Transport du sulfate à travers la double membrane limitante, ou enveloppe, des chloroplastes d'épinard

Evidence is presented for low rates of carriermediated uptake of sulphate, thiosulphate and sulphite into the stroma of the C3 plant Spinacia oleracea. Uptake of sulphate in the dark was followed using two techniques (1) uptake of sulphate [³⁵S] as determined by silicon oil centrifugal filtration and (2) uptake as indicated by inhibition of CO₂-dependant O₂ evolution rates after addition of sulphate.Sulphate, thiosulphate and sulphite were transported across the envelope leading to an accumulation in the chloroplasts. Sulphate transport had saturation kinetics of the Michaelis-Menten type (Vmax : 25 μmoles . mg⁻¹ chl . h⁻¹ at 22°C ; Km : 2.5 mM). The rate of transport for sulphate was not influenced either by illumination or pH change in the external medium. Phosphate was a competitive inhibitor of sulphate uptake by chloroplasts (Ki : 0.7 mM, fig. 1). The rate of transport for phosphate appeared to be much higher than for sulphate. When the chloroplasts were pre-loaded with labelled sulphate, radioactivity was rapidly released after addition of phosphate into the external medium. Consequently, the transport of sulphate occurs by a strict counter-exchange : for each molecule of sulphate entering the chloroplast, one molecule of phosphate leaves the stroma, and vice-versa.The uptake of sulphate by isolated intact chloroplasts exchanging for internal free phosphate induced a lower rate of photophosphorylation, which in turn inhibited CO₂-dependent O₂ evolution.The presence, on the inner membrane of the chloroplast envelope, of a specific sulphate carrier, distinct from the phosphate translocator, is discussed.     

A transport kinetic concept of ion uptake from soil by plants

Summary A suction filter method is described, that allows soil solution to be isolated from soils with varying moisture content and with plant-cover without disturbance of actively growing plant roots. The reproducibility of the method was confirmed by the fact that the relative standard deviations of cation and anion concentrations in isolated soil solutions were of the same order of magnitude as those of corresponding chemical analyses. Evidence for soil solution remaining unchanged by the described isolation procedure was furnished by the close agreement between the thermodynamic solubility product and the calculated solubility product of dehydrated calcium sulphate in soil solution, based on estimated Ca²⁺ and SO ₂₋ ⁴ concentrations in isolated soil solution and corresponding activity coefficients. The uses of the method in connection with studies in plant nutrition are discussed.     

Exogenously applied sulphate as a tool to investigate transport and reduction of chromate in the duckweed Spirodela polyrhiza

The accumulation of chromium in Spirodela polyrhiza was investigated in the presence and absence of exogenously applied sulphate. Precultivation (10 d) at minimum sulphate concentration (0.013 m m versus 1 m m in controls) enhanced the rate of chromium accumulation. This effect was caused by the increased number of sulphate transporters which transport chromate into cells. Chromate and sulphate compete for the available sulphate transporters. The kinetics of reduction Cr(VI)→Cr(V) was investigated by l -band electron paramagnetic resonance (EPR) spectroscopy. The kinetic model developed previously (Appenroth et al., Journal of Inorganic Biochemistry 78, 235–242, 2000) was refined and extended to include chromate transport and reduction in the presence of competing ions. The following conclusions were drawn from the fitting procedure: without simultaneously applied sulphate, the rate constant of Cr(VI) transport from apoplast into plant cells and the rate constant of Cr(VI) to Cr(V) reduction within the apoplast are comparable (7.0 versus 5.7 h⁻¹) demonstrating that these two processes are competing. Moreover, the rate constant of reduction Cr(V)→Cr(III) is much lower within cells than in apoplast (0.39 versus 7.0 h⁻¹) showing that Cr(V) is stabilized in the symplast. The rate of transport of Cr(VI) into plant cells is at least one order of magnitude higher than that of Cr(V) or Cr(III). The treatment with sulphate (10 m m ) decreases the rate constant of the transport of Cr(VI) into cells (2.0 h⁻¹) confirming the competition of chromate and sulphate for the same transporters. Simultaneously, the rate constant of Cr(V)→Cr(III) reduction is increased in the apoplast (by the factor of 3) and decreased in the symplast (by the factor of 5). Treatment with higher sulphate concentrations (100 m m ) increases the accumulation of chromium by enhancing the rate constant of Cr(VI) transport into cells leaving other processes essentially unchanged. We suggest that 100 m m sulphate opens a new pathway for chromate transport into cells.     

Plant sulphate transporters: co-ordination of uptake, intracellular and long-distance transport

Proton/sulphate co-transport in the plasma membrane of root cells is the first step for the uptake of sulphate from the environment by plants. Further intracellular, cell-to-cell and long-distance transport must fulfil the requirements for sulphate assimilation and source/sink demands within the plant. A gene family of sulphate transporters, which may be subdivided into five groups, has been identified with examples from many different plant species. For at least two groups, proton/sulphate co-transport activity has been confirmed. It appears that each group represents sulphate transporters with distinct kinetic properties, patterns of expression, and cell/tissue specificity related to specific roles in the uptake and allocation of sulphate. High-affinity sulphate uptake and low-affinity vascular transport, as well as vacuolar efflux, are controlled by the nutritional status of the plant. Most notably there is an apparent increase in capacity for cellular sulphate uptake and vacuolar efflux when sulphur supply is limiting. Within the groups, the individual sulphate transporters may be further subdivided by differences in temporal, cellular and tissue expression. Many of the transporters are regulated by the nutritional status of the individual tissues, to optimize sulphate movement within and between sink and source organs.     

Potential canopy influences on the isotopic composition of nitrogen and sulphur in atmospheric deposition

Isotopic studies of nitrogen and sulphur inputs to plant/soil systems commonly rely on limited published data for the ¹⁵N/¹⁴N and ³⁴S/³²S ratios of nitrate, ammonium and sulphate in rainfall. For systems with well-developed plant canopies, however, inputs of these ions from dry deposition or particulates may be more important than rainfall. The manner in which isotopic fractionation between ions and gases may lead to dry deposition and particulates having ¹⁵N/¹⁴N or ³⁴S/³²S ratios different from those of rainfall is considered. Data for rainfall and throughfall in coniferous plantations are then discussed, and suggest that: (1) in line with expectations, nitrate washed from the canopy has ¹⁵N/¹⁴N ratios higher than those in rainfall; (2) the ¹⁵N/¹⁴N ratios of ammonium washed from the canopy are variable, with high ratios being found for canopies of higher pH in conditions of elevated ambient ammonia gas concentrations; and (3) in accord with expectations and previous work, ³⁴S/³²S ratios of sulphate washed from the canopy are not substantially different from those in rainfall. The study suggests that if atmospheric inputs are relevant to isotopic studies of the sources of nitrogen for canopied systems, then confident interpretation will require analysis of these inputs. Received: 3 March 1996 / Accepted: 28 September 1996     

Long-term effects of SO2 on plants,SO2 metabolism and regulation of intracellular pH

The impact of SO₂ on the ionic balance of plants and its implications for intracellular pH regulation was studied to find explanations for long-term effects of SO₂. When sulphur, taken up as SO₂ by the shoots of plants, is not assimilated in organic compounds, but stored as sulphate, an equivalent amount of H⁺ is produced. These H⁺ ions are not buffered chemically, but removed by metabolic processes.On the basis of knowledge on metabolic buffering mechanisms a conceptual model is proposed for the removal of shoot-generated H⁺ by (i) OH^- ions, produced in the leaves when sulphate and nitrate are assimilated in organic compounds and/or by (ii) OH^- ions produced by decarboxylation of organic anions (a biochemical pH stat mechanism). The form in which nitrogen is supplied largely determines the potential of the plant to neutralize H⁺ in the leaves during SO₂ uptake by the proposed mechanisms.In field experiments with N₂ fixing Vicia faba L. crops, the increase of sulphate in the shoots of SO₂-exposed plants was equivalent in charge to the decrease of organic anion content, calculated as the difference between inorganic cation content (C) and inorganic anion content (A), indicating that H⁺ ions produced in the leaves following SO₂ uptake were partly removed by OH^- from sulphate reduction and partly by decarboxylation of organic anions.The appearance of chronic SO₂ injury (leaf damage) in the field experiment at the end of the growing period is discussed in relation to the impact of SO₂ on the processes involved in regulation of intracellular pH. It is proposed that the metabolic buffering capacity of leaf cells is related to the rates of sulphate and nitrate reduction and the import rate of organic anions, rather than to the organic anion content in the vacuoles of the leaf cells.     

The oxygen isotopic signature of soil‐ and plant‐derived sulphate is controlled by fertilizer type and water source

The oxygen isotope signature of sulphate (δ¹⁸O_sulphate) is increasingly used to study nutritional fluxes and sulphur transformation processes in a variety of natural environments. However, mechanisms controlling the δ¹⁸O_sulphate signature in soil–plant systems are largely unknown. The objective of this study was to determine key factors, which affect δ¹⁸O_sulphate values in soil and plants. The impact of an ¹⁸O‐water isotopic gradient and different types of fertilizers was investigated in a soil incubation study and a radish (Raphanus sativus L.) greenhouse growth experiment. Water provided 31–64% of oxygen atoms in soil sulphate formed via mineralization of organic residues (green and chicken manures) while 49% of oxygen atoms were derived from water during oxidation of elemental sulphur. In contrast, δ¹⁸O_sulphate values of synthetic fertilizer were not affected by soil water. Correlations between soil and plant δ¹⁸O_sulphate values were controlled by water δ¹⁸O values and fertilizer treatments. Additionally, plant δ³⁴S data showed that the sulphate isotopic composition of plants is a function of S assimilation. This study documents the potential of using compound‐specific isotope ratio analysis for investigating and tracing fertilization strategies in agricultural and environmental studies.     

Sulphur fractionation in calcareous soils and bioavailability to plants

Sulphur fractionation and availability to plants are poorly understood in calcareous soils. Sixty-four calcareous soils containing varying amounts of CaCO₃ were collected from ten provinces in China and their S fractions determined. Organic S was the predominant fraction of S, accounting for on average 77% of the soil total S. The amounts of adsorbed sulphate were found to be negligible. 1 M HCl extracted substantially more sulphate than either 0.01 M CaCl₂ or 0.016 M KH₂PO₄, indicating the existence of water-insoluble but acid-soluble sulphate, probably in the form of sulphate co-precipitated with CaCO₃. The concentrations of water-insoluble sulphate correlated positively with the contents of CaCO₃ and accounted for 0.03–40.3% (mean 11.7%) of soil total S. To test the bioavailability of water-insoluble sulphate, a sulphate-CaCO₃ co-precipitate labelled with ³⁵S was prepared and added to a calcareous soil in a pot experiment with either NH₄⁺ or NO₃^– as the N source. In 29 days, wheat plants took up 10.6% and 3.0% of the ³⁵S added to the soil in the NH₄⁺ and NO₃^– treatments, respectively. At the end of the pot experiment, the decrease of water-insoluble, acid-soluble, sulphate was more apparent in the NH₄⁺ than in the NO₃^– treatment. The results indicate that sulphate co-precipitated with CaCO₃ in calcareous soils may become partly available for plant uptake, depending on rhizosphere pH, if the field precipitate is similar to the laboratory prepared sample studied.     

Phenolsulphotransferase activity in the developing mosquito (Aedes togoi)

Phenolsulphotransferase (PST) activity was measured with N-acetyldopamine (NADA) and harmol as substrates in the larvae, pupae and adult mosquito (Aedes togoi). Only the newly emerged pupae showed high PST activity 1–4 hr after pupation. PST activity could also be measured in each individual pupa, with the female exhibiting significantly higher specific activity (30 ± 3.7 pmol NADA [³⁵S]sulphate/min per mg protein) than the males (13.6 ± 2.9 pmol NADA [³⁵S]sulphate/min per mg protein). The optimum pH for the PST reaction was 9.0. The K_m values for [³⁵S]PAP were 0.55 and 2.5 μM when measured with NADA and harmol as acceptors, respectively; the corresponding K_m values for these two substrates were 2.61 and 16.1 μM. Studies with 2,6-dichloro-4-nitrophenol showed a dose-dependent inhibition of PST. Sulphate conjugation of NADA from ATP and sodium [³⁵S]sulphate was also demonstrated with pupal extracts, with pH optimum between 8.6 and 9.0. The specific activity of this overall sulphate conjugation, measured in the female pupal extract was 5.08 pmol NADA [³⁵S]sulphate/min per mg protein and 1.68 pmol harmol [³⁵S]sulphate/min per mg protein. The importance and possible function of sulphate conjugation of NADA in insects is discussed.     

Sulphate uptake and xylem loading of young pea (Pisum sativum L.) seedlings

Sulphate uptake and xylem loading was analysed in young pea (Pisum sativum) seedlings. The rate of sulphate uptake into intact 8-days-old pea seedlings (determined by a 1 h exposure to radiolabelled sulphate in the nutrient solution) was 585 nmol sulphate g^–1 root fresh weight h^–1. When the cotyledons were removed on day 6 the 8-days-old seedlings took up only 7% of the controls. Interruption of the phloem transport by steam girdling of the stem or the root (1 h before incubation with radiolabelled sulphate) diminished sulphate uptake by approximately 50%. The addition of sucrose to the nutrient solution during incubation did not restore sulphate uptake rates indicating that the decrease was not due to a lack of energy. Apparently, a signal from the shoot and/or the cotyledons is necessary to stimulate sulphate uptake into the roots of pea seedlings. Glutathione fed to the roots for 3 h prior to incubation with radiolabelled sulphate diminished sulphate uptake by approximately 50%. The relative proportion of the sulphate taken up that was loaded into the xylem remained unchanged (between 7 and 9% of total uptake), even when the stem was girdled above the cotyledons or when the seedlings were pre-exposed to glutathione. Only removal of the cotyledons or girdling of the root below the cotyledons increased the proportion of sulphate loaded into the xylem to 13–15% of total uptake upon exposure to glutathione. Apparently, a signal from the cotyledons represses xylem loading to some extent.     

Efficient Plant Regeneration via Shoot Tip Explant in Jatropha curcas L

An efficient regeneration method via shoot tip explant has been developed for Jatropha curcas, which is a medicinally as well as economically important plant. Shoot tips were proliferated on MS medium incorporated with BAP (2.0 mg l^?1) and IAA (0.5 mg l^?1) along with adenine sulphate, glutamine and activated charcoal. In vitro produced shoots were induced to root on IBA (0.5–5.0 mg l^?1) added to half strength MS medium. The highest frequency of root induction was on the medium with 3.0 mg l^?1 IBA. Regenerated plantlets were successfully transferred to field after initial acclimatization.     

Phosphate dependent,ruthenium red insensitive Ca2+ uptake in mung bean mitochondria

Energy linked Ca²⁺ uptake into mung bean mitochondria has been studied. Using arsenazo III as a monitor of extramitochondrial Ca²⁺, we observe a respiration-linked uptake of Ca²⁺ which requires phosphate and is insensitive to ruthenium red. The rate of uptake is of the order of 5 nmol/mg protein/min. Acetate, sulphate and thiosulphate are unable to support Ca²⁺ uptake. The results suggest that although plant mitochondria accumulate Ca²⁺ in an energy dependent fashion, it is not via a simple electrophoretic uniport mechanism.     

Interactive effects of mycorrhization and elevated atmospheric CO2 on sulphur nutrition of young pedunculate oak (Quercus robur L.) trees

Pedunculate oak (Quercus robur L.) was germinated and grown at ambient CO₂ concentration and 650 μmol mol^?1 CO₂ in the presence and absence of the ectomycorrhizal fungus Laccaria laccata for a total of 22 weeks under nonlimiting nutrient conditions. Sulphate uptake, xylem loading and exudation were analysed in excised roots. Despite a relatively high affinity for sulphate (K_M= 1.6 mmol m^?3), the rates of sulphate uptake by excised lateral roots of mycorrhizal oak trees were low as compared to herbaceous plants. Rates of sulphate uptake were similar in mycorrhizal and non-mycorrhizal roots and were not affected by growth of the trees at elevated CO₂. However, the total uptake of sulphate per plant was enhanced by elevated CO₂ and further enhanced by elevated CO₂ and mycorrhization. Sulphate uptake seemed to be closely correlated with biomass accumulation under the conditions applied. The percentage of the sulphate taken up by mycorrhizal oak roots that was loaded into the xylem was an order of magnitude lower than previously observed for herbaceous plants. The rate of xylem loading was enhanced by mycorrhization and, in roots of mycorrhizal trees only, by growth at elevated CO₂. On a whole-plant basis this increase in xylem loading could only partially be explained by the increased growth of the trees. Elevated CO₂ and mycorrhization appeared to increase greatly the sulphate supply of the shoot at the level of xylem loading. For all treatments, calculated rates of sulphate exudation were significantly lower than the corresponding rates of xylem loading of sulphate. Radiolabelled sulphate loaded into the xylem therefore seems to be readily diluted by unlabelled sulphate during xylem transport. Allocation of reduced sulphur from oak leaves was studied by flap-feeding radiolabelled GSH to mature oak leaves. The rate of export of radioactivity from the fed leaves was 4–5 times higher in mycorrhizal oak trees grown at elevated CO₂ than in those grown at ambient CO₂. Export of radiolabel proceeded almost exclusively in a basipetal direction to the roots. From these experiments it can be concluded that, in mycorrhizal oak trees grown at elevated CO₂, the transport of sulphate to the shoot is increased at the level of xylem loading to enable increased sulphate reduction in the leaves. Increased sulphate reduction seems to be required for the enhanced allocation of reduced sulphur to the roots which is observed in trees grown at elevated CO₂. These changes in sulphate and reduced sulphur allocation may be a prerequisite for the positive effect of elevated CO₂ on growth of oak trees previously observed.     

The influence of sulphate on substrate utilization in a thermophilic sewage sludge digestor

Summary Bacterial sulphate reduction and the interaction between sulphate reduction and methane production was studied in an unadapted and sulphate-adapted thermophilic anaerobic sludge digestor. Addition of sulphate to a concentration of 5 mm (100 times the background level) did not influence gas production or volatile fatty acid concentration compared to the control digestor. When sulphate reduction was not limited by the sulphate concentration, the sulphate-adapted digestor had a sulphate reduction rate of 910 mol l^–1 day compared with 17 mol l^–1 day in the control digestor. The results indicate that the potential for sulphate reduction is low in a thermophilic sewage sludge digestor receiving a low sulphate concentration. Counts of sulphate-reducing bacteria and methanogens showed that sulphate-reducing bacteria were found only in significant numbers in the sulphate-adapted digestor and only with H₂/CO₂ as substrate. Only low numbers of acetate-utilizing sulphate-reducing bacteria were found in both digestors. When using radio-labelled acetate, the relative percentage of 2-labelled acetate converted to CO₂ was two to four times higher in the sulphate-adapted digestor compared to the control digestor. These results suggest that oxidation of acetate seems to play a larger role in the sulphate-adapted digestor.Offprint requests to: B. K. Ahring     

A cell line of Nicotiana sylvestris with resistance to kanamycin and streptomycin

Summary Cell lines resistant to 50 g ml^-1 kanamycin sulphate were isolated from cell suspension cultures initiated from a haploid Nicotiana sylvestris plant. One line, KR103, has been studied in detail. Resistance of this line was shown to be stable in the absence of the drug. KR103 was found also to be resistant to streptomycin, another inhibitor of 70S ribosomal protein synthesis.Both KR103 and the sensitive line convert kanamycin, but not streptomycin, to a form which is no longer effective in a bacterial bioassay, while maintaining its toxicity for sensitive plant cells.KR103 is defective in morphogenesis and plastid development.     

The enzymatic sulphation of heparan sulphate by hen's uterus

A sulphotransferase preparation from hen's uterus catalysed the transfer of sulphate from adenosine 3′-phosphate 5′-sulphatophosphate to N-desulphated heparan sulphate, heparan sulphate, N-desulphated heparin and dermatan sulphate. Heparin, chondroitin sulphate and hyaluronic acid were inactive as substrates for the enzyme. N-desulphated heparin was a much poorer substrate for the enzyme than N-desulphated heparan sulphate suggesting that properties of the substrate other than available glucosaminyl residues influenced enzyme activity. N-acetylation of N-desulphated heparin and N-desulphated heparan sulphate reduced their sulphate acceptor properties so it was unlikely that the N-acetyl groups of heparan sulphate facilitated its sulphatiion. Direct evidence for the transfer of [³⁵S]sulphate to amino groups of N-desulphated haparan sulphate was obtained by subsequent isolation of glucosamine $\text{N-[}{}^{35}\text{S}\text{]}\text{sulphate}$ from heparan [³⁵S]sulphate product. This was made possible through the use of a flavobacterial enzyme preparation which contained “heparitinase” activity but had been essentially freed of sulphatases. Attempts to transfer [³⁵S]sulphate to glucosamine or N-acetylglucosamine were unsuccessfull.     

Sulphate activation in higher plants

Summary ATP-sulphurylase was detected in extracts of roots and leaves of several species of higher plant. The enzyme occurs in the supernatant fraction, has a pH optimum of 8.0 and an absolute requirement for Mg²⁺ ions. Sulphurylase activity is inhibited by selenate and molybdate but not by cysteine, methionine, glutathione, or thiol reagents. The synthesis of sulphurylase by turnip, lettuce, tomato, and Lemna plants grown under aseptic conditions is neither induced by sulphate nor repressed by cystine, and in the latter respect sulphate activation in plants differs from than in micro-organisms. APS-kinase could not be detected in extracts of any tissue although its product was stable under the conditions used. Sulphate reduction in higher plants may thus proceed via adenylysulphate and not via phosphoadenylylsulphate as in many micro-organisms.     

Contribution of N2 fixing cyanobacteria to rice production: availability of nitrogen from 15N-labelled cyanobacteria and ammonium sulphate to rice

This study investigate the potential contribution of nitrogen fixation by indigenous cyanobacteria to rice production in the rice fields of Valencia (Spain). N₂-fixing cyanobacteria abundance and N₂ fixation decreased with increasing amounts of fertilizers. Grain yield increased with increasing amounts of fertilizers up to 70 kg N ha^-1. No further increase was observed with 140 kg N ha^-1. Soil N was the main source of N for rice, only 8–14% of the total N incorporated by plants derived from ¹⁵N fertilizer. Recovery of applied ¹⁵N-ammonium sulphate by the soil–plant system was lower than 50%. Losses were attributed to ammonia volatilization, since only 0.3–1% of applied N was lost by denitrification. Recovery of ¹⁵N from labeled cyanobacteria by the soil–plant system was higher than that from chemical fertilizers. Cyanobacterial N was available to rice plant even at the tillering stage, 20 days after N application. This revised version was published online in June 2006 with corrections to the Cover Date.     

Structure and metabolism of rat liver heparan sulphate

Rat liver cells grown in primary cultures in the presence of [³⁵S]sulphate synthesize a labelled heparan sulphate-like glycosaminoglycan. The characterization of the polysaccharide as heparan sulphate is based on its resistance to digestion with chondroitinase ABC or hyaluronidase and its susceptibility to HNO₂ treatment. The sulphate groups (including sulphamino and ester sulphate groups) are distributed along the polymer in the characteristic block fashion. In ³H-labelled heparan sulphate, isolated after incubation of the cells with [³H]galactose, 40% of the radioactive uronic acid units are l-iduronic acid, the remainder being d-glucuronic acid. The location of heparan sulphate at the rat liver cell surface is demonstrated; part of the labelled polysaccharide can be removed from the cells by mild treatment with trypsin or heparitinase. Further, a purified plasma-membrane fraction isolated from rats previously injected with [³⁵S]sulphate contains radioactively labelled heparan sulphate. A proteoglycan macromolecule composed of heparan sulphate chains attached to a protein core can be solubilized from the membrane fraction by extraction with 6m-guanidinium chloride. The proteoglycan structure is degraded by treatment with papain, Pronase or alkali. The production of heparan [³⁵S]sulphate by rat liver cells incubated in the presence of [³⁵S]sulphate was followed. Initially the amount of labelled polysaccharide increased with increasing incubation time. However, after 10h of incubation a steady state was reached where biosynthetic and degradative processes were in balance.