel course - a fact that needs to be taken into account when sampling for isotopic studies.

Methods for xylem sap collection.

Xylem and phloem are essential for the exchange of solutes and signals among organs of land plants. The synergy of both enables the transport and ultimately the partitioning of water, nutrients, metabolic products and signals among the organs of plants. The collection and analysis of xylem sap allow at least qualitative assumptions about bulk transport in the transpiration stream. For quantification of element-, ion-, and compound-flow, the additional estimation of volume flow is necessary. In this chapter we describe methods for collecting xylem sap by (1) root pressure exudate, (2) Scholander-Hammel pressure vessel, (3) root pressurizing method according to Passioura, and (4) (hand/battery) vacuum pump.

Over-expression of gsh1 in the cytosol affects the photosynthetic apparatus and improves the performance of transgenic poplars on heavy metal-contaminated soil.

Recent studies of transgenic poplars over-expressing the genes gsh1 and gsh2 encoding γ-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase, respectively, provided detailed information on regulation of GSH synthesis, enzymes activities and mRNA expression. In this experiment, we studied quantitative parameters of leaves, assimilating tissues, cells and chloroplasts, mesophyll resistance for CO(2) diffusion, chlorophyll and carbohydrate content in wild-type poplar and transgenic plants over-expressing gsh1 in the cytosol after 3 years of growth in relatively clean (control) or heavy metal-contaminated soil in the field. Over-expression of gsh1 in the cytosol led to a twofold increase of intrafoliar GSH concentration and influenced the photosynthetic apparatus at different levels of organisation, i.e., leaves, photosynthetic cells and chloroplasts. At the control site, transgenic poplars had a twofold smaller total leaf area per plant and a 1.6-fold leaf area per leaf compared to wild-type controls. Annual aboveground biomass gain was reduced by 50% in the transgenic plants. The reduction of leaf area of the transformants was accompanied by a significant decline in total cell number per leaf, indicating suppression of cell division. Over-expression of γ-ECS in the cytosol also caused changes in mesophyll structure, i.e., a 20% decrease in cell and chloroplast number per leaf area, but also an enhanced volume share of chloroplasts and intercellular airspaces in the leaves. Transgenic and wild poplars did not exhibit differences in chlorophyll and carotenoid content of leaves, but transformants had 1.3-fold fewer soluble carbohydrates. Cultivation on contaminated soil caused a reduction of palisade cell volume and chloroplast number, both per cell and leaf area, in wild-type plants but not in transformants. Biomass accumulation of wild-type poplars decreased in contaminated soil by more than 30-fold, whereas transformants showed a twofold decrease compared to the control site. Thus, poplars over-expressing γ-ECS in the cytosol were more tolerant to heavy metal stress under field conditions than wild-type plants according to the parameters analysed. Correlation analysis revealed strong dependence of cell number per leaf area unit, chloroplast parameters and mesophyll resistance with the GSH level in poplar leaves.

Chloroplast parameters differ in wild type and transgenic poplars overexpressing gsh1 in the cytosol.

Poplar mutants overexpressing the bacterial genes gsh1 or gsh2 encoding the enzymes of glutathione biosynthesis are among the best-characterised transgenic plants. However, this characterisation originates exclusively from laboratory studies, and the performance of these mutants under field conditions is largely unknown. Here, we report a field experiment in which the wild-type poplar hybrid Populus tremula x P. alba and a transgenic line overexpressing the bacterial gene gsh1 encoding gamma-glutamylcysteine synthetase in the cytosol were grown for 3 years at a relatively clean (control) field site and a field site contaminated with heavy metals. Aboveground biomass accumulation was slightly smaller in transgenic compared to wild-type plants; soil contamination significantly decreased biomass accumulation in both wild-type and transgenic plants by more than 40%. Chloroplasts parameters, i.e., maximal diameter, projection area and perimeter, surface area and volume, surface/volume ratio and a two-dimensional form coefficient, were found to depend on plant type, leaf tissue and soil contamination. The greatest differences between wild and transgenic poplars were observed at the control site. Under these conditions, chloroplast sizes in palisade tissue of transgenic poplar significantly exceeded those of the wild type. In contrast to the wild type, palisade chloroplast volume exceeded that of spongy chloroplasts in transgenic poplars at both field sites. Chlorophyll content per chloroplast was the same in wild and transgenic poplars. Apparently, the increase in chloroplast volume was not connected to changes in the photosynthetic centres. Chloroplasts of transgenic poplar at the control site were more elongated in palisade cells and close to spherical in spongy mesophyll chloroplasts. At the contaminated site, palisade and spongy cell chloroplasts of leaves from transgenic trees and the wild type were the same shape. Transgenic poplars also had a smaller chloroplast surface/volume ratio, both at the control and the contaminated site. Chloroplast number per cell did not differ between wild and transgenic poplars at the control site. Soil contamination led to suppression of chloroplast replication in wild-type plants. From these results, we assume that overexpressing the bacterial gsh1 gene in the cytosol interacts with processes in the chloroplast and that sequestration of heavy metal phytochelatin complexes into the vacuole may partially counteract this interaction in plants grown at heavy metal-contaminated field sites. Further experiments are required to test these assumptions.

The effect of drought on C and N stable isotopes in different fractions of leaves, stems and roots of sensitive and tolerant beech ecotypes.

Beech seedlings from 11 German climatic provenances were exposed to a realistically timed drought treatment in a greenhouse experiment. The stable isotope composition of carbon (C) and nitrogen (N) was analysed in pooled bulk material of roots, stems and leaves, as well as in the aqueous extracts and starch fractions. The delta 13C values increased in bulk samples (BS) of roots, stems and leaves by drought, although no leaf growth occurred during the experimental period. A clear drought effect on delta 13C in aqueous extracts was detected in leaves. In aqueous extracts of stems and roots as well as in starch fractions of all organs, abundance of delta 13C also tended to be increased by drought, but this effect was not statistically significant. For both delta 13C and delta 15N, enrichment was observed from the site of uptake/ source to the site of use/sink. A gradient for delta 13C in all fractions from leaves (-29.49, -28.89 and -27.85 per thousand) to stems (-28.81, -27.48 and -26.98 per thousand) and to roots (-27.60, -26.37 and -26.48 per thousand) was detected in BS, aqueous extracts and starch, respectively. An opposite gradient for delta 15N was found in BS: 1.59 per thousand, 1.84 per thousand and 3.05 per thousand in roots, stems and leaves, respectively. delta 15N was neither affected by drought in the BS nor in aqueous extracts, but an effect of provenance was observed. Particularly in roots and stems, drought-sensitive provenances showed the strongest shifts in delta 13C induced by drought and the lowest delta 15N values. In the present experiment, delta 13C values were more affected by the environmental factor drought, while delta 15N values were more affected by the genetic factor provenance.

Identification of drought-sensitive beech ecotypes by physiological parameters.

• The effects of drought on European beech (Fagus sylvatica) were assessed in a pot experiment under controlled conditions. • Plants from 11 autochthonous provenances originating from regions in Germany, which differed in annual precipitation, were exposed to a 3-wk drought period in a glasshouse after the first stage of shoot growth had been completed. • Drought reduced the water content to 97% of control in leaves and axes and to 92% in the roots. A strong reduction of predawn water potential in roots and shoots, as well as on transpiration rate, was found. In the roots, the effect on water potential was the same for all provenances, but differences were observed in the shoot water potential. Leaf concentrations of abscisic acid (ABA), proline and sucrose increased in the drought-treated plants compared with the controls. • Two extreme clusters from opposite climatic sites were identified by cluster analysis. A drought-sensitive cluster, originating from regions with high annual precipitation, had low water potential and transpiration rates, as well as high concentrations of fructose, ABA and proline after drought. Water potential and transpiration rates were less affected by drought in the other cluster, which comprised two provenances of relatively dry habitats, and concentrations of hexose, ABA and proline were low.

Dynamic studies of phloem and xylem flow in fully differentiated plants by fast nuclear-magnetic-resonance microimaging.

A fast nuclear-magnetic-resonance imaging method was developed in order to measure simultaneously and quantitatively the water flow velocities in the xylem and the phloem of intact and transpiring plants. Due to technical improvements a temporal resolution of 7 min could be reached and flow measurements could be performed over a time course of 12-30 h. The novel method was applied to the hypocotyl of 35- to 40-day-old, leafy plants of Ricinus communis which were subjected to different light-dark regimes. The results showed that the xylem flow velocities and the xylem volume flow responded immediately to light on-off changes. Upon illumination the flow velocity and the volume flow increased as expected in respect to literature. In contrast, the phloem flow velocity did not change in response to the light-dark regimes. Interestingly, though, the volume flow in the phloem increased during darkness. These findings can be explained by assuming that the conducting area of the phloem becomes enlarged during the dark period due to opening of sieve pores.

The effects of SO2 fumigation on the nitrogen metabolism of aseptically grown spruce seedlings.

Aseptically grown spruce seedlings were cultivated in a hydroponic system, where the roots were separated from the shoots by a gastight, silicone material. The plants were fumigated with four SO(2) concentrations (93, 190, 270 and 530 microg m(-3)) for nine weeks. Up to 270 microg m(-3) of SO(2), an inhibition of nitrogen metabolism (enzyme activities of nitrate reductase (NR) and glutamine sythetase (GS) and nitrate content) in the shoot was compensated by a stimulation in the root, while nitrogen uptake was unaffected. Only the treatment with 530 microg m(-3) of SO(3) decreased enzyme activities, nitrate content in both roots and shoots as well as nitrate uptake, and inhibited the growth of plants. Increases in the content of thiols and superoxidismutase activity are discussed in terms of SO(2) detoxification.