Remediation of atmospheric sulfur and ammonia by wetland plants: development of a study method.

In the context of S and N pollutant remediation, this study aimed to develop a methodology to test the ability of wetland plants to reduce atmospheric pollution by S and N. A methodology using 34S and 15N-labeled Sinapsis alba compost and five species (trap plants) used to fix volatile compounds was developed. 18.66% of 34S and 40.63% of 15N produced by Sinapsis alba compost, equivalent to 67 mg of S and 1611 mg of N, were recovered in trap plants, a negligible proportion of the labeling was found in the culture substrate. 34S and 15N atom% excess were two to ten times higher in leaves than in roots. Agrostis stolonifera, Symphytum officinale, and Lythrum salicaria were more efficient to use atmospheric inorganic sources of S and N than Mentha aquatica and Carex riparia. A low concentration of sulfate in the leaf laminas, a high specific leaf area, and a low leaf dry mass content could represent trait patterns that explain higher abilities to fix pollutants. This study confirms that plants can be used to remediate inorganic atmospheric pollution and highlights the importance of plant screening for this environmental function.Novelty statementThe removal efficiency of botanical biofiltration is well-documented for Volatile Organic pollutants, but little is known concerning Volatile Inorganic pollutants, such as SO2 and NH3 which can also constitute plant nutrients.We developed a methodology based on the use of 34S and 15N-labeled mustard compost to study the ability of wetland plant species to fix volatile N and S pollutants. This methodology was effective as 19% of 34S and 41% of 15N lost by mustard compost were recovered in trap plants. Among the species used as "trap plants" Agrostis stolonifera, Symphytum officinale, and Lythrum salicaria appeared more efficient to use atmospheric inorganic sources of S and N than Mentha aquatica and Carex riparia.

Is white clover able to switch to atmospheric sulphur sources when sulphate availability decreases?

Sulphur (S) is one of the very few nutrients that plants can absorb either through roots as sulphate or via leaves in a gas form such as SO2 or H2S. This study was realized in a non-S-enriched atmosphere and its purpose was to test whether clover plants can increase their ability to use atmospheric S when sulphate availability decreases. A novel methodology measuring the dilution of (34)S provided from a nutrient solution by atmospheric (32)S was developed to measure S acquisition by Trifolium repens L. Clones of white clover were grown for 140 d in a hydroponic system with three levels of sulphate concentrations. S concentration in plants decreased with S deficiency and plant age. In the experimental conditions used here, S derived from atmospheric deposition (Sdad) constituted from 36% to 100% of the total S. The allocation of S coming from atmospheric and pedospheric sources depends on organs and compounds. Nodules appeared as major sinks for sulphate. A greater proportion of atmospheric S was observed in buffer-soluble proteins than in the insoluble S fraction. Decreasing the S concentration in the nutrient solution resulted in an increase in the Sdad:leaf area ratio and in an increase in the leaf:stolon and root:shoot mass ratios, suggesting that a plasticity in the partitioning of resources to organs may allow a higher gain of S by both roots and leaves. This study shows that clover can increase its ability to use atmospheric S even at low concentration when pedospheric S availability decreases.

How does sulphur availability modify N acquisition of white clover (Trifolium repens L.)?

The role of S in legume growth, N uptake, and N2 fixation was investigated using white clover (Trifolium repens L.) as a model species. We examined whether the effect of sulphate addition on N fixation resulted from a stimulation of host plant growth, a specific effect of S on nodulation, or a specific effect of S on nodule metabolism. Clones of white clover, inoculated with Rhizobium leguminosarum, were grown for 140 d in a hydroponic system with three levels of sulphate concentration (0 mM, 0.095 mM, and 0.380 mM). Nodule morphological and biochemical traits, such as root length, nodule biomass and volume, nodule protein contents (nitrogenase and leghaemoglobin obtained by an immunological approach), and root amino acid concentrations, were used to analyse the effect of sulphate availability on N2 fixation. The application of sulphate increased whole plant dry mass, root length, and nodule biomass, expressed on a root-length basis. N uptake proved less sensitive than N2 fixation to the effects of S-deficiency, and decreased as a consequence of the lower root length observed in S-deficient plants. N2 fixation was drastically reduced in S-deficient plants as a consequence of a low nodule development, but also due to low nitrogenase and leghaemoglobin production. This effect is likely to be due to down-regulation by a N-feedback mechanism, as, under severe S-deficiency, the high concentration of whole plant N and the accumulation of N-rich amino acids (such as asparagine) indicated that the assimilation of N exceeded the amount required for plant growth.

Increased proline loading to phloem and its effects on nitrogen uptake and assimilation in water-stressed white clover (Trifolium repens).

The aim of this study was to investigate the physiological significance of increased proline loading to phloem caused by water-deficit stress in relation to nitrogen (N) uptake and assimilation. N uptake and N assimilation were quantified by 15N tracing in well-watered (control) and water deficit-stressed white clover (Trifolium repens). De novo proline synthesis and proline loading to the phloem were also compared between treatments. The relationships among proline concentrations in phloem exudates, N uptake, and assimilation of newly absorbed N were assessed. The newly synthesized proline in the phloem exudates increased rapidly after 3 d of water deficit. The water-deficit treatment significantly reduced the maximum nitrate reductase activity (NRA), and also attenuated de novo synthesis of amino acids and proteins in the roots. The increase in proline concentrations in phloem exudates was closely related to reductions in NRA in the roots, N uptake, and the assimilation of newly absorbed N. The accumulation of proline induced in roots by exogenous proline and NH4Cl treatments was closely associated with the decrease in NRA. These results indicate that increased proline transport to roots via phloem caused by water deficit has a significant influence on the down-regulation of N uptake and the assimilation of newly absorbed N.

Concerted modulation of alanine and glutamate metabolism in young Medicago truncatula seedlings under hypoxic stress.

The modulation of primary nitrogen metabolism by hypoxic stress was studied in young Medicago truncatula seedlings. Hypoxic seedlings were characterized by the up-regulation of glutamate dehydrogenase 1 (GDH1) and mitochondrial alanine aminotransferase (mAlaAT), and down-regulation of glutamine synthetase 1b (GS1b), NADH-glutamate synthase (NADH-GOGAT), glutamate dehydrogenase 3 (GDH3), and isocitrate dehydrogenase (ICDH) gene expression. Hypoxic stress severely inhibited GS activity and stimulated NADH-GOGAT activity. GDH activity was lower in hypoxic seedlings than in the control, however, under either normoxia or hypoxia, the in vivo activity was directed towards glutamate deamination. (15)NH(4) labelling showed for the first time that the adaptive reaction of the plant to hypoxia consisted of a concerted modulation of nitrogen flux through the pathways of both alanine and glutamate synthesis. In hypoxic seedlings, newly synthesized (15)N-alanine increased and accumulated as the major amino acid, asparagine synthesis was inhibited, while (15)N-glutamate was synthesized at a similar rate to that in the control. A discrepancy between the up-regulation of GDH1 expression and the down-regulation of GDH activity by hypoxic stress highlighted for the first time the complex regulation of this enzyme by hypoxia. Higher rates of glycolysis and ethanol fermentation are known to cause the fast depletion of sugar stores and carbon stress. It is proposed that the expression of GDH1 was stimulated by hypoxia-induced carbon stress, while the enzyme protein might be involved during post-hypoxic stress contributing to the regeneration of 2-oxoglutarate via the GDH shunt.

Effect of the arbuscular mycorrhizal fungus Glomus fasciculatum on the uptake of amino nitrogen by Lolium perenne.

The ability of ryegrass (Lolium perenne L.) to take up and utilize aspartic acid (Asp) and serine (Ser,), and the effect of colonization of the roots by the arbuscular mycorrhizal (AM) fungus Glomus fasciculatum (Thax. sensu Gerd.) were studied. The seedlings were grown under controlled conditions in a series of micro-lysimeters. All plants were fed with a nutrient solution containing either nitrate, Asp or Ser as the sole N source. After 49 d, they were supplied with 15 N labeled nitrate. Asp or Ser for 1 h and harvested. AM colonization increased the growth and total N content of the plants in all cases. Similarly, the amount of Asp or Ser taken up was higher in AM than in control plants. There were no differences in biomass production between the nitrate and Ser-fed plants. However uptake rates were lower for Ser than for nitrate. Growth of the Asp-fed plants was significantly less than the other two treatments, and uptake of 15 N-Asp was lower than uptake of 15 N-Ser. Analysis of 15 N incorporation into the amino acids extracted from the roots suggests the hydrolysis of Ser followed by re-assimilation of the resulting ammonia via the GS-GOGAT cycle. There were no differences in the patterns of accumulation of amino acids in the root-zone of control and AM-ryegrass. The implication of these results for the pathway of nitrogen transfer between plants is discussed.

Active root-inhabiting microbes identified by rapid incorporation of plant-derived carbon into RNA.

Plant roots harbor a large diversity of microorganisms that have an essential role in ecosystem functioning. To better understand the level of intimacy of root-inhabiting microbes such as arbuscular mycorrhizal fungi and bacteria, we provided (13)CO(2) to plants at atmospheric concentration during a 5-h pulse. We expected microbes dependent on a carbon flux from their host plant to become rapidly labeled. We showed that a wide variety of microbes occurred in roots, mostly previously unknown. Strikingly, the greatest part of this unsuspected diversity corresponded to active primary consumers. We found 17 bacterial phylotypes co-occurring within roots of a single plant, including five potentially new phylotypes. Fourteen phylotypes were heavily labeled with the (13)C. Eight were phylogenetically close to Burkholderiales, which encompass known symbionts; the others were potentially new bacterial root symbionts. By analyzing unlabeled and (13)C-enriched RNAs, we demonstrated differential activity in C consumption among these root-inhabiting microbes. Arbuscular mycorrhizal fungal RNAs were heavily labeled, confirming the high carbon flux from the plant to the fungal compartment, but some of the fungi present appeared to be much more active than others. The results presented here reveal the possibility of uncharacterized root symbioses.

Characterization of alanine aminotransferase (AlaAT) multigene family and hypoxic response in young seedlings of the model legume Medicago truncatula.

Four alanine aminotransferases (AlaATs) are expressed in Medicago truncatula. In adult plants, two genes encoding mitochondrial isoforms m-AlaAT and alanine-glyoxylate aminotransferase (AGT), catalysing, respectively, reversible reactions of alanine/oxoglutarate<==>glutamate/pyruvate and alanine/glyoxylate<==>glycine/pyruvate, were expressed in roots, stems, and leaves. A gene encoding a cytosolic (c-AlaAT) isoform, catalysing the same reaction as m-AlaAT, was expressed specifically in leaves, while a gene encoding an isoform involved in branched chain amino acid metabolism was expressed in stems and roots. In young seedlings, only m-AlaAT and AGT were expressed in embryo axes. In hypoxic embryo axes, the amounts of transcript and putative protein of m-AlaAT (EC 2.6.1.2) increased while those of AGT (EC 2.6.1.44) decreased and in vivo enzyme activities changed as revealed by [(15)N]alanine and [(15)N]glutamate labelling. Under hypoxia, m-AlaAT catalysed only alanine synthesis while glutamate synthesis using alanine as amino donor was inhibited. As a result, alanine accumulated as the major amino acid in hypoxic seedlings instead of asparagine, in agreement with the involvement of the fermentative AlaAT pathway in hypoxia tolerance. Regulation of m-AlaAT at both the transcriptional and post-translational levels allowed for an increase in gene expression and orientation of the activity of the product of its transcription towards alanine synthesis under hypoxia. Labelling experiments showed that glycine synthesis occurred at the expense of either alanine or glutamate as amino donor, indicating that a glutamate-glyoxylate aminotransferase was operating together with AGT in Medicago truncatula seedlings. Both enzymes seemed to be inhibited by hypoxia, resulting in a very low amount of glycine in hypoxic seedlings.

Respective roles of the glutamine synthetase/glutamate synthase cycle and glutamate dehydrogenase in ammonium and amino acid metabolism during germination and post-germinative growth in the model legume Medicago truncatula.

Our objective was to determine the respective roles of the couple glutamine synthetase/glutamate synthase (GS/GOGAT) and glutamate dehydrogenase (GDH) in ammonium and amino acid metabolism during germination and post-germinative growth in the model legume Medicago truncatula Gaertn. For this aim, amino acids were analyzed by HPLC and changes in gene expression of several enzymes involved in N and C metabolism were studied by real-time quantitative reverse transcription-polymerase chain reaction. Among the enzymes studied, GDH showed the highest increase in gene expression (80-fold), specifically in the embryo axis and concomitant with the increase in ammonium content during post-germinative growth. In cotyledons, GDH gene expression was very low. Although in vitro GDH aminating activity was several times higher than its deaminating activity, in vivo 15NH4 incorporation into amino acids was completely inhibited by methionine sulfoximine, a GS inhibitor, indicating that GDH is not involved in ammonium assimilation/detoxification. Changes in the expressions of GS and GOGAT isoforms revealed that GS1b (EC 6.3.1.2) in concert with NADH-dependent GOGAT (EC 1.4.1.14) constitute the major route of assimilation of ammonium derived from reserve mobilization and glutamic acid/glutamine synthesis in germinating M. truncatula seeds. However, during post-germinative growth, although germination was held in darkness, expression of GS2 and Fd-GOGAT (EC 1.4.7.1) increased and expression of GS1b decreased in cotyledons but not in the embryo axis. 2-Oxoglutarate, the substrate of the transamination reaction, was provided by the cytosolic isoform of isocitrate dehydrogenase (EC 1.1.1.42). We suggest that GDH during post-germinative growth, specifically in the developing embryo axis, contributes to ammonium delivery to GS for glutamine synthesis in the absence of primary NO3- assimilation. Interestingly, this reaction also produces reducing power (NADH) in organs deprived of photosynthesis.