Journal of Experimental Botany, Vol. 52, No. 360, pp. 1519-1526,
July 1, 2001
© 2001 Oxford University Press
Original Papers |
Sulphur nutrition affects delivery and metabolism of S in developing endosperms of wheat
1 School of Botany, La Trobe University, Bundoora 3083, Victoria, Australia
2 Institute of Sustainable Irrigated Agriculture, Agriculture Victoria, Tatura 3616, Victoria, Australia
Received 12 December 2000; Accepted 15 March 2001
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Abstract |
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Experiments were conducted to investigate the effect of S nutrition and availability on the forms of S and N in the endosperm cavity and endosperm of wheat, and on the capacity of the endosperm to utilize those compounds for the synthesis of proteins. Plants were grown in solution culture with 2 mM N and either 200 µM S (high-S) or 50 µM S (low-S) and all nutrients were withdrawn at various times from booting until 8 d post-anthesis. Sulphate was the major form of soluble S in the endosperm cavity and endosperm of high-S plants during the time of rapid grain development. By contrast, glutathione (GSH) was the major form of soluble S in the endosperm cavity and in the endosperm in low-S plants. Crude extracts of endosperm tissue from both high-S and low-S plants supported (i) the hydrolysis of GSH to
-glutamyl cysteine and glycine, and of -glutamyl cysteine to glutamate and cysteine, and (ii) sulphate-dependent PPi-ATP exchange and the sulphydration of O-acetylserine catalysed by ATP sulphurylase and cysteine synthase, respectively. High-S nutrition enhanced the in vitro rates of ATP sulphurylase and cysteine synthase. Key words: Endosperm cavity, endosperm, glutathione, methionine, sulphate, sulphur metabolism, wheat grains.
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Introduction |
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The developing grains of generative wheat plants have a substantial demand for S for the synthesis of storage proteins. The transport of N and S to the endosperm of developing grains involves several complete exchanges of material between tissues, each site offering the opportunity for selective transfer of metabolites. Firstly, material travelling in the xylem must be transferred into the phloem. This transfer probably occurs at the base of the rachilla, where there is an apoplastic discontinuity (Wang et al., 1994
). Secondly, material travelling in the crease vascular bundle must be transferred from the phloem in the maternal generation to the apoplastic endosperm cavity in the filial generation (Ugalde and Jenner, 1990a). Recruitment of material from the endosperm cavity into the endosperm, in theory, provides yet another site for selective transfer. Passage through the endosperm cavity is the only way that N and C reach the endosperm (Ugalde and Jenner, 1990a; Wang et al., 1994), and it seems likely that the transport of other compounds, like S, follows the same path.
When plants receive adequate S and N during vegetative growth, they develop substantial reserves of sulphate in the root and glutathione (GSH) in the leaves (Fitzgerald et al., 1999b). In the absence of exogenous sources of N and S during generative growth, these reserves can be sufficient to supply the S demand of the developing grains (Fitzgerald et al., 1999a). If, however, S supply during vegetative growth is insufficient to support the build-up of these reserves, then S in leaf protein becomes the dominant source of S for grain protein (Fitzgerald et al., 1999b).
Here, several hypotheses that stem from these observations were tested: (i) that sulphate and glutathione are the main forms of S delivered to the endosperm cavity; (ii) that S nutrition affects the forms of S delivered to the endosperm; and (iii) that the endosperm can take up sulphate and glutathione and utilize this S for the synthesis of cysteine and methionine for incorporation into grain proteins. It is reported here that sulphate is the major form of S delivered to the endosperm cavity and endosperm in plants grown in S-rich conditions but as plants receive less S, GSH (derived from the hydrolysis of leaf protein) is the predominant form of S delivered to the endosperm cavity and endosperm. In addition, endosperm appears to contain enzymes that support sulphate assimilation and GSH catabolism at rates consistent with a role in the incorporation of sulphate-S and GSH-S into grain proteins.
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Materials and methods |
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Growth and harvest of material
Experiment 1:
This experiment was designed to try to simulate nutrient availability during generative growth in conditions of limited water availability during grain-filling. When water availability is limited, exogenous nutrients are not available, so the plant must redistribute endogenous nutrients accumulated earlier. However, it is possible that other factors could promote redistribution when nutrients are removed in the presence of water. The aim of this experiment was to determine the effect of S nutrition on the forms of S redistributed from vegetative tissues to the endosperm cavity, and to the endosperm. Triticum æstivum cv. Condor, was grown in liquid culture and the supply of all nutrients in the culture was terminated at four different stages of generative growth: booting, ear emergence, anthesis, and 8 days post-anthesis (dpa). Thus, in all cases the requirements of the generative sink could be met only by endogenous nutrients. Two concentrations of S nutrition were used: high-S (200 mM S and a molar ratio of N : S of 10) and low-S (40 mM S and a molar ratio of N : S of 50). All growth conditions for Experiment 1 are as described earlier (Fitzgerald et al., 1999
a).
Three plants were harvested at 13 dpa to correspond with the start of grain-filling, and another three plants were harvested at 25 dpa, which was midway through grain-filling (details are in Fitzgerald et al., 1999a). Endosperms (10) were collected from three plants from each nutritional treatment, but the fluid of the endosperm cavity was collected from 10 endosperms of each plant from which nutrients were withdrawn at anthesis. The endosperms were peeled by carefully removing the inner and outer pericarps and the small strip of pericarp containing the crease vascular bundle. After the endosperms were completely peeled, taking care to preserve the aleurone layer, the fluid of the endosperm cavity was collected by gently delivering deionized water (20 µl) from a syringe into the endosperm cavity, then withdrawing the water, and the contents of the cavity, into the syringe. For each endosperm, the process was repeated five times. The syringe was rinsed with water (20 µl) between each flush, and the rinses added to the same vial. Fluid from 10 endosperms from each plant was collected this way, and for each plant, the fluid from the 10 cavities was pooled. The amount of cavity fluid was calculated from the difference in weight between the pooled fluid and rinses and the amount of deionized water delivered.
Experiment 2:
In this experiment, wheat plants were grown with an adequate supply of exogenous S, N and water during vegetative and generative growth to simulate wheat grown under irrigation. The molar ratio of N and S supplied was 8. The aim of this experiment was to determine the form of S delivered to the endosperm cavity and endosperm from S sourced directly from the soil, rather than from sources within the plant. Each week, 13 seeds of T. æstivum cv. Condor were sown in potting mix, in pots (20 cm diameter) in a growth cabinet as described for Experiment 2 previously (Fitzgerald et al., 1999a).
Four plants were harvested at anthesis, 12, 18, 25, and 35 dpa, and both the endosperm and fluid of the endosperm cavity were collected as described above.
Experiment 3:
Seeds of Triticum æstivum (cv. Condor) were sown in 20 cm pots (10 seeds per pot), containing a mix of 1 part vermiculite and 2 parts sand. Plants were raised in a growth cabinet as in Experiment 2 (Fitzgerald et al., 1999a). Pots were supplied with high-S nutrient solution (1.0 l week-1) (N : S of 10) or low-S nutrient solution (1.0 l week-1) (N : S of 50). The composition of the nutrient solution was given for Experiment 1 by Fitzgerald et al. (Fitzgerald et al., 1999a). The nutrient supply to both the low-S and high-S plants was terminated at anthesis. These conditions were such that each plant received the same amount of nutrients as those high-S and low-S plants in Experiment 1 for which nutrients were terminated at anthesis, i.e. all plants received 18 mmol N, the low-S plants received 0.36 mmol S and the high-S plants received 1.8 mmol S. Grains were harvested at 25 dpa for enzyme assays. Endosperms were isolated (as above), weighed, immediately frozen in LN2 and stored at -20 °C. The S-status of the low-S and high-S plants was determined by measuring the amount of ethanol-insoluble S in the leaves and grains by ICP-OES as described earlier (Fitzgerald et al., 1999a).
Measurement of soluble S in endosperms and cavities (Experiments 1 and 2)
For each sample, frozen endosperms (10) were ground to a powder in liquid N, with a precooled mortar and pestle, without thawing. Ethanolic extracts were prepared by repeatedly extracting with 80% ethanol (1 ml g-1 FW) at 70 °C (2x20 min, 1x10 min). After centrifugation (3000 g, 10 min), the supernatants were pooled.
Ethanol was evaporated from aliquots of ethanolic extracts of endosperms in a Savant® Speedvac Concentrator connected to a refrigerated condensation trap. The residue was dissolved in distilled water (1 ml). The volume was adjusted to 4 ml with 5 mM nitric acid. Total soluble S in the adjusted extracts was measured by ICP-OES as described previously (Sunarpi and Anderson, 1995).
An aliquot (100 µl) of endosperm cavity fluid was retained for amino acid and anion analysis. The volume of the remainder was adjusted to 4 ml with 5 mM nitric acid. Total soluble S in the adjusted extracts was measured by ICP-OES as described earlier (Sunarpi and Anderson, 1995).
GSH and soluble amino acids in the extracts of endosperms and in the fluid of the endosperm cavities were derivatized with 6 aminoquinolyl-N-succinimidyl carbamate (AQC), resolved by High Performance Liquid Chromatography (HPLC), and detected with ultraviolet absorption using the following procedures. An aliquot of either the ethanolic extract or fluid of endosperm cavities (20 µl) was dried and redissolved in 20 mM HCl (10 µl). The samples were derivatized with AQC by adjusting the pH to 8 with borate buffer (0.2 M, 70 µl), then adding 20 µl AQC reagent (Waters Corporation, Milford USA). After 1 min, derivatization mixtures (100 µl) were transferred to autosampler vials and heated (55 °C, 10 min). Amino acids were separated on a Waters AccQ.Tag C18 bonded silica column (300x3.9 mm) at 37 °C using the multi-step gradient (Waters Corporation, Milford USA) and detected spectrophotometrically (254 nm) as described previously (Fitzgerald et al., 1999b). The gradient was controlled, and data analysed, using the Millennium 2010 Chromatography Manager (Waters Corporation USA).
Inorganic sulphate and nitrate were measured in aliquots (50 µl) of the ethanolic extracts and of the fluid of the endosperm cavity using ion exchange HPLC. Ions were separated on a 150x4.6 mm Universal Anion column (Alltech, Aust.) using an isocratic mobile phase (1.7 mM NaHCO3 and 1.8 mM Na2CO3), a GBC LC 1110 pump, and a 350 Conductivity Detector fitted with a 335 Suppressor Unit (Alltech Australia).
Extraction and assay of enzymes of S metabolism in endosperms of Experiment 3
Enzyme extracts were prepared in duplicate from 10 frozen endosperms of high-S and low-S plants.
Enzymes of GSH catabolism:
Endosperms were ground in 2 ml 50 mM MOPS buffer pH 7.8, containing 1 mM EDTA and 2 mM DTT. The supernatant solution was recovered by centrifugation (10 000 g, 20 min). Incubation mixtures contained endosperm extract (200 µl or 100 µl), 50 µmol MOPS buffer (pH 7.8), 1 µmol EDTA, 2 µmol DTT, and 4 µmol of either GSH, cysteinylglycine or -glutamylcysteine in a final volume of 1 ml at 30 °C. Either GSH, cysteinylglycine, or -glutamylcysteine was omitted from control assays. Reactions were initiated by adding one of the appropriate S substrates. Samples (50 µl) were removed after 0 (t0) and 20 min (t20), mixed with 50 µl 5% TCA, and centrifuged, dried and redissolved in 20 mM HCl (10 µl). Amino acids were derivatized with AQC and analysed for the AQC derivatives of GSH, cysteinylglycine, -glutamylcysteine, cysteine, glycine, and glutamate by HPLC as described above. Activity was determined for the difference between the amount of metabolite in the t0 and t20 samples corrected for activity in the absence of the appropriate substrate.
Enzymes of S anabolism:
ATP sulphurylase was extracted and assayed as described earlier (Shaw and Anderson, 1972) using 10 endosperms per 0.5 ml 0.1 M TRIS buffer containing 20 mM MgCl2 pH 7.8. After dialysis, the volume was adjusted to 2 ml with buffer and the activity of aliquots (250 µl or 500 µl) was measured by sulphate-dependent [32P]PPi-ATP exchange in a final volume of 1 ml (Shaw and Anderson, 1972).
Cysteine synthase was extracted and assayed as described previously (Ng and Anderson, 1978) using 10 frozen endosperms per 2 ml 0.1 M potassium phosphate buffer pH 7.8 containing 1 mM EDTA and 1 mM DTT. After passing through a Sephadex G25 column the activity of samples (25 µl or 50 µl) of the desalted extract was measured by sulphide-dependent production of cysteine in the presence of O-acetylserine in a final volume of 1 ml (Ng and Anderson, 1978).
Cystathionine synthase was extracted from 10 endosperms in 2 ml 50 mM MES buffer pH 7.8 containing 1 mM EDTA and 2 mM DTT. The supernatant solution was recovered by centrifugation (10 000 g, 20 min) and passed through a Sephadex G25 column. Enzyme activity was determined by the production of cystathionine in incubation mixtures containing extract (100 µl or 200 µl), 5 µmol cysteine, 5 µmol homoserine-4-P, 50 µmol MES buffer (pH 7.8), 1 µmol EDTA, and 2 µmol DTT, in a final volume of 1 ml at 30 °C. The reaction was initiated by adding cysteine. Aliquots (50 µl) were withdrawn after 0 (t0) and 20 min (t20) and mixed with 50 µl 5% TCA. The supernatant solution was recovered by centrifugation, dried and redissolved in 20 mM HCl (10 µl). Amino acids in the acidified extract (10 µl), were derivatized with AQC and analysed for cystathionine (and other amino acids) by HPLC by the procedures given above. Activity was determined from the amount of cystathionine formed between t0 and t20 corrected for activity in the absence of homoserine-4-P.
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Results |
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Experiment 1 simulates the conditions where the capacity of the plant to take up nutrients from the soil becomes restricted by increasing soil water deficits during generative growth. Experiment 2 simulates conditions where there is sufficient moisture in the soil for plants to take up nutrients during grain development.
Delivery of S and N to endosperm cavity and endosperms
The composition of organic compounds in the fluid of the endosperm cavity was similar for high-S plants in Experiments 1 and 2 (Table 1). Sulphate accounted for most of the soluble S in the endosperm cavities of high-S plants in Experiments 1 and 2 (Table 1). Also in these plants, GSH accounted for 8–20% and methionine 4–5% of the soluble S in the endosperm cavity. Cysteine was not detected in the endosperm cavity of any of the plants. The fluid of the endosperm cavity of low-S plants from which nutrients were withdrawn at anthesis (Experiment 1) contained more GSH, at both harvests than was detected in cavities of high-S plants from which nutrients were withdrawn at anthesis (Table 1). Sulphate was not detected in the fluid of the endosperm cavity of the low-S plants. Nitrate was not detected in the endosperm cavity of plants in Experiment 1, but in Experiment 2, it accounted for 2% of the total N in the cavity fluid. In all endosperm cavities, glutamate, asparagine, glutamine, serine, and alanine accounted for most of the measured N (Table 1). At 13 dpa, the weight of fluid extracted from one endosperm cavity was 11 mg, and at 25 dpa it was 8 mg.
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The amount of each form of ethanol-soluble S in the endosperms of plants in Experiments 1 and 2 was measured at 13 dpa and at 25 dpa (Table 2
). Endosperms of all high-S plants in Experiments 1 and 2 contained sulphate at 13 dpa, but when nutrients were withdrawn at booting, the endosperms did not contain sulphate at 25 dpa (Table 2). Endosperms from low-S plants in Experiment 1 did not contain sulphate, but when nutrients were supplied until 8 dpa, sulphate was detected in endosperms at 13 dpa (Table 2). Endosperms of all plants in Experiments 1 and 2 contained similar sized pools of GSH at both 13 dpa and 25 dpa (Table 2). However, endosperms of low-S plants in Experiment 1 contained more methionine at 25 dpa than the other endosperms (Table 2). Cysteine was not detectable in endosperms at either 13 dpa or 25 dpa. No other S-containing compounds were detected in the endosperms, and the total S measured by ICP-OES in endosperms and cavity fluid was accounted for by sulphate, glutathione and methionine (data not shown).
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The pools of soluble S-metabolites in endosperms of wheat plants that received adequate S and water (Experiment 2) during generative growth were monitored from anthesis until maturity (60 dpa). The data (Fig. 1
) show that the amount of sulphate in the endosperms did not change from anthesis to 13 dpa, increased to a maximum at about 18 dpa, declined rapidly until 35 dpa, then declined more slowly to an undetectable level at maturity. GSH reached a maximum at about 35 dpa and thereafter decreased until maturity (Fig. 1). Each endosperm contained approximately 8 nmol GSH at maturity. Methionine remained at a very low level throughout endosperm development, and was not detectable at maturity (Fig. 1). Cysteine was not detectable at any stage of development.
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Catabolism of GSH by endosperm extracts (Experiment 3)
Crude extracts supported the production of -glutamylcysteine in the presence of 4 mM GSH. Activity was not detected in the absence of crude extract or GSH (Table 3 ). The production of -glutamylcysteine was accompanied by the stoichiometric production of glycine and the stoichiometric consumption of GSH. Production of -glutamylcysteine was proportional to the volume of crude extract up to 200 µl in standard incubations using the specified extraction procedure. The rate of production of -glutamylcysteine was constant for at least 20 min but thereafter declined. The rate of GSH-dependent production of -glutamylcysteine exhibited substrate saturation at a concentration of 2 mM; higher concentrations of GSH, (up to 4 mM) were not inhibitory. The apparent KM was approximately 0.5 mM (Fig. 2). The S nutrition of the plants from which the endosperms were derived did not affect the rate of GSH-dependent formation of -glutamylcysteine (Table 3). Crude extracts passed through a Sephadex G25 column did not support the production of -glutamylcysteine from GSH perhaps indicating the removal of an essential cofactor.
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Crude extracts also supported the consumption of
-glutamylcysteine with the stoichiometric production of glutamate and of cysteine (Table 3
). Activity did not occur in the absence -glutamylcysteine or in the absence of extract. S nutrition did not affect the rate of -glutamylcysteine dependent formation of glutamate and cysteine (Table 3). The rates of hydrolysis of -glutamylcysteine were about 70–80% of the rate for GSH hydrolysis, Thus, the minimum rate of GSH catabolism as calculated from the slowest constituent step (hydrolysis of -glutamylcysteine in low-S plants) and measured in crude extracts under optimum conditions in vitro and expressed per endosperm, was about 62 nmol endosperm-1 d-1. Extracts passed through Sephadex G25 were inactive towards -glutamylcysteine.
Neither crude extracts nor desalted endosperm extracts catalysed the hydrolysis of cysteinylglycine as measured by production of glycine or cysteine or by consumption of the substrate, regardless of the S nutrition of the plants (Table 3).
Effect of S nutrition on enzymes of S assimilation in endosperms
Activity of ATP sulphurylase:
Dialysed crude extracts did not support significant PPi-ATP exchange in the absence of sulphate and ATP. Sulphate-dependent PPi-ATP exchange was proportional to the volume of crude extract up to at least 500 µl in standard incubations using the specified extraction procedure, but the activity was low in comparison to the other enzymes of S assimilation studied (Table 4 ). S nutrition influenced the activity of ATP sulphurylase; the in vitro activity in endosperms from high-S plants was about 4-fold greater than in endosperms from low-S plants (Table 4).
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Activity of cysteine synthase:
Cysteine synthase was the most active of the enzymes of S assimilation studied in endosperms. Activity was not detected in the absence of sulphide and O-acetylserine. Substrate-dependent formation of cysteine was proportional to the volume of crude extract up to at least 50 µl. S nutrition influenced the in vitro activity of cysteine synthase; the activity in endosperms from high-S plants was approximately 2-fold greater than in those from low-S plants (Table 4).
Activity of cystathionine synthase:
Cystathionine synthesis was only detected in the presence of cysteine, homoserine-4-P and endosperm extract, so activity can be attributed to cystathionine synthase. The rate of the reaction as determined by the production of cystathionine was constant for at least 20 min. The substrate homoserine-4-P is also a substrate for the formation of threonine, catalysed by threonine synthase. The latter activity can be measured simultaneously with cystathionine synthase by analysis of the AQC derivatives of any products formed in the reaction mixture. Despite the presence of threonine synthase activity, the concentration of homoserine-4P did not decrease sufficiently to become rate-limiting for cystathionine synthase. Cystathionine, in turn, is the substrate for the formation of homocysteine, catalysed by cystathionine ß-lyase. Although the incubation mixtures used to assay cystathionine synthase activity would not provide optimum conditions for the production of homocysteine, the latter activity can, if present, be detected by formation of the AQC derivative of homocysteine. Using this approach, cystathionine synthase, threonine synthase and cystathionine ß-lyase activities were all detected in the incubation mixtures for the assay of cystathionine synthase; the activity of cystathionine ß-lyase was dependent on the formation of cystathionine by cystathionine synthase. The total cystathionine synthase given in Table 4 is the sum of the rate of cystathionine and of homocysteine formation.
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Delivery of S and N compounds to the endosperm
Sulphate and GSH in soluble pools of roots and leaves, and protein-S in leaves are all exported during reproductive growth, and the losses can be accounted for by commensurate gains by the ear over the same interval (Fitzgerald et al., 1999
a, b). Previous measurements of the contents of the endosperm cavity of wheat have shown that the amounts of cysteine and methionine in the cavity are unlikely to meet the needs for protein synthesis in the endosperm (Fisher and MacNicol, 1986; Blumenthal et al., 1990; Ugalde and Jenner, 1990b). Fisher and MacNicol proposed that S is transported to the endosperm cavity in a form other than as cysteine and methionine (Fisher and MacNicol, 1986). In rice, GSH has been found in the phloem sap of vegetative plants (Kuzuhara et al., 2000), but that study does not extend to the generative phase of plant growth. Studies by Bourgis et al. show that S-methylmethionine is a transport form of methionine that travels in the phloem to wheat ears (Bourgis et al., 1999). In that study S-methylmethionine was detected only in the phloem below the developing ear (Bourgis et al., 1999). In this study, transport compounds were collected from the endosperm cavity and the endosperm. It is entirely possible that S-methylmethionine could contribute to the transport of S to the developing ear, and it is also possible that a methyltransferase recovers the methionine before it reaches the endosperm. The data in Table 1 indicate that sulphate is the main form of S delivered to the endosperm cavity in plants receiving adequate S, but GSH is the main form of S delivered to the cavity in conditions of low S nutrition.
The GSH and methionine detected in the endosperm cavity are most likely to be derived from the hydrolysis of leaf-protein. The proportion of GSH to methionine, and the absence of cysteine, in the cavity (Table 1) suggests that all of the cysteine and much of the methionine from protein hydrolysis was incorporated into cysteinyl residues of GSH. This is consistent with the proposed role of GSH for the long-distance transport of reduced S (Rennenberg, 1984). No other compounds were detected, and the GSH, sulphate and methionine measured in each treatment account for the total S measured by ICP-OES for each tissue in each treatment.
Effect of S nutrition on sulphate and GSH in endosperms
The data in Table 2 indicate that sulphate and GSH accounted for most of the soluble S in developing wheat endosperms and that sulphate was barely detectable in the endosperms of low-S plants, except in those that continued to receive S up to 8 dpa, though even this was consumed in the period between 13 and 25 dpa. The data in Table 2, and that reported in Fitzgerald et al. (Fitzgerald et al., 1999a, b) provide the information to calculate, for the period between 13 and 25 dpa: the amount of each form of S delivered, the amount of each form of S that accumulated in the steady-state pool (unmetabolized), and the amount of each form of S utilized, presumably for the synthesis of storage proteins. The results of those calculations are presented in Table 5.
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For Experiment 1, the amount of sulphate, GSH and methionine delivered to endosperms depended on both the duration of the nutrient supply and the level of S nutrition (Table 5
). Endosperms of low-S plants did not import any sulphate between 13 and 25 dpa, but endosperms from high-S plants did. Taken together with the data in Table 1, in conditions of adequate S, sulphate is a significant source of S for endosperm growth between 13 and 25 dpa. All endosperms received a similar amount of GSH during this period. GSH comprised the main form of S delivered to the endosperms of low-S plants (Table 5) and this was almost entirely consumed in supporting endosperm growth, implying that GSH must have served as the principal source for the synthesis of both cysteine and methionine for the synthesis of grain proteins in the low-S plants.
The changes in the concentration of sulphate and GSH in the endosperms as shown in Experiment 2 (Fig. 1) perhaps reflect assimilation of the pool of sulphate within the endosperm concomitant with a changing composition of nutrients delivered to the endosperm cavity. The decrease in the amount of sulphate in the endosperms of plants as they mature (Fig. 1) indicates that endosperms do not acquire and accumulate sulphate indefinitely, but eventually delivery and assimilation of sulphate cease.
The data in Table 5 have implications with respect to uptake of sulphate and GSH into endosperms from the endosperm cavity and the regulation of S metabolism in the endosperm. Comparing the concentration of S (calculated from Tables 1 and 2) in the endosperm cavity with that in endosperms shows that both GSH and sulphate are taken up against concentration gradients, thereby raising the question of specific transporters at this site. Three sulphate transporters have been described in Stylosanthes hamata (Hawkesford and Smith, 1997), one of which is not confined to root tissue. Also of interest is that neither GSH nor sulphate accumulated in the endosperm cavity, the endosperm cavities of all the high-S plants contained the same amount of GSH and sulphate at any one time, and the endosperm cavities of all the low-S plants contained the same amount of GSH at any one time (Table 1). This indicates that endosperms from each nutritional treatment had different capacities to take up both GSH and sulphate from the endosperm cavity to the endosperm.
Enzymes of GSH catabolism in endosperms
The hypothesis that endosperms have the biochemical capacity to catabolize GSH and form cysteine and metabolize cysteine to methionine is supported by the study of GSH catabolism by crude endosperm extracts (Tables 3, 4).
The pathway of GSH-hydrolysis in wheat endosperms appears to be a reversal of the synthetic pathway, with glycine being cleaved in the first step to form -glutamylcysteine which is then cleaved in a second reaction to its constituent amino acids, glutamate and cysteine (Table 3) consistent with the action of enzymes such as GSH hydrolase (-glutamylcysteine forming) and -glutamylcysteine hydrolase, respectively. The rates of the two activities are similar, but there are no data on the number of enzymes involved, and both are inactivated by passage through Sephadex G25. The in vitro rates of hydrolysis of GSH and of -glutamylcysteine (Table 3) are sufficient to account for the theoretical maximum rate of GSH hydrolysis (c. 42 nmol endosperm-1 d-1) based on the data in Table 5. Given the rate of GSH hydrolysis (Table 3), the in vitro activities of cystathionine synthase and cystathionine ß-lyase (Table 4) imply that these enzymes would not be rate-limiting in the formation of methionine from the cysteine formed from GSH.
Effect of S nutrition on assimilation of S in endosperms
The presence of enzymes of the sulphate assimilation pathway (ATP sulphurylase and cysteine synthase) and of methionine synthesis (cystathionine synthase and cystathionine ß-lyase) in extracts of developing endosperms (Table 4) is consistent with the proposal that endosperms can assimilate sulphate-S into cysteine and methionine for incorporation into grain proteins. Since the two enzymes involved in the assimilation of sulphate-S into cysteine exhibit higher activity in high-S plants than in low-S plants (Table 4), and the endosperms of high-S plants contained endogenous sulphate but those of low-S plants did not (Table 2), this suggests that the capacity of the sulphate assimilation pathway in endosperms was enhanced by sulphate or by a product derived from it. The data in Table 4 suggest that, of the enzymes studied, ATP sulphurylase was the one most likely to be rate-limiting. Although the relevance of the in vitro rates of enzyme activity (Tables 3, 4) to the rates under in vivo conditions is uncertain, perhaps control of the activity of ATP sulphurylase could be important in determining the flux of S through the sulphate assimilation pathway in endosperms.
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Acknowledgments |
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The authors sincerely thank Agriculture Victoria for the Nancy Millis Award to MAF that supported this work, Annabel Good for conducting the ICP-OES analyses, and Jerry Kanellos and Stuart Morrison of Waters Australia for allowing the use of the HPLC equipment in their laboratory.
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Notes |
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3 Present address and to whom correspondence should be sent: Yanco Agricultural Institute, NSW Agriculture, PMB, Yanco 2703, NSW, Australia. Fax: +61 2 69 512 719. E-mail: melissa.fitzgerald{at}agric.nsw.gov.au
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