Glutathione-Mediated Regulation of ATP Sulfurylase Activity,SO42- Uptake,and Oxidative Stress Response in Intact Canola Roots

The dual role of glutathione as a transducer of S status (A.G. Lappartient and B. Touraine [1996] Plant Physiol 111: 147-157) and as an antioxidant was examined by comparing the effects of S deprivation, glutathione feeding, and H2O2 (oxidative stress) on SO42- uptake and ATP sulfurylase activity in roots of intact canola (Brassica napus L.). ATP sulfurylase activity increased and SO42- uptake rate severely decreased in roots exposed to 10 mM H2O2, whereas both increased in S-starved plants. In split-root experiments, an oxidative stress response was induced in roots remote from H2O2 exposure, as revealed by changes in the reduced glutathione (GSH) level and the GSH/oxidized glutathione (GSSG) ratio, but there was only a small decrease in SO42- uptake rate and no effect on ATP sulfurylase activity. Feeding plants with GSH increased GSH, but did not affect the GSH/GSSG ratio, and both ATP sulfurylase activity and SO42- uptake were inhibited. The responses of the H2O2-scavenging enzymes ascorbate peroxidase and glutathione reductase to S starvation, GSH treatment, and H2O2 treatment were not to glutathione-mediated S demand regulatory process. We conclude that the regulation of ATP sulfurylase activity and SO42- uptake by S demand is related to GSH rather than to the GSH/GSSG ratio, and is distinct from the oxidative stress response.     

Overexpression of ATP Sulfurylase in Indian Mustard Leads to Increased Selenate Uptake, Reduction, and Tolerance

In earlier studies, the assimilation of selenate by plants appeared to be limited by its reduction, a step that is thought to be mediated by ATP sulfurylase. Here, the Arabidopsis APS1 gene, encoding a plastidic ATP sulfurylase, was constitutively overexpressed in Indian mustard (Brassica juncea). Compared with that in untransformed plants, the ATP sulfurylase activity was 2- to 2.5-fold higher in shoots and roots of transgenic seedlings, and 1.5- to 2-fold higher in shoots but not roots of selenate-supplied mature ATP-sulfurylase-overexpressing (APS) plants. The APS plants showed increased selenate reduction: x-ray absorption spectroscopy showed that root and shoot tissues of mature APS plants contained mostly organic Se (possibly selenomethionine), whereas wild-type plants accumulated selenate. The APS plants were not able to reduce selenate when shoots were removed immediately before selenate was supplied. In addition, Se accumulation in APS plants was 2- to 3-fold higher in shoots and 1.5-fold higher in roots compared with wild-type plants, and Se tolerance was higher in both seedlings and mature APS plants. These studies show that ATP sulfurylase not only mediates selenate reduction in plants, but is also rate limiting for selenate uptake and assimilation.     

Efficiency of the first steps of sulfate utilization by maize hybrids in relation to their productivity

The genetic variability of the efficiency of the first steps of sulfate utilization and its correlation with productivity were evaluated in nine maize hybrids. ³⁵SO₄²⁻ uptake by excised roots, uptake by intact plant roots, translocation to leaves, and ATP sulfurylase in leaves were taken into account. Uptake rate by roots of intact plants did not show any pulse within 7 to 12 days from emergence, in contrast with the previously observed behaviour of excised roots during root elongation. The uptake rate of intact plants was positively correlated with that of excised roots, but the variability within the nine genotypes tested was less. Productivity was positively correlated with sulfate uptake by both intact plant and excised roots, the level of significance being higher in the first case. Translocation to leaves and ATP sulfurylase activity were not correlated to productivity. Therefore, in the case of sulfate, the grain yield of commonly cultivated maize hybrids appeared to be controlled more by the root uptake step than by the activation and translocation steps.     

Flux control of sulphate assimilation in Arabidopsis thaliana: adenosine 5'-phosphosulphate reductase is more susceptible than ATP sulphurylase to negative control by thiols

The effect of externally applied L-cysteine and glutathione (GSH) on ATP sulphurylase and adenosine 5'-phosphosulphate reductase (APR), two key enzymes of assimilatory sulphate reduction, was examined in Arabidopsis thaliana root cultures. Addition of increasing L-cysteine to the nutrient solution increased internal cysteine, gamma-glutamylcysteine and GSH concentrations, and decreased APR mRNA, protein and extractable activity. An effect on APR could already be detected at 0.2 mm L-cysteine, whereas ATP sulphurylase was significantly affected only at 2 mm L-cysteine. APR mRNA, protein and activity were also decreased by GSH at 0.2 mm and higher concentrations. In the presence of L-buthionine-S, R-sulphoximine (BSO), an inhibitor of GSH synthesis, 0.2 mm L-cysteine had no effect on APR activity, indicating that GSH formed from cysteine was the regulating substance. Simultaneous addition of BSO and 0.5 mm GSH to the culture medium decreased APR mRNA, enzyme protein and activity. ATP sulphurylase activity was not affected by this treatment. Tracer experiments using (35)SO(4)(2-) in the presence of 0.5 mm L-cysteine or GSH showed that both thiols decreased sulphate uptake, APR activity and the flux of label into cysteine, GSH and protein, but had no effect on the activity of all other enzymes of assimilatory sulphate reduction and serine acetyltransferase. These results are consistent with the hypothesis that thiols regulate the flux through sulphate assimilation at the uptake and the APR step. Analysis of radioactive labelling indicates that the flux control coefficient of APR is more than 0.5 for the intracellular pathway of sulphate assimilation. This analysis also shows that the uptake of external sulphate is inhibited by GSH to a greater extent than the flux through the pathway, and that the flux control coefficient of APR for the pathway, including the transport step, is proportionately less, with a significant share of the control exerted by the transport step.     

Increased Zinc Tolerance in Silene vulgaris (Moench) Garcke Is Not Due to Increased Production of Phytochelatins

The concentration of acid-soluble thiols other than reduced glutathione (SH - GSH) increases in the roots of zinc-sensitive and zinc-tolerant Silene vulgaris (Moench) Garcke after exposure to zinc for 1 to 3 d. The concentration of SH - GSH in the roots is higher in the sensitive plants than in the tolerant ones, both at equal external zinc concentrations and at zinc concentrations causing the same level of root-length growth inhibition. High performance liquid chromatography analyses show that the increase in the concentration of SH - GSH is not only due to the production of phytochelatins, but is also due to an increase in the concentration of cysteine and the production of nonidentified thiols. The cysteine concentration increases equally in the roots of sensitive and tolerant plants. The accumulation of phytochelatins is higher in the roots of the sensitive plants, whereas the chain length distribution of phytochelatins is the same in sensitive and tolerant plants. It is concluded that increased zinc tolerance in S. vulgaris is not due to increased production of phytochelatins.     

Influence of age and sulfur metabolism on ATP sulfurylase activity in the soybean and a survey of selected species

ATP sulfurylase activity varied greatly among different leaves on the soybean plant [Glycine max (L.) Meer.], and high levels of activity did not appear in the leaves until the seedlings were about 3 weeks old. In general, leaves from the top of the plant had a higher activity than leaves from the bottom of the plant. A much greater activity was found in soybean leaves than in soybean roots. The absence of sulfate in the nutrient solution resulted in higher enzyme activity in leaves from young plants and in lower activity in leaves from older plants. Over the growing season, however, ATP sulfurylase activity appeared to be related to sulfur content of the leaf. Several other plant species also had measurable levels of ATP sulfurylase.     

Cysteine, gamma-Glutamylcysteine, and Glutathione Levels in Maize Seedlings : Distribution and Translocation in Normal and Cadmium-Exposed Plants

The levels of cysteine (Cys), γ-glutamylcysteine (γEC), and glutathione (GSH) were measured in the endosperms, scutella, roots, and shoots of maize (Zea mays L.) seedlings. GSH was the major thiol in roots, shoots, and scutella, Cys predominated in endosperms. The endosperm, scutellum, and functional phloem translocation were required for maintenance of GSH pools in roots and shoots of 6-day-old seedlings. Exposure of roots to 3 micromolar Cd, besides causing a decline in GSH, caused an accumulation of γEC, as if the activity of GSH synthetase was reduced in vivo. [³⁵S]Cys injected into endosperms of seedlings was partly metabolized to [³⁵S]sulfate. The scutella absorbed both [³⁵S]sulfate and [³⁵S]Cys and transformed 68 to 87% of the radioactivity into [³⁵S]GSH. [³⁵S]GSH was translocated to roots and shoots in proportion to the tissue fresh weight. Taken together, the data supported the hypothesis that Cys from the endosperm is absorbed by the scutellum and used to synthesize GSH for transfer through the phloem to the root and shoot. The estimated flux of GSH to the roots was 35 to 60 nanomoles per gram per hour, which totally accounted for the small gain in GSH in roots between days 6 and 7. For Cd-treated roots the GSH influx was similar, yet the GSH pool did not recover to control levels within 24 hours. The estimated flux of GSH to the entire shoot was like that to the roots; however, it was low (11-13 nanomoles per gram per hour) to the first leaf and high (76-135 nanomoles per gram per hour) to the second and younger leaves.     

Phloem transport of sulfur in Ricinus

Mature leaves of Ricinus communis fed with ³⁵SO ₄ ^2- in the light export labeled sulfate and reduced sulfur compounds by phloem transport. Only 1–2% of the absorbed radiosulfur is exported to the stem within 2–3 h, roughly 12% of ³⁵S recovered was in reduced form. The composition of phloem translocate moving down the stem toward the root was determined from phloem exudate: 20–40% of the ³⁵S moved in the form of organic sulfur compounds, however, the bulk of sulfur was transported as inorganic sulfate. The most important organic sulfur compound translocated was glutathione, carrying about 70% of the label present in the organic fraction. In addition, methionine and cysteine were involved in phloem sulfur transport and accounted for roughly 10%. Primarily, the reduced forms of both, glutathione and cysteine are prsent in the siever tubes.Abbreviations CySH cysteine - GSH glutathione - GSSG glutathione disulfide - NEM N-ethylmaleimide - CyS-SCy cystine     

Rhizobium Meliloti Genes Involved in Sulfate Activation: The Two Copies of Nodpq and a New Locus, Saa

The nitrogen-fixing symbiont Rhizobium meliloti establishes nodules on leguminous host plants. Nodulation (nod) genes used for this process are located in a cluster on the pSym-a megaplasmid of R. meliloti. These genes include nodP and nodQ (here termed nodPQ), which encode ATP sulfurylase and APS kinase, enzymes that catalyze the conversion of ATP and SO(4)2- into the activated sulfate form 3'-phosphoadenosine 5'-phosphosulfate (PAPS), an intermediate in cysteine synthesis. In Rhizobium, PAPS is also a precursor for sulfated and N-acylated oligosaccharide Nod-factor signals that cause symbiotic responses on specific host plants such as alfalfa. We previously found a highly conserved second copy of nodPQ in R. meliloti. We report here the mapping and cloning of this second copy, and its location on the second megaplasmid, pSym-b. The function of nodP2Q2 is equivalent to that of nodP1Q1 in complementation tests of R. meliloti and Escherichia coli mutants in ATP sulfurylase and adenosine 5'-phosphosulfate (APS) kinase. Mutations in nodP2Q2 do not have as severe an effect on symbiosis or plant host range as do those in nodP1Q1, however, possibly reflecting differences in expression and/or channeling of metabolites to specific enzymes involved in sulfate transfer. Strains mutated or deleted for both copies of nodQ are severely defective in symbiotic phenotypes, but remain prototrophic. This suggests the existence in R. meliloti of a third locus for ATP sulfurylase and APS kinase activities. We have found a new locus saa (sulfur amino acid), which may also encode these activities.     

Cloning of a cDNA encoding ATP sulfurylase from Arabidopsis thaliana by functional expression in Saccharomyces cerevisiae.

ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate. Here we report the cloning of a cDNA encoding ATP sulfurylase (APS1) from Arabidopsis thaliana. APS1 was isolated by its ability to alleviate the methionine requirement of an ATP sulfurylase mutant strain of Saccharomyces cerevisiae (yeast). Expression of APS1 correlated with the presence of ATP sulfurylase enzyme activity in cell extracts. APS1 is a 1748-bp cDNA with an open reading frame predicted to encode a 463-amino acid, 51,372-D protein. The predicted amino acid sequence of APS1 is similar to ATP sulfurylase of S. cerevisiae, with which it is 25% identical. Two lines of evidence indicate that APS1 encodes a chloroplast form of ATP sulfurylase. Its predicted amino-terminal sequence resembles a chloroplast transit peptide; and the APS1 polypeptide, synthesized in vitro, is capable of entering isolated intact chloroplasts. Several genomic DNA fragments that hybridize with the APS1 probe were identified. The APS1 cDNA hybridizes to three species of mRNA in leaves (1.85, 1.60, and 1.20 kb) and to a single species of mRNA in roots (1.85 kb).     

Atmospheric H(2)S as sulfur source for Brassica oleracea: kinetics of H(2)S uptake and activity of O-acetylserine (thiol)lyase as affected by sulfur nutrition

The uptake of hydrogen sulfide (H(2)S) by shoots of curly kale (Brassica oleracea) showed saturation kinetics with respect to the atmospheric concentration. The kinetics are largely determined by the rate of metabolism of the absorbed H(2)S into cysteine, catalyzed by O-acetylserine (thiol)lyase, and can be described by the Michaelis-Menten equation. When B. oleracea was grown under sulfate (SO(4)(2-))-deprived conditions, plants developed sulfur (S) deficiency symptoms and H(2)S uptake kinetics were substantially altered. Shoots of SO(4)(2-)-deprived plants had a lower affinity to H(2)S uptake, whereas the maximal H(2)S uptake rate was higher. When SO(4)(2-)-deprived plants were simultaneously exposed to 0.2 &mgr;l l(-1) H(2)S all S deficiency symptoms disappeared and H(2)S uptake kinetics returned rapidly to values observed for S-sufficient shoots. The activity of the H(2)S-fixating enzyme O-acetylserine (thiol)lyase was hardly affected upon either prolonged H(2)S exposure or SO(4)(2-) deprivation. Evidently, the activity of O-acetylserine (thiol)lyase was not the rate-limiting step in the H(2)S uptake by shoots. The significance of the in situ availability and rate of synthesis of the substrate O-acetylserine for O-acetylserine (thiol)lyase as determining factor in the uptake kinetics of H(2)S needs further evaluation.     

The roles of three functional sulphate transporters involved in uptake and translocation of sulphate in Arabidopsis thaliana

To investigate the uptake and long-distance translocation of sulphate in plants, we have characterized three cell-type-specific sulphate transporters, Sultr1;1, Sultr2;1 and Sultr2;2 in Arabidopsis thaliana. Heterologous expression in the yeast sulphate transporter mutant indicated that Sultr1;1 encodes a high-affinity sulphate transporter (Km for sulphate 3.6 +/- 0.6 microM), whereas Sultr2;1 and Sultr2;2 encode low-affinity sulphate transporters (Km for sulphate 0.41 +/- 0.07 mM and >/= 1.2 mM, respectively). In Arabidopsis plants expressing the fusion gene construct of the Sultr1;1 promoter and green fluorescent protein (GFP), GFP was localized in the lateral root cap, root hairs, epidermis and cortex of roots. beta-glucuronidase (GUS) expressed with the Sultr2;1 promoter was specifically accumulated in the xylem parenchyma cells of roots and leaves, and in the root pericycles and leaf phloem. Expression of the Sultr2;2 promoter-GFP fusion gene showed specific localization of GFP in the root phloem and leaf vascular bundle sheath cells. Plants continuously grown with low sulphate concentrations accumulated high levels of Sultr1;1 and Sultr2;1 mRNA in roots and Sultr2;2 mRNA in leaves. The abundance of Sultr1;1 and Sultr2;1 mRNA was increased remarkably in roots by short-term stress caused by withdrawal of sulphate. Addition of selenate in the sulphate-sufficient medium increased the sulphate uptake capacity, tissue sulphate content and the abundance of Sultr1;1 and Sultr2;1 mRNA in roots. Concomitant decrease of the tissue thiol content after selenate treatment was consistent with the suggested role of glutathione (GSH) as a repressive effector for the expression of sulphate transporter genes.     

Morphological and physiological responses of rice (Oryza sativa) to limited phosphorus supply in aerated and stagnant solution culture

BACKGROUND AND AIMS: Rain-fed lowland rice commonly encounters stresses from fluctuating water regimes and nutrient deficiency. Roots have to acquire both oxygen and nutrients under adverse conditions while also acclimating to changes in soil-water regime. This study assessed responses of rice roots to low phosphorus supply in aerated and stagnant nutrient solution. METHODS: Rice (Oryza sativa 'Amaroo') was grown in aerated solution with high P (200 micro m) for 14 d, then transferred to high or low (1.6 micro m) P supply in aerated or stagnant solution for up to 8 d. KEY RESULTS: After only 1 d in stagnant conditions, root radial oxygen loss (ROL) had decreased by 90 % in subapical zones, whereas near the tip ROL was maintained. After 4 d in stagnant conditions, maximum root length was 11 % less, and after 8 d, shoot growth was 25 % less, compared with plants in aerated solution. The plants in stagnant solution had up to 19 % more adventitious roots, 24 % greater root porosity and 26 % higher root/shoot ratio. Rice in low P supply had fewer tillers in both stagnant and aerated conditions. After 1-2 d in stagnant solution, relative P uptake declined, especially at low P supply. Aerated roots at low P supply maintained relative P uptake for 4 d, after which uptake decreased to the same levels as in stagnant solution. CONCLUSIONS: Roots responded rapidly to oxygen deficiency with decreased ROL in subapical zones within 1-2 d, indicating induction of a barrier to ROL, and these changes in ROL occurred at least 2 d before any changes in root morphology, porosity or anatomy were evident. Relative P uptake also decreased under oxygen deficiency, showing that a sudden decline in root-zone oxygen adversely affects P nutrition of rice.     

不同磷效率小麦品种对缺磷胁迫反应的比较

在营养液培养条件下,以根据相对产量为指标筛选出的6个不同磷效率的小麦(Triticum aestivum L.)品种为材料,对其苗期在缺磷条件下生长、根冠磷含量及其分配,以及叶片韧皮部汁液中磷浓度等进行了比较研究。结果表明,缺磷抑制植株地上部生长,但刺激根系生长,导致植株根／冠比增加。无论在供磷或缺磷条件下,磷高效品种的根冠生长速率都低于磷低效品种。缺磷导致植株体内的磷含量下降与根系相比,地上部磷含量的下降速率更快。但在缺磷条件下,不同磷效率的小麦品种根冠间的磷分配变化没有差异。研究发现,在正常供磷条件下,磷高效小麦品种的叶片韧皮部汁液中磷浓度较低,而磷低效品种的叶片韧皮部汁液中磷浓度较高。但开始缺磷后,磷高效品种的叶片韧皮部汁液中的磷浓度下降较慢,使其相对磷浓度较高。缺磷后10天,磷低效品种叶片韧皮部汁液中的磷浓度为供磷对照的35．9％,而磷高效品种叶片韧皮部汁液中的磷浓度为供磷对照的59％。     

香草醛对杉木幼苗养分吸收的影响

 通过模拟实验研究了不同浓度的香草醛对杉木（Cunninghamia lanceolata）幼苗养分吸收和根系活力的影响,结果发现,当浓度为1 mmol·L-1时,香草醛能够显著抑制杉木幼苗对NO－3、NH+4、SO24-及HPO24-离子的吸收和根系活力（p<0.01）,而浓度为1×10-2 mmol·L-1时香草醛却促进了杉木幼苗对HPO24-离子的吸收（p<0.01）。这说明高浓度的香草醛能够通过化感作用对杉木幼苗产生影响,降低杉木幼苗的根系活力,进而减少了杉木幼苗对养分离子的吸收,从而影响了杉木幼苗的生长。