Interaction of heavy metals with the sulphur metabolism in angiosperms from an ecological point of view.

The metabolism of sulphur in angiosperms is reviewed under the aspect of exposure to ecologically relevant concentrations of sulphur, heavy metals and metalloids. Because of the inconsistent use of the term 'metal tolerance', in this review the degree of tolerance to arsenic and heavy metals is divided into three categories: hypotolerance, basal tolerance and hypertolerance. The composition of nutrient solutions applied to physiological experiments let see that the well-known interactions of calcium, sulphate and zinc supply with uptake of heavy metals, especially cadmium are insufficiently considered. Expression of genes involved in reductive sulphate assimilation pathway and enzyme activities are stimulated by cadmium and partially by copper, but nearly not by other heavy metals. The synthesis of the sulphur-rich compounds glucosinolates, metallothioneins and phytochelatins is affected in a metal-specific way. Phytochelatin levels are low in all metal(loid)-hypertolerant plant species growing in the natural environment on metal(loid)-enriched soils. If laboratory experiments mimic the natural environments, especially high Zn/Cd ratios and good sulphur supply, and chemical analyses are extended to more mineral elements than the single metal(loid) under investigation, a better understanding of the impact of metal(loid)s on the sulphur metabolism can be achieved.

Changed isoflavone levels in red clover (Trifolium pratense L.) leaves with disturbed root nodulation in response to waterlogging.

The effect of disturbed root nodulation on the quantitative and qualitative composition of the main isoflavonoid glucoside malonates, glucosides, and aglycones in the leaves of Trifolium pratense L. grown under waterlogging conditions was investigated. Isoflavonoids are involved in the regulation of root nodule activity and the establishment of the mycorrhizal association. Isoflavonoid determination was performed using reversed-phase liquid chromatography coupled to mass spectrometric and UV absorbance detection. In response to waterlogging, the concentrations of biochanin A and biochanin A-7-O-glucoside malonate, biochanin A-7-O-glucoside, and genistein-7-O-glucoside in the leaves increased two- to threefold after a lag period of 3 wk because of disturbed root nodulation. The other isoflavones detected formononetin, formononetin-7-O-glucoside malonate, and formononetin-7-O-glucoside-did not show any significant changes related to waterlogging. After restoring normal soil water conditions, the concentrations of biochanin A and its glucoside and glucoside-malonate rapidly returned to the initial values, whereas the concentration of genistein-7-O-glucoside remained high.

Extraction of isoflavone malonylglucosides from Trifolium pratense L.

Extraction of isoflavone malonylglucosides from red clover (Trifolium pratense L.) is a complicated procedure. This is due to the relatively unstable character of the thermolabile glucoside malonates as well as by action of native beta-glucosidases, resulting in a rapid degradation of malonylated glucosides into their corresponding aglucones. In this study, Tris was identified as a suitable beta-glucosidase inhibitor in red clover extracts, optimized at 350 mM Tris in 80% ethanol at pH 7.2. Extraction of fresh red clover leaves using Tris increased the concentration of malonate conjugated isoflavones approximately 13 to 24 times as opposed to extraction without Tris. A comparison of isoflavone profiles obtained after extraction with and without Tris of different plant organs of red clover and several species within the family Fabaceae suggests that the amount and/or activity of the degenerative beta-glucosidase enzymes vary for the different plant parts of red clover and among the species studied. Therefore, the use of standard extraction methods may well result in overestimation of the concentration of aglucones and consequently underestimation of the malonylglucoside isoflavones concentration depending on the plant species and plant part studied.

Differential metal-specific tolerance and accumulation patterns among Thlaspi caerulescens populations originating from different soil types.

• Here, Thlaspi caerulescens populations from contrasting soil types (serpentine, calamine and nonmetalliferous) were characterized with regard to tolerance, uptake and translocation of zinc (Zn), cadmium (Cd) and nickel (Ni) in hydroponic culture. • Results showed that high-level tolerances were apparently metal-specific and confined to the metals that were enriched at toxic levels in the soil at the population site. • With regard to metal accumulation, results suggested that, unlike Zn hyperaccumulation, Cd and Ni hyperaccumulation were not constitutive at the species level in T. caerulescens. • In general, the populations under study exhibited a pronounced uncorrelated and metal-specific variation in uptake, root to shoot translocation, and tolerance of Zn, Cd and Ni. The distinct intraspecific variation of these characters provides excellent opportunities for further genetic and physiological dissection of the hyperaccumulation trait.

A cosegregation analysis of zinc (Zn) accumulation and Zn tolerance in the Zn hyperaccumulator Thlaspi caerulescens.

• To analyse the relation between zinc (Zn) accumulation and Zn tolerance in the Zn hyperaccumulator, Thlaspi caerulescens, a cross was made between a plant from a nonmetallicolous population (LE: high accumulation, low tolerance) and one from a calamine population (LC: low accumulation, high tolerance). • More or less homogeneous F3 lines with contrasting extreme accumulation phenotypes were selected and phenotyped for tolerance, using the threshold exposure level for chlorosis as a tolerance measure. Zn accumulation and tolerance segregated largely independently, although there was a significant degree of association between low accumulation and high tolerance. • Plants from an F2 family were phenotyped for Zn tolerance and their Zn accumulation rates were compared. The plants with low Zn tolerance exhibited significantly higher Zn accumulation than did the more tolerant plants. • The results suggest that the superior Zn tolerance in LC plants compared with LE plants results from a superior plant-internal Zn sequestration capacity and, although to a lower degree, a reduced rate of Zn accumulation. It is argued that the relatively low Zn accumulation capacity levels found in LC and several other calamine T. caerulescens populations might represent an adaptive response to Zn-toxic soil.

Mechanisms of arsenate tolerance in Cytisus striatus.

• Arsenate tolerance, uptake and arsenate-induced phytochelatin (PC) accumulation were compared at different phosphorus supply rates in two populations of the broom, Cytisus striatus , one from an arsenic-enriched gold mine and one from a nonmetalliferous site. • After 7 d of exposure, arsenate tolerance was higher in the mine population. Arsenate uptake was phosphate-suppressible, and much lower in the mine plants. When compared at equal levels of stress, the mine plants and the nonmetallicolous plants exhibited similar arsenic accumulation, suggesting that reduced arsenate uptake is mainly responsible for superior tolerance. • Arsenate-induced PC accumulation occurred in both plant types. The γ-glutamylcysteine synthetase inhibitor, L-buthioninesulfoximine, caused arsenate hypersensitivity in both plant types, suggesting that PC-based arsenic sequestration is essential for both normal and enhanced arsenate tolerance. Mine plants produced longer PCs than the nonmetallicolous plants, possibly due to a differential temporal pattern of arsenate accumulation. • Our results are consistent with a similar mechanism underlying arsenate hypertolerance in C. striatus and grasses, that is reduced arsenate uptake through suppression of phosphate transporter activity.

Enhanced ATP-dependent copper efflux across the root cell plasma membrane in copper-tolerant Silene vulgaris.

We studied copper uptake in inside-out plasma membrane vesicles derived from roots of copper-sensitive, moderately copper-tolerant and highly copper-tolerant populations of Silene vulgaris (Amsterdam, Marsberg and Imsbach, respectively). Plasma membrane vesicles were isolated using the two-phase partitioning method and copper efflux was measured using direct filtration experiments. Vesicles derived from Imsbach plants accumulated two and three times more copper than those derived from Marsberg and Amsterdam plants, respectively. This accumulation was ATP-dependent. Also, 9-amino-6-chloro-2-methoxyacridine fluorescence quenching rates upon copper addition decreased in the order Imsbach>Marsberg>Amsterdam. Our results support the hypothesis that efflux of copper across the root plasma membrane plays a role in the copper tolerance mechanism in S. vulgaris.