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• Sulphur export and redistribution from the cotyledons of pea (Pisum sativum) seedlings was investigated to determine the role of cotyledons as a sulphur source during root–shoot axis development.
• Thiols and sulphate were analysed using standard biochemical techniques, and 35S fed to cotyledons by injection.
• After 35S-cysteine injection, c. 50% of the labelled S in the cotyledon was metabolized to 35S-sulphate. This reaction was partly inhibited by aminooxyacetic acid, an inhibitor of cysteine-desulfhydrase. After 35S-sulphate application, c. 1% of the radiolabel was found in cysteine and glutathione in the cotyledon. After 2 h, c. 20% of the 35S was transported into the root–shoot axis independently of whether 35S-sulphate or 35S-cysteine was injected into the cotyledon. After 4 h, 40% of 35S was found outside the cotyledon.
• Cotyledons of pea seedlings are capable of sulphate assimilation and cysteine degradation. Both sulphate and reduced sulphur were allocated from the cotyledons to the developing tissues of the pea seedlings.
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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.
Materials and Methods
Seeds of Pisum sativum L. cv. ‘Kleine Rheinländerin’ (Fellmann, Graz, Austria) were soaked in aerated water for 24 h and germinated in moist paper towels (Rauser et al., 1991). Uniform seedlings were selected 3 d later, and then transferred to hydroponic cultures and were grown under controlled environmental conditions, i.e. at 25°C, 65 ± 5% rh and under permanent illumination with 48–56 µmol m−2 s−1 PAR at plant level (HPS L65W/150 ‘ultrawhite’, L Fluora 35 W/77R, Osram, München, Germany). The roots were immersed in an aerated one-tenth strength complete nutrient solution (Hoagland & Arnon, 1950) containing a total of 260 µmol l−1 sulphate. At harvest on day 8, two leaves were completely expanded in addition to the cotyledons, the average main root length was 19.1 ± 2.7 cm and the shoot length was 5.4 ± 0.4 cm 33% of f. wt and 63% of d. wt of the seedlings was accounted for by the cotyledons (Table 1). The water content of the cotyledons amounted to c. 76%, the water content of the root-shoot-axis to 88–95%.
Table 1. Fresh and dry weight of 8-day-old-pea seedlings
F. wt [mg]
D. wt [mg]
Data are means ± SD of 10 seedlings.
First leaf developed (leaf 1)
129 ± 15
13 ± 2
Second developed leaf plus the apex (2)
116 ± 17
14 ± 2
147 ± 19
10 ± 1
475 ± 85
113 ± 21
556 ± 69
28 ± 2
HCl soluble low molecular weight thiols were determined fluorimetrically after reduction with DTE, derivatization with monobromobimane (mBBr) and separation by reversed-phase HPLC using a modification of the method of Schupp & Rennenberg (1988) and Rauser et al. (1991). Thiols were extracted from frozen tissues homogenized in 1 ml ice-cold 0.1 M HCl, 1 mM EDTA in small mortars on ice. Homogenates were transferred into 2 ml microfuge tubes and mortars as well as pestles were rinsed with 1 ml extraction solution. The combined extracts were centrifugated at 13 000 g at 4°C for 15 min. 500 µl of the supernatant was mixed with 150 µl of 500 mM 2-(N-Cyclohexylamino)ethanesulphonic acid (CHES-buffer, pH 10.25) to obtain a pH of 8.3 ± 0.2. After reduction of the thiols at room temperature for 60 min with 20 µl of 10 mM DTE, 20 µl of 30 mM mBBr in acetonitrile was added. After 15 min at dim light, conjugation of thiols with mBBr was completed and 60 µl of 0.5% (v/v) methanesulphonic acid was added to stabilize mBBr derivatives. After centrifugation at 13 000 g for 15 min 250 µl of the mixture was subjected to HPLC analysis (Shimadzu Corp., Kyoto, Japan).
Thiol derivatives were separated on a C-18 column (ODS-Hypersil 250 × 4.6 mm id, 5 µm particle size; Bischoff, Leonberg, Germany) at a flow rate of 1.5 ml min−1. A binary gradient from 0% solution B to 10% (v/v) solution B within 25 min was applied (Solution B: 0.25% (v/v) acetic acid, 90% (v/v) Methanol, pH 3.9). This composition was maintained for 2 min; thereafter the column was washed with 100% solution B for 5 min and re-equilibrated with 100% solution A (0.25% (v/v) acetic acid, 10% (v/v) methanol, pH 3.9) for 5 min. Thiol derivatives were quantified by fluorescence detection (Fluorimeter RF 530, excitation at 380, emission at 480 nm). A standard solution of 0.2 mM Cys, 0.1 mM γ-glutamylcysteine (γ-EC) and 1 mM GSH was used for quantification. Retention times were 4 min for sulphate (from radiolabel analysis), 9 min for Cys, 11 min for γ-EC, 16 min for GSH and 22 min for homoglutathione (hGSH). Recoveries for GSH, Cys, and γ-EC were 91.2 ± 11.4, 84.1 ± 9.5, and 87.8 ± 7.7%, respectively (n = 8; means ± SD).
For identification of the 35S-labelled thiols the eluate was collected (FRAC-100, Pharmacia, Sweden) at one minute intervals. Each fraction (1.5 ml) was mixed with 2.5 ml of a scintillation fluid (Quickszint FLOW 306; Zinsser Analytic, Frankfurt a.M., Germany) and radioactivity was determined by liquid scintillation counting.
Sulphate was determined in HCl extracts by anion exchange HPLC. 100µl of the extraction solution was injected by an autosampler (Marathon Autosampler, Spark Holland, Emmen, The Netherlands) and sulphate was seperated on an anion exchange column (Chrompack Ionosphere 5 A 250 × 4.6 mm id, Varian Inc., Middelburg, The Netherlands) coupled to a precolumn (Chrompack anion exchange 50 × 4.6 mm, Varian Inc., Middelburg, The Netherlands). Sulphate was eluted with 2 mM potassium hydrogenphthalate (pH 5.5) at a flow rate of 1.5 ml over 12 min. The column was heated to 40°C. Sulphate was detected with a conductivity detector (Model 5400, ESA, Chelmsford, MA, USA) and quantified by comparison with a 12.5–100 µl aliquot of a standard solution containing 833 µM sulphate (= 80 µg SO4 ml−1).
35S-labelled sulphate was quantified in the same way as described for thiols.
35S feeding experiments
Pea seedlings were transferred to the laboratory 1 h before the start of the experiments and were transplanted into test tubes with 20 ml fresh nutrient solution containing 260 µmol l−1 sulphate. 35S Cys or 35S sulphate was injected into the parenchyma of the cotyledon of 8-d-old-pea seedlings. For this purpose, a 3-mm deep channel was drilled with a pin into the centre of one of the cotyledons. This channel could absorb 1 µl without wetting the epidermis of the cotyledon. Either 1 µl of 100 µmol l−1 Cys (specific radioactivity 6.8–7.4 × 109 Bq mmol−1) or 1 µl carrier-free sulphate (specific radioactivity 1.6–2.1 × 109 Bq mmol−1; NEN DuPONT, Dreieich, Germany) was injected. When required 1 µl of 5 mM aminooxyacetic acid (AOA) was injected 1 h before the 35S-Cys injection. Distribution of the injected solutions within the cotyledon was uniform as demonstrated by the injection of 1 µl of blue ink as a test solution: 2 min after injection the storage tissue of the cotyledon was stained homogenously. Pea seedlings were harvested after 2 and 4 h of incubation with radiolabel at room temperature (25–30°C) and a PAR of 120 µmol m−2 s−1 at plant level. At the end of the experiment 400 µl of the nutrient solution was mixed with 2.5 ml of a scintillation fluid (Quicksave A, Zinsser Analytic, Frankfurt a.M., Germany) to determine the release of 35S by the roots. Appreciable radioactivity was not detected in the nutrient solution. The roots, the stem, each of the cotyledons, and the leaves (leaf 1: the first developed leaf; leaf 2: the second developed leaf plus the apex) were separated, weighed, frozen in liquid nitrogen, and stored at −25°C until analyses.
Determination of total 35S
Total acid-soluble low molecular weight 35S compounds were determined in all tissues. For this purpose the frozen tissues were extracted for thiol analysis (see above). The remaining pellets from the extractions were re-suspended three times with 1.5 ml 0.1 M HCl, shaken for 30 min and centrifuged. An aliquot of 400 µl of the combined supernatants were mixed with 2.5 ml scintillation fluid (Quicksave A, Zinsser Analytic, Frankfurt a.M., Germany). Less than 1% of the total soluble radioactivity was recovered in the fourth supernatant. Radioactivity was determined by liquid scintillation counting at an efficiency of approx. 90% (LS 3801, Beckmann, München, Germany).
Acid insoluble 35S was determined in the pellets remaining after HCl extraction. First the pellets were bleached with 200 µl 30% (v/v) H2O2 during drying at 50°C for 2 d. Subsequently, 1 ml tissue solubilizer (TS-1, Zinsser Analytic, Frankfurt a.M., Germany) was added and incubated overnight. The mixture was acidified with 50 µl glacial acetic acid and 10 ml scintillation fluid (Quickszint 501, Zinsser Analytic, Frankfurt a.M., Germany) were added. Radioactivity was determined by liquid scintillation counting and was corrected for quenching.
The data shown are means of three to six independent experiments for each treatment. Statistical analysis was done by the Mann–Whitney U-test.
Sulphur composition of pea tissues
An 8-d-old-pea seedling contained a total of 81 ± 19 nmol Cys, 13 ± 3 nmol γ-EC, 517 ± 121 nmol GSH, and 45 ± 11 nmol hGSH. On a f. wt basis the highest thiol concentrations were detected in the youngest developed leaf plus the apex and in the first developed leaf (Table 2). In the roots the thiol concentration was 4.7 times lower and in the cotyledons the total thiol concentration (499 ± 128 nmol g−1 f. wt) was half of that found in the newly developed leaves. The dominant low-molecular weight thiol in all tissues was glutathione (Table 2). Only in the roots was homoglutathione (hGSH) found in considerable amounts (approx. 50% of GSH). In the stem hGSH was found only in trace amounts (0.6% of the GSH content). Remarkably high Cys contents (88% of the plant’s total Cys content) were observed in the cotyledons, that is, cysteine concentration was 56% of GSH concentration in cotyledons. In all other tissues Cys contents were 8–21 times lower and were comparable to those found in tissues from other herbaceous plants (Herschbach & Rennenberg, 1994). In addition to reduced sulphur, sulphate was found in considerable amounts (Table 2). The ratio between inorganic and organic sulphur was lowest in the youngest leaf plus the apex and the cotyledons and highest in the roots.
Table 2. Concentrations of low molecular weight thiols [nmol g−1 f. wt] and sulphate [µmol g−1 f. wt] in different plant parts of 8-d-old-pea seedlings
Inorganic/organic sulphur ratio
Data are means ± SD of 27 seedlings.
First leaf developed
10.1 ± 2.3
6.2 ± 0.8
992.2 ± 170.5
17.18 ± 2.06
2.2 ± 0.6
21.6 ± 4.3
22.4 ± 4.3
1121 ± 172
14.85 ± 1.63
1.1 ± 0.7
leaf plus apex
8.2 ± 2.0
12.2 ± 2.7
326.5 ± 81.6
2.0 ± 0.7
3.10 ± 0.37
2.9 ± 1.0
174.4 ± 22.1
12.3 ± 2.9
312.0 ± 76.2
8.40 ± 2.03
1.4 ± 0.3
9.0 ± 2.3
4.5 ± 1.1
151.1 ± 34.2
80.9 ± 18.0
17.94 ± 0.47
4.8 ± 0.2
35S fed to a cotyledon: identification of the 35S labelled compounds in the cotyledon
35S was injected into one of the two cotyledons. In none of the experiments were considerable amounts of 35S radioactivity (above 0.05%) detected in the untreated cotyledon. That means the intercotyledon exchange of S was zero. 82% of the radioactivity was still found 2 h after the injection of 35S into the cotyledon. This amount decreased to between 61 and 69% after 4 h of incubation.
After injection of 35S-Cys c. 50% of the 35S in the cotyledon was found in 35S-sulphate, 17% remained as 35S-Cys and 5% was detected as 35S-GSH after 2 h of incubation (Table 3). After 4 h incubation the amount of 35S detected as sulphate further increased and the radiolabelled Cys pool further decreased. The distribution pattern of 35S between γ-EC, GSH and insoluble 35S sulphur was not significantly different after 4 h incubation. Thus, half of the 35S-Cys injected into the cotyledon was converted into 35S-sulphate. Aminooxyacetic acid (AOA), an inhibitor of the cysteine-desulfhydrase (Rennenberg & Filner, 1982) partially prevented sulphate formation when injected into the cotyledon 1 h before 35S-Cys injection (Table 3). In the AOA pretreated cotyledons only c. 30% and 40% of the radioactivity was identified as 35S-sulphate after 2 and 4 h, respectively.
Table 3. Identification of the 35S compounds in the cotyledon 2 or 4 h after [35S]Cys or [35S]sulphate injection into the cotyledon of 8-d-old-pea seedlings
Injection of [35S]Cys one hour after AOA preinjection
After 2 h
After 4 h
After 2 h
After 4 h
After 2 h
After 4 h
Data are percentages of the total 35S detected in the fed cotyledon and are means ± SD of 6 pea seedlings of at least two independent experiments. UT, not identified HCl soluble bimane-conjugated thiols appearing in the chromatogram. γ-EC and hGSH were not detectable in these experiments.
50.1 ± 12.5
58.8 ± 14.7
30.2 ± 7.6
40.7 ± 10.2
98.2 ± 24.6
98.4 ± 24.6
17.0 ± 2
10.9 ± 1.3
46.4 ± 5.5
30.6 ± 3.7
0.5 ± 0.1
0.4 ± 0.1
5.2 ± 1.3
7.1 ± 1.8
7.2 ± 1.8
11.1 ± 2.8
0.6 ± 0.2
0.7 ± 0.2
14.7 ± 3.8
12.4 ± 3.2
10.9 ± 2.8
10.3 ± 2.7
0.2 ± 0.1
0.2 ± 0.1
13.0 ± 3.1
10.8 ± 2.6
5.3 ± 1.3
7.3 ± 1.8
0.4 ± 0.1
0.2 ± 0.1
To study the fate of sulphate in the cotyledons 35S-sulphate was injected into one of the cotyledons. After 2 and 4 h, respectively, < 1% of the injected 35S-sulphate was reduced to 35S-Cys, and subsequently incorporated into 35S-GSH (Table 3).
35S feeding to the cotyledon: export from the cotyledon
Of the injected 35S 18% to 23% was exported out of the cotyledon after 2 h. After 4 h of incubation two times higher amounts of 35S were detected in the root-shoot axis. The main sink for the exported 35S was the youngest leaf including the apex. Approx. 58–75% of the exported 35S was detected in these organs (Fig. 1). The first developed leaf contained only 3–8% and the roots 8–14% of the exported 35S. This distribution pattern was independent of the incubation time and the 35S compound fed.
35S fed to the cotyledon: identification of the 35S in the root-shoot axis
The main proportion of the 35S in the tissues of the root-shoot axis was identified as sulphate (Figs 2, 3) independently of whether pea seedlings were harvested 2 or 4 h after 35S-Cys injection. Enhancing the incubation time from 2 to 4 h resulted in a decreased relative labelling of GSH in the roots and the first developed leaf (Fig. 3), whereas the relative labelling as 35S sulphate increased in all tissues (Fig. 3). The proportion of labelled 35S found as insoluble sulphur slightly decreased in the roots and the stem with increasing incubation time. The hGSH fraction in the stem and roots was labelled, but only in trace amounts.
Pre-incubation with AOA resulted in a decreased sulphate and an increased GSH labelling in all tissues of the root-shoot axis in experiments where 35S-Cys was fed to a cotyledon. In the roots a higher 35S labelling of hGSH was also found (Fig. 2).
When 35S-sulphate was injected into the cotyledon, the proportion of the 35S detected in the sulphate fraction was higher in the roots and in the first developed leaf by c. 85% as well as in the stem by c. 93% compared to the 35S-Cys injection. The proportion of radiolabelled sulphate in the youngest leaf was similar after 35S-Cys and 35S-sulphate injection. However, the relative proportion found as 35S-GSH was lower in all tissues when 35S-sulphate rather than 35S-Cys was injected. In the youngest developed leaf plus the apex the proportion of 35S found as sulphate was comparable to that found for the 35S-Cys injection (Fig. 3). The 35S labelling of unidentified soluble low molecular weight sulphur compounds was significantly higher after 35S-sulphate injection.
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.
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.
This work was supported by grants from the Austrian Science Foundation (Fonds zur Förderung der wissenschaftlichen Forschung).