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Sulphur flux through the sulphate assimilation pathway is differently controlled by adenosine 5'-phosphosulphate reductase under stress and in transgenic poplar plants overexpressing gamma-ECS, SO, or APR.

Scheerer U, Haensch R, Mendel RR, Kopriva S, Rennenberg H, Herschbach C - J. Exp. Bot. (2009)

Bottom Line: Sulphate assimilation provides reduced sulphur for the synthesis of cysteine, methionine, and numerous other essential metabolites and secondary compounds.The key step in the pathway is the reduction of activated sulphate, adenosine 5'-phosphosulphate (APS), to sulphite catalysed by APS reductase (APR).Thereby the sulphur flux into GSH or protein remained unaffected. (iii) Cd treatment increased APR activity without any changes in sulphur flux but lowered sulphate uptake.

View Article: PubMed Central - PubMed

Affiliation: Albert-Ludwigs-University Freiburg, Institute of Forest Botany and Tree Physiology, Chair of Tree Physiology, Georges-Köhler-Allee 053/054, Freiburg, Germany.

ABSTRACT
Sulphate assimilation provides reduced sulphur for the synthesis of cysteine, methionine, and numerous other essential metabolites and secondary compounds. The key step in the pathway is the reduction of activated sulphate, adenosine 5'-phosphosulphate (APS), to sulphite catalysed by APS reductase (APR). In the present study, [(35)S]sulphur flux from external sulphate into glutathione (GSH) and proteins was analysed to check whether APR controls the flux through the sulphate assimilation pathway in poplar roots under some stress conditions and in transgenic poplars. (i) O-Acetylserine (OAS) induced APR activity and the sulphur flux into GSH. (ii) The herbicide Acetochlor induced APR activity and results in a decline of GSH. Thereby the sulphur flux into GSH or protein remained unaffected. (iii) Cd treatment increased APR activity without any changes in sulphur flux but lowered sulphate uptake. Several transgenic poplar plants that were manipulated in sulphur metabolism were also analysed. (i) Transgenic poplar plants that overexpressed the gamma-glutamylcysteine synthetase (gamma-ECS) gene, the enzyme catalysing the key step in GSH formation, showed an increase in sulphur flux into GSH and sulphate uptake when gamma-ECS was targeted to the cytosol, while no changes in sulphur flux were observed when gamma-ECS was targeted to plastids. (ii) No effect on sulphur flux was observed when the sulphite oxidase (SO) gene from Arabidopsis thaliana, which catalyses the back reaction of APR, that is the reaction from sulphite to sulphate, was overexpressed. (iii) When Lemna minor APR was overexpressed in poplar, APR activity increased as expected, but no changes in sulphur flux were observed. For all of these experiments the flux control coefficient for APR was calculated. APR as a controlling step in sulphate assimilation seems obvious under OAS treatment, in gamma-ECS and SO overexpressing poplars. A possible loss of control under certain conditions, that is Cd treatment, Acetochlor treatment, and in APR overexpressing poplar, is discussed.

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Sulphur metabolite contents and 35S flux into different metabolite pools of fine roots from poplar plants overexpressing APR from Lemna. Excised fine roots from the WT (A, n=4) and from different transgenic poplar lines overexpressing Lemna APR (B, n=16) were selected and exposed to [35S]sulphate for 4 h to measure sulphate uptake and 35S flux into different metabolite pools. Sulphate (μmol g−1 FW), GSH (nmol g−1 FW), and protein (mg g−1 FW) contents were determined (pink squares). 35S flux into internal sulphate, GSH, and protein is given as pmol 35S g−1 FW h−1 (light blue squares). APR activity is indicated in a green square as nmol mg−1 protein min−1. Significant differences between WT and transgenic poplar roots at P <0.05 are indicated by asterisks.
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fig6: Sulphur metabolite contents and 35S flux into different metabolite pools of fine roots from poplar plants overexpressing APR from Lemna. Excised fine roots from the WT (A, n=4) and from different transgenic poplar lines overexpressing Lemna APR (B, n=16) were selected and exposed to [35S]sulphate for 4 h to measure sulphate uptake and 35S flux into different metabolite pools. Sulphate (μmol g−1 FW), GSH (nmol g−1 FW), and protein (mg g−1 FW) contents were determined (pink squares). 35S flux into internal sulphate, GSH, and protein is given as pmol 35S g−1 FW h−1 (light blue squares). APR activity is indicated in a green square as nmol mg−1 protein min−1. Significant differences between WT and transgenic poplar roots at P <0.05 are indicated by asterisks.

Mentions: Fifty independent kanamycin-resistant plantlets that overexpressed Lemna minor APR were grown in tissue culture and were tested for APR activity in the leaves. Four lines (303, 304, 391, and 404) with 17- to 72-fold increased foliar APR activity (data not shown) were selected and further propagated. Initially, the plants did not show any obvious morphological differences compared with WT poplars when grown on sandy soil, although with prolonged growth on perlite, sand, and humus soil, some alterations in leaf shape and increased branching were observed (Rennenberg et al., 2007). The increase in foliar APR activity was accompanied by a significant increase in foliar thiol levels (data not shown). In leaves, Cys and GSH were increased 2- to 4-fold in the transgenic plants, with the highest thiol levels in line 303 that also possessed the highest APR activity (data not shown). No consistent changes in the levels of sulphate were detected (data not shown). While the leaves of line 391 accumulated ∼50% more sulphate than the WT, sulphate levels in roots seemed, although not significantly, doubled in line 304 and 391, but remained unaffected in the other lines (Fig. 4D). All transgenic lines revealed constant GSH contents in fine roots (Fig. 4F) although the APR activity was up to 10-fold higher compared with the WT (Figs 4E, 6). Surprisingly, the high APR activity did not affect the flux of 35S into sulphate, thiols, and proteins. Also sulphate uptake rates of roots from APR overexpressing poplars did not differ from those of the WT (Fig. 6, Table 1).


Sulphur flux through the sulphate assimilation pathway is differently controlled by adenosine 5'-phosphosulphate reductase under stress and in transgenic poplar plants overexpressing gamma-ECS, SO, or APR.

Scheerer U, Haensch R, Mendel RR, Kopriva S, Rennenberg H, Herschbach C - J. Exp. Bot. (2009)

Sulphur metabolite contents and 35S flux into different metabolite pools of fine roots from poplar plants overexpressing APR from Lemna. Excised fine roots from the WT (A, n=4) and from different transgenic poplar lines overexpressing Lemna APR (B, n=16) were selected and exposed to [35S]sulphate for 4 h to measure sulphate uptake and 35S flux into different metabolite pools. Sulphate (μmol g−1 FW), GSH (nmol g−1 FW), and protein (mg g−1 FW) contents were determined (pink squares). 35S flux into internal sulphate, GSH, and protein is given as pmol 35S g−1 FW h−1 (light blue squares). APR activity is indicated in a green square as nmol mg−1 protein min−1. Significant differences between WT and transgenic poplar roots at P <0.05 are indicated by asterisks.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2803220&req=5

fig6: Sulphur metabolite contents and 35S flux into different metabolite pools of fine roots from poplar plants overexpressing APR from Lemna. Excised fine roots from the WT (A, n=4) and from different transgenic poplar lines overexpressing Lemna APR (B, n=16) were selected and exposed to [35S]sulphate for 4 h to measure sulphate uptake and 35S flux into different metabolite pools. Sulphate (μmol g−1 FW), GSH (nmol g−1 FW), and protein (mg g−1 FW) contents were determined (pink squares). 35S flux into internal sulphate, GSH, and protein is given as pmol 35S g−1 FW h−1 (light blue squares). APR activity is indicated in a green square as nmol mg−1 protein min−1. Significant differences between WT and transgenic poplar roots at P <0.05 are indicated by asterisks.
Mentions: Fifty independent kanamycin-resistant plantlets that overexpressed Lemna minor APR were grown in tissue culture and were tested for APR activity in the leaves. Four lines (303, 304, 391, and 404) with 17- to 72-fold increased foliar APR activity (data not shown) were selected and further propagated. Initially, the plants did not show any obvious morphological differences compared with WT poplars when grown on sandy soil, although with prolonged growth on perlite, sand, and humus soil, some alterations in leaf shape and increased branching were observed (Rennenberg et al., 2007). The increase in foliar APR activity was accompanied by a significant increase in foliar thiol levels (data not shown). In leaves, Cys and GSH were increased 2- to 4-fold in the transgenic plants, with the highest thiol levels in line 303 that also possessed the highest APR activity (data not shown). No consistent changes in the levels of sulphate were detected (data not shown). While the leaves of line 391 accumulated ∼50% more sulphate than the WT, sulphate levels in roots seemed, although not significantly, doubled in line 304 and 391, but remained unaffected in the other lines (Fig. 4D). All transgenic lines revealed constant GSH contents in fine roots (Fig. 4F) although the APR activity was up to 10-fold higher compared with the WT (Figs 4E, 6). Surprisingly, the high APR activity did not affect the flux of 35S into sulphate, thiols, and proteins. Also sulphate uptake rates of roots from APR overexpressing poplars did not differ from those of the WT (Fig. 6, Table 1).

Bottom Line: Sulphate assimilation provides reduced sulphur for the synthesis of cysteine, methionine, and numerous other essential metabolites and secondary compounds.The key step in the pathway is the reduction of activated sulphate, adenosine 5'-phosphosulphate (APS), to sulphite catalysed by APS reductase (APR).Thereby the sulphur flux into GSH or protein remained unaffected. (iii) Cd treatment increased APR activity without any changes in sulphur flux but lowered sulphate uptake.

View Article: PubMed Central - PubMed

Affiliation: Albert-Ludwigs-University Freiburg, Institute of Forest Botany and Tree Physiology, Chair of Tree Physiology, Georges-Köhler-Allee 053/054, Freiburg, Germany.

ABSTRACT
Sulphate assimilation provides reduced sulphur for the synthesis of cysteine, methionine, and numerous other essential metabolites and secondary compounds. The key step in the pathway is the reduction of activated sulphate, adenosine 5'-phosphosulphate (APS), to sulphite catalysed by APS reductase (APR). In the present study, [(35)S]sulphur flux from external sulphate into glutathione (GSH) and proteins was analysed to check whether APR controls the flux through the sulphate assimilation pathway in poplar roots under some stress conditions and in transgenic poplars. (i) O-Acetylserine (OAS) induced APR activity and the sulphur flux into GSH. (ii) The herbicide Acetochlor induced APR activity and results in a decline of GSH. Thereby the sulphur flux into GSH or protein remained unaffected. (iii) Cd treatment increased APR activity without any changes in sulphur flux but lowered sulphate uptake. Several transgenic poplar plants that were manipulated in sulphur metabolism were also analysed. (i) Transgenic poplar plants that overexpressed the gamma-glutamylcysteine synthetase (gamma-ECS) gene, the enzyme catalysing the key step in GSH formation, showed an increase in sulphur flux into GSH and sulphate uptake when gamma-ECS was targeted to the cytosol, while no changes in sulphur flux were observed when gamma-ECS was targeted to plastids. (ii) No effect on sulphur flux was observed when the sulphite oxidase (SO) gene from Arabidopsis thaliana, which catalyses the back reaction of APR, that is the reaction from sulphite to sulphate, was overexpressed. (iii) When Lemna minor APR was overexpressed in poplar, APR activity increased as expected, but no changes in sulphur flux were observed. For all of these experiments the flux control coefficient for APR was calculated. APR as a controlling step in sulphate assimilation seems obvious under OAS treatment, in gamma-ECS and SO overexpressing poplars. A possible loss of control under certain conditions, that is Cd treatment, Acetochlor treatment, and in APR overexpressing poplar, is discussed.

Show MeSH
Related in: MedlinePlus