Lemna extractions

Increased the sensitivity of this bloassay to 0.5 nM concentrations of allelochemlcals. The Lemna assays were also useful for determining bloactlve fractions extracted from allelopathic plants. 

Other kinds of bloassays have been used to detect the presence of specific allelochemical effects (8), effects on N2 fIxatlon (9), the presence of volatile compounds (10) and of Inhibitory substances produced by marine microalgae (11). Putnam and Duke (12) have summarized the extraction techniques and bioassay methods used In allelopathy research. Recent developments In high performance liquid chromatography (HPLC) separation of allelochemlcals from plant extracts dictates the need for bloassays with sensitivity to low concentrations of compounds contained In small volumes of eluent. Einhellig at al. (13) described a bloassay using Lemna minor L. growing In tissue culture cluster dish wells that maximizes sensitivity and minimizes sample requirements. 

Lemna obscura fronds contain high quantities of anthocyanln that are extractable by soaking the fronds In 0.1 M HC1. L. obscura growth in the culture dish bloassay was similar to that of L. minor and appeared to be more sensitive to low levels of allelochemlcals. Anthocyanln concentration was affected by low concentrations of salicylic acid (Table VIII). Final frond number and dry weight were consistently reduced by 100 pM and 500 pM concentrations of salicylic acid whereas anthocyanln formation was inhibited by 50 UM concentrations of salicylic acid and stimulated by 0.5 VM. 

Three bioassays a) 8d growth rate aquatic plant test (Lemna minor) b) 72h germination plant test (Lepidium sativum) and 8d root elongation plant test (Lepidium sativum) c) 72h germination plant test (Brassica rapa) and 8d root elongation plant test (Brassica rapa) Different industrial solid waste leachates Saline extraction... 

The importance of zinc for a normal functioning of the Cu-Zn-SOD was shown in Lemna gibba. In zinc-deficient culture media the activity of Cu-Zn-SOD was strongly inhibited whereas in copper-deficient media little change was found in the enzyme activity (Vaughan et al., 1982). In extracts of zinc-deficient plants, restoration of enzyme activity was possible by supplying zinc to the enzyme assay medium. 

Smith et al. (1989, 1991) exposed Lemna minor to 5lCr(VI) in the form of chromate and demonstrated that extracts showed Cr associated with complexes of about 2000 D and > 100000 D. Aqueous extracts were subjected to gel permeation chromatography (Sephadex G50) and both 51Cr and protein activity were followed. Although the Cr fractions corresponded to protein fractions, the authors were unable to demonstrate that these were Cr-induced proteins, since the protein profiles for Cr-treated and untreated plants were the same. Clearly more work is required on the chemical identification of metal complexes in such extracts. 

Cysteine has a strong inhibitory effect on the level of extractable activity of APS sulfotransferase in Lemna (Brunold and Schmidt, 1978) and Phaseolus... 

The level of sulfotransferase in Lemna and in Phaseolus vulgaris is also subject to strong inhibition by gaseousH2S(Brunoldand Schmidt, 1976,1978 Wyss and Brunold, 1979). However, the extractable acti vity of cysteine synthase is not similarly affected. Removal of H2S firom the gas phase results in rapid restoration of activity which, based on a study of labeling of the enzyme (von Arb and Brunold, 1980), was attributed to synthesis ofthe enzyme de novo. HjS also inhibits the level of APS sulfotransferase in cell suspension cultures of Nicotiana sylvestris in this tissue neither the ATP-sulfiirylase or cysteine synthase activity was affected by H2S or cysteine (Brunold etal., 9Sl). Importantly, the inhibition of APS sulfotransferase by H2S was correlated with an enhanced level of cysteine, suggesting that the H2S inhibition could have been mediated via this reaction product. Uptake of exogenous sulfate was also inhibited by H2S in this system (Brunold et al., 1981). 

In tobacco cell cultures the extractable levels of ATP-sulfurylase and cysteine synthase are very low when the cells are subject to nitrogen stress but increase rapidly upon alleviation of the stress, suggesting that a product of nitrogen assimilation derepresses the levels of these enzymes. In Lemna (and possibly in cultured Rosa cells) it appears that this role is fulfilled by APS sulfotransferase and that ATP-sulfurylase and cysteine play unimportant roles in coordinating the sulfate assimilation pathway with the nitrate assimilation pathway. A further regulatory mechanism known to occur in cultured tobacco cells is that excessively high concentrations of cysteine induce the synthesis of cysteine desulfiiydrase (see Section VI). 

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