Florence Paynel

Root amino acid exudation: measurement of high efflux rates of glycine and serine from six different plant species

Amino acid concentration in the rhizosphere results from fluxes between plant roots, soil and microorganisms. In this context, root amino acid exudation process, composed of both efflux and influx, remains unclear. One main issue is to understand the selectivity of amino acid exudation resulting mainly in high proportions of glycine and serine in exudates compared to low proportions inside the root. To reach this point, a quantitative analysis of exudation with dissociated measurements of efflux from influx is needed. We measured efflux and influx by supplying 15N-labelled glycine or serine for a short time of exposure at ecologically relevant concentrations to plants of white clover (Trifolium repens L.), perennial ryegrass (Lolium perenne L.), maize (Zea mays L.), oilseed rape (Brassica napus L.), tomato (Lycopersicon esculentum Mill.) and alfalfa (Medicago sativa L.). Efflux was estimated by the increase of 14N content of amino acids in root exudates and influx was estimated by the increase of 15N content in plant tissue. Glycine efflux exceeded influx for all six species and was much higher in Fabaceae than in Poaceae. Serine efflux exceeded influx in alfalfa, white clover and rape. We conclude that presence of glycine and serine in root bathing solutions results from high glycine and serine efflux rates, observed in all six species studied here. The physiological and ecological significances of these high efflux rates are discussed in the context of N metabolism and plant–soil–microorganisms interactions.

A study of 15N transfer between legumes and grasses

The overuse of classical N fertilisers contributes substantially to environmental degradation by pollution of groundwater by nitrates. This leaching of N in waters is also an economic flaw for farmers because only a part of the fertiliser is used by the plants. Here, systems involving mixtures of legumes and grasses represent a sustainable alternative because legumes can fix atmospheric N2 using symbiotic microbes. N transfer in those mixtures has been thoroughly investigated but little is known concerning the effect of N fertiliser on N transfer between N-fixing legumes and companion grasses. In white clover (Trifolium repens L.) — perennial ryegrass (Lolium perenne L.) associations, N is transferred mostly through rhizodeposition into the soil by clover followed by re-uptake by ryegrass. Rhizodeposition of N occurs through senescence and decomposition of legume tissue or through exudation of N compounds by living cells. Ammonium and amino acids are the main compounds exuded and their exudation is thought to occur by passive diffusion attributed to a concentration gradient from root to soil. In this study, we test the hypothesis that greater N transfer from clover to grass, as seen in N-rich soils or nutrient solutions, is due to greater N rhizodeposition brought about by higher ammonium and amino acid content of roots. The relations between N input, root N content, N net exudation and N transfer between legumes and grasses were investigated using 15N by growing white clover and perennial ryegrass with increasing N application in axenic microlysimeters or in pots. Ammonium and amino acid concentrations were measured in root tissues, in root bathing solutions and in soils. We found that mineral N application strongly reduced atmospheric N fixation by clover, from 3.0 to 0.9 mg per plant, and root amino acid content, from 164 to 49 nmoles per g dry weight, but had no effect on ammonium and amino acid concentrations in sterile exudates, showing for the first time that amino acid net exudation is independent of root content. In contrast, ammonium and amino acid concentrations in clover soils increased with N fixation, showing the link between N fixation and N rhizodeposition in soils. Nitrate application increased ryegrass root growth by 7–8 times, and transfer of N between clover and ryegrass (by 3 times). It is concluded that N fertiliser does not modify N exudation but decreases N fixation and ammonium rhizodeposition in soil by clover. N fertiliser increases N transfer between clover and ryegrass by increasing soil exploration by ryegrass and giving a better access to different available N sources, including the N compounds exuded from clover.

Characteristics of cadmium tolerance in 'Hermes' flax seedlings: contribution of cell walls.

Most flax (Linum usitatissimum) varieties are described as tolerant to high concentrations of Cd. The aim of the present paper was to better characterize this tolerance, by studying the responses of flax plantlets, cv Hermes, to 18d growth on 0.5mM Cd. In Cd-treated seedlings, the majority of Cd was compartmentalized in the roots. Analysis of other elements showed that only Fe concentration was reduced, while Mn increased. Growth parameters of Cd treated flax were only moderately altered, with similar mass tolerance-indices for roots and shoots. Tissue anatomy was unaffected by treatment. The effect on lipid peroxidation, protein carbonylation and antioxidative activities appeared low but slightly higher in roots. The most important impacts of Cd were, in all organs, cell expansion, cell-wall thickening, pectin cross-linking and increase of cell-wall enzymatic activities (pectin methylesterase and peroxidase). Thus, the role of the cell wall in Cd tolerance might be important at two levels: (i) in the reinforcement of the tissue cohesion and (ii) in the sequestration of Cd.

Temporal regulation of cell-wall pectin methylesterase and peroxidase isoforms in cadmium-treated flax hypocotyl

BACKGROUND AND AIMS In hypocotyls of flax (Linum usitatissimum) cadmium-induced reorientation of growth (i.e. an increase in expansion and a decrease in elongation) coincides with marked changes in the methylesterification and cross-linking of homogalacturonans within various cell-wall (CW) domains. The aim of the present study was to examine the involvement of pectin methylesterase (PME) and peroxidase (PER) in this cadmium-induced CW remodelling. METHODS CW proteins were extracted from hypocotyls of 10- and 18-d-old flax that had been treated or not treated with 0.5 mm Cd(NO(3))(2). PME and PER expression within these extracts was detected by LC/MS, by isoelectric focusing and enzyme activity assays. Transcript expression by RT-PCR of known flax PME and PER genes was also measured in corresponding samples. KEY RESULTS In cadmium-treated seedlings, PME activity increased as compared with controls, particularly at day 10. The increased activity of PME was accompanied by increased abundance of both a basic protein isoform (B2) and a particular transcript (Lupme5). In contrast, induction of PER activity by cadmium was highest at day 18. Among the four reported PER genes, Flxper1 and 3 increased in abundance in the presence of cadmium at day 18. CONCLUSIONS The temporal regulation of Lupme and Flxper genes and of their respective enzyme activities fits the previously reported cadmium-induced structural changes of homogalacturonans within the CWs. After PME-catalysed de-esterification of homogalacturonans, their cross-linking would depend on the activity of PERs interacting with calcium-dimerized blocks and reinforce the cell cohesion during the cadmium-induced swelling.

Characterization of mucilage polysaccharides, arabinogalactanproteins and cell-wall hemicellulosic polysaccharides isolated from flax seed meal: A wealth of structural moieties.

The present study aimed at analyzing the structural features of seed mucilage and cell-wall polysaccharides which accounted for 41% of the mass of flax meal (FM). A combination of high molar-mass mucilage-like polysaccharides (rhamnogalacturonan and arabinoxylan) was released from FM in water, together with arabinogalactan proteins and glucans. About half of FM homogalacturonans was extracted using a calcium chelator and boiling water. Hemicellulosic xyloglucans and xylans were further extracted with 1M KOH, in ∼13% FM-sugars yield. Structural characterization of the xyloglucan using specific enzyme hydrolysis, ion exchange chromatography (HPAEC) and matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectroscopy showed the presence of XXXG type xyloglucan, but also that of XXGG-structure, possibly characteristic of flax seeds. Hydrolysis of xylans with endo-(1→4)-β-D-xylanase, and analysis of the neutral and acidic oligosaccharides by MALDI-TOF-MS showed that xylan consisted of β-(1→4)-linked-D-xylopyranose backbone with some zones (DP 5-7) substituted with 4-O-MeGlcA\GlcA\Glc residues.

Kiwi fruit PMEI inhibits PME activity, modulates root elongation and induces pollen tube burst in Arabidopsis thaliana

Pectins are major components of primary cell wall that play a crucial role in plant development. After biosynthesis, pectins are secreted in the cell wall by Golgi-derived vesicles under a highly methylesterified form and are de-methylesterified by pectin methylesterases (PME). It is hypothesized that PME might be regulated by pectin methylesterase inhibitor (PMEI). In this paper, we show by isoelectric focalisation and subsequent zymogram that kiwi PMEI was able to inhibit Arabidopsis PME activity by forming a complex. The complexes were stable under a wide range of ionic strength and pH. Moreover, PMEI might be able to form a complex with basic PMEs including three PMEs strongly expressed in root and four PMEs expressed in pollen grains. Finally, exogenous treatment with kiwi PMEI was able to reduce the activity of cell wall resident PMEs with persistent effects such as an increase of the root growth and a dramatic effect on pollen tube stability.

Variability of seed traits and properties of soluble mucilages in lines of the flax genetic collection of Vavilov Institute

Upon hydration, flax seeds secrete mucilages whose content and physico-chemical properties vary according to the genotype and environment. The aim of the work was to investigate the complex genetic relationships between the vegetative period, colour, size and production of seed, the composition (polysaccharides and proteins) and physico-chemical properties of soluble mucilages collected at 28 °C from seeds of 18 lines grown in St Petersburg area. The vegetative period duration was found to impact the size and production of seeds, the yield of mucilages, including the polysaccharides, and the galactosidase enzymes, as well as their composition (mainly the rhamnogalacturonan I moieties) and some of their properties (mainly viscosity). Data allowed to significantly distinguish 6 fibre lines with mucilages enriched in rhamnogalacturonan I, 6 lines with mucilages enriched in arabinoxylan including 5 linseeds and 1 mutated fibre-line, and 5 lines with mucilages enriched in homogalacturonan-like polymer including 4 fibre lines and 1 brown linseed. Seven fibre lines had mucilages particularly rich in galactose. High to very high variability was found for 14 traits. Relatively independent characters (form/shape, protein and galactosidase) were identified and could be combined by breeding, with a focus on mucilage yield, composition and properties. Main-component analyses of line characters showed a large diversity in linseeds mainly due to their different origin but small variation in Russian fibre lines with brown seeds.

AtPME3, a ubiquitous cell wall pectin methylesterase of Arabidopsis thaliana, alters the metabolism of cruciferin seed storage proteins during post-germinative growth of seedlings

&NA; AtPME3 (At3g14310) is a ubiquitous cell wall pectin methylesterase. Atpme3‐1 loss‐of‐function mutants exhibited distinct phenotypes from the wild type (WT), and were characterized by earlier germination and reduction of root hair production. These phenotypical traits were correlated with the accumulation of a 21.5‐kDa protein in the different organs of 4‐day‐old Atpme3‐1 seedlings grown in the dark, as well as in 6‐week‐old mutant plants. Microarray analysis showed significant down‐regulation of the genes encoding several pectin‐degrading enzymes and enzymes involved in lipid and protein metabolism in the hypocotyl of 4‐day‐old dark grown mutant seedlings. Accordingly, there was a decrease in proteolytic activity of the mutant as compared with the WT. Among the genes specifying seed storage proteins, two encoding CRUCIFERINS were up‐regulated. Additional analysis by RT‐qPCR showed an overexpression of four CRUCIFERIN genes in the mutant Atpme3‐1, in which precursors of the &agr;‐ and &bgr;‐subunits of CRUCIFERIN accumulated. Together, these results provide evidence for a link between AtPME3, present in the cell wall, and CRUCIFERIN metabolism that occurs in vacuoles.