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- Materials and Methods
The tripeptide glutathione (GSH) has been studied intensively regarding its significance for sulphur nutrition of plants (Rennenberg et al., 1979; Bonas et al., 1982; Herschbach & Rennenberg, 2001a). Many studies investigated transport, regulation, and compartmentation of sulphur metabolites at the cellular (Davidian et al., 2000) and at the whole plant level (Herschbach & Rennenberg, 2001a). However, these experiments investigated almost exclusively whole plant aspects in adult or young plants (Herschbach & Rennenberg, 1994; Herschbach et al., 1995a,b; Lappartient & Touraine, 1996; Lappartient et al., 1999). Only one study on seedlings analysed the role of storage tissues for sulphur nutrition during seedling development. This study demonstrated that sulphur from the endosperm of maize seeds was absorbed by the scutellum of the embryo and distributed within the seedling as GSH that was synthesized in the scutellum (Rauser et al., 1991).
During the germination of seeds, reserve compounds, for example starch or proteins, are mobilized and the metabolites, mostly sucrose and amino acids, are transported into the developing root-shoot axis to support early growth. Seeds of Fabaceae contain two major protein classes, globulins and albumins. During germination, the globulins are broken down rapidly to provide the major source of nitrogen (Higgins, 1984). The albumin fraction of pea cotyledons contains most of the enzymatic and metabolic proteins as well as the major sulphur-rich component (Jakubek & Przybylska, 1979). The albumin comprises only 4.5% of total pea seed protein, but contributes 23% of the seed’s sulphur amino acids (Schroeder, 1984). It is degraded within the first 8 d of germination (Higgins et al., 1986) and high amounts of S-containing amino acids become available. High contents of cysteine (Cys) were also found in the partly liquefied endosperm of 6 and 7 d old-maize seedlings (Rauser et al., 1991). Apparently, mobilization of Cys from storage proteins exceeds its transfer from the cotyledons to the axis. However, high contents of L-Cys do not reflect the normal metabolic situation of plant cells. Excess cysteine may be present in storage tissues due to imbalances between mobilization and export.
Pea seedlings exhibit distinct morphological differences compared to maize seedlings. The storage tissue in the cotyledons of pea is directly connected to the developing seedling whereas the scutellum connects the maize embryo with the storage tissue of the seed. Pea cotyledons contain the enzymes of the assimilatory sulphate reduction pathway (Brunold & Suter, 1989) and glutathione synthesis (Schlunz, 1991). Still, it is unknown whether cotyledons of pea seedlings can use Cys to synthesize GSH for reduced-sulphur transport into the developing axis in a similar way observed for the endosperm and the scutellum of maize seedlings. The present study was undertaken to characterize the role of pea cotyledons as sulphur source for the developing root-shoot axis. For this purpose 35S labelled sulphur was injected as Cys or sulphate into the centre of the storage tissue of one of the two cotyledons and the partitioning of radiolabelled sulphur was analysed in all parts of the seedlings.
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- Materials and Methods
The present results show that in pea cotyledons Cys from the storage proteins can be oxidized to sulphate, which can then be transported to the developing seedling. Similarly, in maize seedlings after 6 h incubation 66% of the 35S label injected as Cys into the endosperm was found as sulphate, 30% as Cys and 4% as GSH (Rauser et al., 1991). None of the 35S-Cys stock solution was oxidized to sulphate within 8 h at room temperature (Rauser et al., 1991). Therefore, free sulphide must have been produced by Cys degradation through cysteine-desulfhydrase yielding sulphide. This product was oxidized via sulfite to sulphate (Sekiya et al., 1982). Aminooxyacetic acid (AOA), an inhibitor of the cysteine-desulfhydrase (Rennenberg & Filner, 1982), partially prevented the formation of sulphate from 35S-Cys in the present study and therefore supports the hypothesis of a cysteine-desulfhydrase catalysed Cys degradation. Although pea cotyledons contain the enzymes of the sulphate assimilatory reduction pathway (Brunold & Suter, 1989) and synthesize GSH (Schlunz, 1991), the amount of sulphate reduced was minute. Only < 2% of the label remaining in the cotyledons was recovered in reduced S compounds, but > 30% of the injected label was already exported to the shoot-root axis. This points to sulphate itself as an important sulphur transport form for the export from the cotyledons.
This finding contradicts observations on maize seedlings that have a completely different seed structure (Rauser et al., 1991). By contrast to pea seeds where the storage tissues of the cotyledons are directly connected to the developing seedling by the vascular system, the maize embryo is separated from the endosperm which is the storage tissue for proteins and starch by the scutellum. Both starch and protein degradation in the endosperm need the secretion of hydrolases and proteinases by cells of the scutellar epithelium and the aleurone layer. The metabolites are then absorbed by the scutellum and distributed within the developing embryo via phloem transport afterwards. 35S-Cys injected into the endosperm of maize seeds was used for GSH synthesis in the scutellum (Rauser et al., 1991). Cys and sulphate were labelled in the scutellum, but GSH was the only radiolabelled sulphur compound in the roots and the major labelled compound in the shoot. This indicates that GSH synthesized in the scutellum is the dominant sulphur compound transported from storage tissues after degradation of the stored material into the developing root-shoot axis of maize seedlings. Apparently, the evolution of certain morphological seed structures is only economic with specific types of sulphur metabolism and transport.
From the present experiments it cannot be distinguished whether the reduced sulphur compounds determined in the target organs originate from sulphur reduction therein or from reduced sulphur exported from the cotyledon. It can only be speculated which sulphur compound was transported out of the cotyledons after 35S-Cys or 35S-sulphate injection into the root-shoot axis. Nevertheless, the proportion of labelled sulphate in different pea parts, that is the roots, the stem, the first developed and the second developed leaf including the apex (Fig. 3), is correlated with the sulphate content in the cotyledon (Table 3). Such a close correlation, in particluar between the cotyledon and the stem, corroborates the role of sulphate as S transport form.
Since the roots are the only part of pea plants where homoglutathione (hGSH) is synthesized by a hGSH synthetase (Schlunz, 1991) and labelled hGSH was found in considerable amounts, it is clear that sulphate reduction and assimilation of the sulphate transported from the cotyledons into different parts of the plant takes place in the sink organ. 35S exported from the cotyledons was also incorporated into insoluble material for growth and development, chiefly into proteins. From the label in these compounds (a maximum of approx. 30% of the total label in roots) the magnitude of synthesis may be estimated. The percentage of radiolabel incorporated in reduced sulphur is greater after Cys was fed to the cotyledon than after 35S-sulphate supply, which points to a contribution of reduced sulphur compounds to the S-transport from cotyledons (Fig. 3). Transport of reduced sulphur compounds is further corroborated by the fact that preincubation with AOA enhanced the availability of reduced sulphur in the cotyledons and also increased the percentage of the 35S incorporated into insoluble sulphur compounds in target organs.
Several studies with a variety of plants showed that sulphur is transported in the phloem (Rennenberg et al., 1979; Bonas et al., 1982; Smith & Lang, 1988; Larsson et al., 1991; Lappartient & Touraine, 1996; Herschbach et al., 1998; Herschbach & Rennenberg, 2001b) as well as in the xylem (Schupp et al., 1991; Köstner et al., 1998; Herschbach et al., 2000) in the form of inorganic or organic, reduced sulphur. As also found in soybeans (Smith & Lang, 1988; Adiputra & Anderson, 1992) the youngest pea leaf plus the apex was the main sink for the radiolabelled sulphur from the cotyledon. In soybean, the cotyledons supplied the first and the second developed leaf with sulphur, but the proportion of sulphur supplied by the nutrient solution increased from the first to the third developed leaf (Sunarpi & Anderson, 1995). Sulphate uptake experiments on pea seedlings demonstrated that a signal from the cotyledons, possibly O-acetylserine, stimulates sulphate uptake (C. Herschbach et al., unpublished). Thus, in addition to the S-storage function, the cotyledons contribute to the sulphur budget for growth and development of young, 8-d-old-pea seedlings by their regulation function.
Although the transported compounds could not be determined directly in the vascular system, the present data strongly support the possibility of both sulphate and reduced sulphur export from pea cotyledons.