Impact of Rhamnolipids (RLs), Natural Defense Elicitors, on Shoot and Root Proteomes of Brassica napus by a Tandem Mass Tags (TMTs) Labeling Approach.

The rapeseed crop is susceptible to many pathogens such as parasitic plants or fungi attacking aerial or root parts. Conventional plant protection products, used intensively in agriculture, have a negative impact on the environment as well as on human health. There is therefore a growing demand for the development of more planet-friendly alternative protection methods such as biocontrol compounds. Natural rhamnolipids (RLs) can be used as elicitors of plant defense mechanisms. These glycolipids, from bacteria secretome, are biodegradable, non-toxic and are known for their stimulating and protective effects, in particular on rapeseed against filamentous fungi. Characterizing the organ responsiveness to defense-stimulating compounds such as RLs is missing. This analysis is crucial in the frame of optimizing the effectiveness of RLs against various diseases. A Tandem Mass Tags (TMT) labeling of the proteins extracted from the shoots and roots of rapeseed has been performed and showed a differential pattern of protein abundance between them. Quantitative proteomic analysis highlighted the differential accumulation of parietal and cytoplasmic defense or stress proteins in response to RL treatments with a clear effect of the type of application (foliar spraying or root absorption). These results must be considered for further use of RLs to fight specific rapeseed pathogens.

Integument-Specific Transcriptional Regulation in the Mid-Stage of Flax Seed Development Influences the Release of Mucilage and the Seed Oil Content.

Flax (Linum usitatissimum L.) seed oil, which accumulates in the embryo, and mucilage, which is synthesized in the seed coat, are of great economic importance for food, pharmaceutical as well as chemical industries. Theories on the link between oil and mucilage production in seeds consist in the spatio-temporal competition of both compounds for photosynthates during the very early stages of seed development. In this study, we demonstrate a positive relationship between seed oil production and seed coat mucilage extrusion in the agronomic model, flax. Three recombinant inbred lines were selected for low, medium and high mucilage and seed oil contents. Metabolite and transcript profiling (1H NMR and DNA oligo-microarrays) was performed on the seeds during seed development. These analyses showed main changes in the seed coat transcriptome during the mid-phase of seed development (25 Days Post-Anthesis), once the mucilage biosynthesis and modification processes are thought to be finished. These transcriptome changes comprised genes that are putatively involved in mucilage chemical modification and oil synthesis, as well as gibberellic acid (GA) metabolism. The results of this integrative biology approach suggest that transcriptional regulations of seed oil and fatty acid (FA) metabolism could occur in the seed coat during the mid-stage of seed development, once the seed coat carbon supplies have been used for mucilage biosynthesis and mechanochemical properties of the mucilage secretory cells.

Physiological and Biochemical Traits of Two Major Arabidopsis Accessions, Col-0 and Ws, Under Salinity.

Salinity affects plant growth and development as shown with the glycophyte model plant, Arabidopsis thaliana (Arabidopsis). Two Arabidopsis accessions, Wassilewskija (Ws) and Columbia (Col-0), are widely used to generate mutants available from various Arabidopsis seed resources. However, these two ecotypes are known to be salt-sensitive with different degrees of tolerance. In our study, 3-week-old Col-0 and Ws plants were treated with and without 150 mM NaCl for 48, 72, or 96 h, and several physiological and biochemical traits were characterized on shoots to identify any specific traits in their tolerance to salinity. Before salt treatment was carried out, a different phenotype was observed between Col-0 and Ws, whose main inflorescence stem became elongated in contrast to Col-0, which only displayed rosette leaves. Our results showed that Col-0 and Ws were both affected by salt stress with limited growth associated with a reduction in nutrient uptake, a degradation of photosynthetic pigments, an increase in protein degradation, as well as showing changes in carbohydrate metabolism and cell wall composition. These traits were often more pronounced in Col-0 and occurred usually earlier than in Ws. Tandem Mass Tags quantitative proteomics data correlated well with the physiological and biochemical results. The Col-0 response to salt stress was specifically characterized by a greater accumulation of osmoprotectants such as anthocyanin, galactinol, and raffinose; a lower reactive oxygen detoxification capacity; and a transient reduction in galacturonic acid content. Pectin degradation was associated with an overaccumulation of the wall-associated kinase 1, WAK1, which plays a role in cell wall integrity (CWI) upon salt stress exposure. Under control conditions, Ws produced more antioxidant enzymes than Col-0. Fewer specific changes occurred in Ws in response to salt stress apart from a higher number of different fascilin-like arabinogalactan proteins and a greater abundance of expansin-like proteins, which could participate in CWI. Altogether, these data indicate that Col-0 and Ws trigger similar mechanisms to cope with salt stress, and specific changes are more likely related to the developmental stage than to their respective genetic background.

A Tandem Mass Tags (TMTs) labeling approach highlights differences between the shoot proteome of two Arabidopsis thaliana ecotypes, Col-0 and Ws.

Arabidopsis has become a powerful model to study morphogenesis, plant growth, development but also plant response to environmental conditions. Over 1000 Arabidopsis genomes are available and show natural genetic variations. Among them, the main reference accessions Wassilewskija (Ws) and Columbia (Col-0), originally growing at contrasted altitudes and temperatures, are widely studied, but data contributing to their molecular phenotyping are still scarce. A global quantitative proteomics approach using isobaric stable isotope labeling (Tandem Mass Tags, TMT) was performed on Ws and Col-0. Plants have been hydroponically grown at 16 h/8 h (light/dark cycle) at 23°C day/19°C night for three weeks. A TMT labeling of the proteins extracted from their shoots has been performed and showed a differential pattern of protein abundance between them. These results have allowed identifying several proteins families possibly involved in the differential responses observed for Ws and Col-0 during plant development and upon environmental changes. In particular, Ws and Col-0 mainly differ in photosynthesis, cell wall-related proteins, plant defense/stress, ROS scavenging enzymes/redox homeostasis and DNA/RNA binding/transcription/translation/protein folding.

Cytological Approaches Combined With Chemical Analysis Reveals the Layered Nature of Flax Mucilage.

The external seed coat cell layer of certain species is specialized in the production and extrusion of a polysaccharide matrix called mucilage. Variations in the content of the released mucilage have been mainly associated with genetically regulated physiological modifications. Understanding the mucilage extrusion process in crop species is of importance to gain deeper insight into the complex cell wall biosynthesis and dynamics. In this study, we took advantage of the varying polysaccharide composition and the size of the flax mucilage secretory cells (MSCs) to study mucilage composition and extrusion in this species of agricultural interest. We demonstrate herein that flax MSCs are structured in four superimposed layers and that rhamnogalacturonans I (RG I) are firstly synthesized, in the upper face, preceding arabinoxylan and glucan synthesis in MSC lower layers. Our results also reveal that the flax mucilage release originates from inside MSC, between the upper and deeper layers, the latter collaborating to trigger polysaccharide expansion, radial cell wall breaking and mucilage extrusion in a peeling fashion. Here, we provide evidence that the layer organization and polysaccharide composition of the MSCs regulate the mucilage release efficiency like a peeling mechanism. Finally, we propose that flax MSCs may represent an excellent model for further investigations of mucilage biosynthesis and its release.

MuSeeQ, a novel supervised image analysis tool for the simultaneous phenotyping of the soluble mucilage and seed morphometric parameters.

BACKGROUND: The mucilage is a model to study the polysaccharide biosynthesis since it is produced in large amounts and composed of complex polymers. In addition, it is of great economic interest for its technical and nutritional value. A fast method for phenotyping the released mucilage and the seed morphometric parameters will be useful for fundamental, food, pharmaceutical and breeding researches. Current strategies to phenotype soluble mucilage are restricted to visual evaluations or are highly time-consuming. RESULTS: Here, we developed a high-throughput phenotyping method for the simultaneous measurement of the soluble mucilage content released on a gel and the seed morphometric parameters. Within this context, we combined a biochemical assay and an open-source computer-aided image analysis tool, MuSeeQ. The biochemical assay consists in sowing seeds on an agarose medium containing the dye toluidine blue O, which specifically stains the mucilage once it is released on the gel. The second part of MuSeeQ is a macro developed in ImageJ allowing to quickly extract and analyse 11 morphometric data of seeds and their respective released mucilages. As an example, MuSeeQ was applied on a flax recombinant inbred lines population (previously screened for fatty acids content.) and revealed significant correlations between the soluble mucilage shape and the concentration of some fatty acids, e.g. C16:0 and C18:2. Other fatty acids were also found to correlate with the seed shape parameters, e.g. C18:0 and C18:2. MuSeeQ was then showed to be used for the analysis of other myxospermous species, including Arabidopsis thaliana and Camelina sativa. CONCLUSIONS: MuSeeQ is a low-cost and user-friendly method which may be used by breeders and researchers for phenotyping simultaneously seeds of specific cultivars, natural variants or mutants and their respective soluble mucilage area released on a gel. The script of MuSeeQ and video tutorials are freely available at http://MuSeeQ.free.fr.

Combined Experimental and Computational Approaches Reveal Distinct pH Dependence of Pectin Methylesterase Inhibitors.

The fine-tuning of the degree of methylesterification of cell wall pectin is a key to regulating cell elongation and ultimately the shape of the plant body. Pectin methylesterification is spatiotemporally controlled by pectin methylesterases (PMEs; 66 members in Arabidopsis [Arabidopsis thaliana]). The comparably large number of proteinaceous pectin methylesterase inhibitors (PMEIs; 76 members in Arabidopsis) questions the specificity of the PME-PMEI interaction and the functional role of such abundance. To understand the difference, or redundancy, between PMEIs, we used molecular dynamics (MD) simulations to predict the behavior of two PMEIs that are coexpressed and have distinct effects on plant development: AtPMEI4 and AtPMEI9. Simulations revealed the structural determinants of the pH dependence for the interaction of these inhibitors with AtPME3, a major PME expressed in roots. Key residues that are likely to play a role in the pH dependence were identified. The predictions obtained from MD simulations were confirmed in vitro, showing that AtPMEI9 is a stronger, less pH-independent inhibitor compared with AtPMEI4. Using pollen tubes as a developmental model, we showed that these biochemical differences have a biological significance. Application of purified proteins at pH ranges in which PMEI inhibition differed between AtPMEI4 and AtPMEI9 had distinct consequences on pollen tube elongation. Therefore, MD simulations have proven to be a powerful tool to predict functional diversity between PMEIs, allowing the discovery of a strategy that may be used by PMEIs to inhibit PMEs in different microenvironmental conditions and paving the way to identify the specific role of PMEI diversity in muro.

PME58 plays a role in pectin distribution during seed coat mucilage extrusion through homogalacturonan modification.

Pectins are major components of plant primary cell walls. They include homogalacturonans (HGs), which are the most abundant pectin and can be the target of apoplastic enzymes like pectin methylesterases (PMEs) that control their methylesterification level. Several PMEs are expressed in the seed coat of Arabidopsis thaliana, particularly in mucilage secretory cells (MSCs). On the basis of public transcriptomic data, seven PME genes were selected and checked for their seed-specific expression by quantitative reverse transcription PCR. Of these, PME58 presented the highest level of expression and was specifically expressed in MSCs at the early stages of seed development. pme58 mutants presented two discrete phenotypes: (i) their adherent mucilage was less stained by ruthenium red when compared to wild-type seeds, but only in the presence of EDTA, a Ca(2+)chelator; and (ii) the MSC surface area was decreased. These phenotypes are the consequence of an increase in the degree of HG methylesterification connected to a decrease in PME activity. Analysis of the sugar composition of soluble and adherent mucilage showed that, in the presence of EDTA, sugars of adherent mucilage were more readily extracted in pme58 mutants. Immunolabelling with LM19, an antibody that preferentially recognizes unesterified HGs, also showed that molecular interactions with HGs were modified in the adherent mucilage of pme58 mutants, suggesting a role of PME58 in mucilage structure and organization. In conclusion, PME58 is the first PME identified to play a direct role in seed mucilage structure.

The role of nitrogen availability for the salt-tolerance of two different varieties of durum wheat.

Salt stress tolerance of durum wheat was assessed in control and 200 and 300 mM NaCl-exposed seed of two cultivars (BidiAP4 and Azizi). These salt treatments were accompanied by different levels of nitrate (Ca(NO3)2) added to the media (0.1, 3, 10 mM). The data showed that NaCl stress increased Na(+) and Cl(-) contents and lowered K(+) and NO3 (-) levels in seeds of BidiAP4 cultivar. In Azizi seeds exposed to NaCl, Na(+) and K(+) were highly accumulated while low levels of NO3 (-) and Cl(-) were detected. Those findings highlight the difference in the salt stress tolerance of these two durum wheat cultivars also depending on nitrogen (N) availability, Azizi cultivar being less sensitive to NaCl treatment than BidiAP4. These data also suggested a relationship between salt tolerance capacity and enhancement or maintenance of nitrogen and carbon metabolisms enzyme activity.

Further insights into the isoenzyme composition and activity of glutamate dehydrogenase in Arabidopsis thaliana.

Following the discovery that in Arabidopsis, a third isoenzyme of NADH-dependent glutamate dehydrogenase (GDH) is expressed in the mitochondria of the root companion cells, we have re-examined the GDH isoenzyme composition. By analyzing the NADH-GDH isoenzyme composition of single, double and triple mutants deficient in the expression of the three genes encoding the enzyme, we have found that the α, ß and γ polypeptides that comprise the enzyme can be assembled into a complex combination of heterohexamers in roots. Moreover, we observed that when one or two of the three root isoenzymes were missing from the mutants, the remaining isoenzymes compensated for this deficiency. The significance of such complexity is discussed in relation to the metabolic and signaling function of the NADH-GDH enzyme. Although it has been shown that a fourth gene encoding a NADPH-dependent enzyme is present in Arabidopsis, we were not able to detect corresponding enzyme activity, even in the triple mutant totally lacking NADH-GDH activity.

A quantitative genetic study for elucidating the contribution of glutamine synthetase, glutamate dehydrogenase and other nitrogen-related physiological traits to the agronomic performance of common wheat.

To better understand the genetic variability for nitrogen use efficiency in winter wheat is a necessity in the frame of the present economic and ecological context. The objective of this work was to investigate the role of the enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH), and other nitrogen (N)-related physiological traits in the control of agronomic performance in wheat. A quantitative genetics approach was developed using the Arche x Récital population of doubled haploid lines grown for 3 years in the field. GS and GDH activities, ammonium, amino acid and protein contents were measured at different stages of plant development in different organs after flowering. Significant genotypic effects were observed for all measured physiological and agronomical traits. Heading date was negatively correlated with ammonium, amino acid, protein contents and GS activity in the flag leaf lamina. Grain protein content was positively correlated with both ammonium and amino acid content, and to a lesser extent with soluble protein content and GS activity. A total of 148 quantitative trait loci (QTLs) were detected, 104 QTLs for physiological traits and 44 QTLs for agronomic traits. Twenty-six QTLs were detected for GDH activity spread over 13 chromosomes and 25 QTLs for GS activity spread over 12 chromosomes. We found only a co-localization between a QTL for GS activity and GSe, a structural gene encoding cytosolic GS on chromosome 4B. A coincidence between a QTL for GDH activity and a gene encoding GDH was also found on chromosome 2B. QTL regions combining both physiological and agronomical QTLs were mainly identified on linkage groups 2A, 2B, 2D, 5A, 5B and 5D. This approach allowed us to propose possible functions of physiological traits to explain the variation observed for agronomic traits including yield and its components.

The plant nitrogen mobilization promoted by Colletotrichum lindemuthianum in Phaseolus leaves depends on fungus pathogenicity.

Nitrogen plays an essential role in the nutrient relationship between plants and pathogens. Some studies report that the nitrogen-mobilizing plant metabolism that occurs during abiotic and biotic stress could be a 'slash-and-burn' defence strategy. In order to study nitrogen recycling and mobilization in host plants during pathogen attack and invasion, the Colletotrichum lindemuthianum/Phaseolus vulgaris interaction was used as a model. C. lindemuthianum is a hemibiotroph that causes anthracnose disease on P. vulgaris. Non-pathogenic mutants and the pathogenic wild-type strain were used to compare their effects on plant metabolism. The deleterious effects of infection were monitored by measuring changes in chlorophyll, protein, and amino acid concentrations. It was shown that amino acid composition changed depending on the plant-fungus interaction and that glutamine accumulated mainly in the leaves infected by the pathogenic strain. Glutamine accumulation correlated with the accumulation of cytosolic glutamine synthetase (GS1 alpha) mRNA. The most striking result was that the GS1 alpha gene was induced in all the fungus-infected leaves, independent of the strain used for inoculation, and that GS1 alpha expression paralleled the PAL3 and CHS defence gene expression. It is concluded that a role of GS1 alpha in plant defence has to be considered.

Combined agronomic and physiological aspects of nitrogen management in wheat highlight a central role for glutamine synthetase.

In wheat the period of grain filling is characterized by a transition for all vegetative organs from sink to source status. To study this transition, the progression of physiological markers and enzyme activities representative of nitrogen metabolism was monitored from the vegetative stage to maturity in different leaf stages and stem sections of two wheat (Triticum aestivum) cultivars grown at high and low levels of N fertilization. In the two cultivars examined, we found a general decrease of the metabolic and enzyme markers occurred during leaf ageing, and that this decrease was enhanced when plants were N-limited. Both correlation studies and principal components analysis (PCA) showed that there was a strong relationship among total N, chlorophyll, soluble protein, ammonium, amino acids and glutamine synthetase (GS) activity. The use of a marker such as GS activity to predict the N status of wheat, as a function of both plant development and N availability, is discussed with the aim of selecting wheat genotypes with better N-use efficiency.

Glutamine synthetase-glutamate synthase pathway and glutamate dehydrogenase play distinct roles in the sink-source nitrogen cycle in tobacco.

Glutamate (Glu) metabolism and amino acid translocation were investigated in the young and old leaves of tobacco (Nicotiana tabacum L. cv Xanthi) using [15N]ammonium and [2-15N]Glu tracers. Regardless of leaf age, [15N]ammonium assimilation occurred via glutamine synthetase (GS; EC 6.1.1.3) and Glu synthase (ferredoxin [Fd]-GOGAT; EC 1.4.7.1; NADH-GOGAT; EC 1.4.1.14), both in the light and darkness, and it did not depend on Glu dehydrogenase (GDH; EC 1.4.1.2). The [15N]ammonium and ammonium accumulation patterns support the role of GDH in the deamination of [2-15N]Glu to provide 2-oxoglutarate and [15N]ammonium. In the dark, excess [15N]ammonium was incorporated into asparagine that served as an additional detoxification molecule. The constant Glu levels in the phloem sap suggested that Glu was continuously synthesized and supplied into the phloem regardless of leaf age. Further study using transgenic tobacco lines, harboring the promoter of the GLU1 gene (encoding Arabidopsis [Arabidopsis thaliana] Fd-GOGAT) fused to a GUS reporter gene, revealed that the expression of Fd-GOGAT remained higher in young leaves compared to old leaves, and higher in the veins compared to the mesophyll. Confocal laser-scanning microscopy localized the Fd-GOGAT protein to the phloem companion cells-sieve element complex in the leaf veins. The results are consistent with a role of Fd-GOGAT in supplying Glu for the synthesis and transport of amino acids. Taken together, the data provide evidence that the GS-GOGAT pathway and GDH play distinct roles in the source-sink nitrogen cycle of tobacco leaves.

The two senescence-related markers, GS1 (cytosolic glutamine synthetase) and GDH (glutamate dehydrogenase), involved in nitrogen mobilization, are differentially regulated during pathogen attack and by stress hormones and reactive oxygen species in Nicotiana tabacum L. leaves.

To investigate the role of stress in nitrogen management in plants, the effect of pathogen attack, elicitors, and phytohormone application on the expression of the two senescence-related markers GS1 (cytosolic glutamine synthetase EC 6.3.1.2) and GDH (glutamate dehydrogenase, EC 1.4.1.2) involved in nitrogen mobilization in senescing leaves of tobacco (Nicotiana tabacum L.) plants, was studied. The expression of genes involved in primary nitrogen assimilation such as GS2 (chloroplastic glutamine synthetase) and Nia (nitrate reductase, EC 1.6.1.1) was also analysed. The Glubas gene, coding a beta-1,3-glucanase, was used as a plant-defence gene control. As during natural senescence, the expression of GS2 and Nia was repressed under almost all stress conditions. By contrast, GS1 and GDH mRNA accumulation was increased. However, GS1 and GDH showed differential patterns of expression depending on the stress applied. The expression of GS1 appeared more selective than GDH. Results indicate that the GDH and GS1 genes involved in leaf senescence are also a component of the plant defence response during plant-pathogen interaction. The links between natural plant senescence and stress-induced senescence are discussed, as well as the potential role of GS1 and GDH in a metabolic safeguard process.

Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.).

GLU1 encodes the major ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in Arabidopsis thaliana (ecotype Columbia). With the aim of providing clues on the role of Fd-GOGAT, we analyzed the expression of Fd-GOGAT in tobacco (Nicotiana tabacum L. cv. Xanthi). The 5' flanking element of GLU1 directed the expression of the uidA reporter gene in the palisade and spongy parenchyma of mesophyll, in the phloem cells of vascular tissue and in the roots of tobacco. White light, red light or sucrose induced GUS expression in the dark-grown seedlings in a pattern similar to the GLU1 mRNA accumulation in Arabidopsis. The levels of GLU2 mRNA encoding the second Fd-GOGAT and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) were not affected by light. Both in the light and in darkness, (15)NH4(+) was incorporated into [5-(15)N]glutamine and [2-(15)N]glutamate by glutamine synthetase (GS, EC 6.3.1.2) and Fd-GOGAT in leaf disks of transgenic tobacco expressing antisense Fd-GOGAT mRNA and in wild-type tobacco. In the light, low level of Fd-glutamate synthase limited the [2-(15)N]glutamate synthesis in transgenic leaf disks. The efficient dark labeling of [2-(15)N]glutamate in the antisense transgenic tobacco leaves indicates that the remaining Fd-GOGAT (15-20% of the wild-type activity) was not the main limiting factor in the dark ammonium assimilation. The antisense tobacco under high CO2 contained glutamine, glutamate, asparagine and aspartate as the bulk of the nitrogen carriers in leaves (62.5%), roots (69.9%) and phloem exudates (53.2%). The levels of glutamate, asparagine and aspartate in the transgenic phloem exudates were similar to the wild-type levels while the glutamine level increased. The proportion of these amino acids remained unchanged in the roots of the transgenic plants. Expression of GLU1 in mesophyll cells implies that Fd-GOGAT assimilates photorespiratory and primary ammonium. GLU1 expression in vascular cells indicates that Fd-GOGAT provides amino acids for nitrogen translocation.

Cadmium toxicity induced changes in nitrogen management in Lycopersicon esculentum leading to a metabolic safeguard through an amino acid storage strategy.

Tomato (Lycopersicon esculentum) seedlings were grown in the presence of cadmium. After 1 week of Cd treatment, a sharp decline in biomass accumulation in the leaves and roots was observed, together with a decrease in the rate of photosynthetic activity due to both Rubisco and chlorophyll degradation and stomata closure. Cadmium induced a significant decrease in nitrate content and inhibition of the activities of nitrate reductase, nitrite reductase, glutamine synthetase (GS) and ferredoxin-glutamate synthase. An increase in NADH-glutamate synthase and NADH-glutamate dehydrogenase activity was observed in parallel. The accumulation of ammonium into the tissues of treated plants was accompanied by a loss of total protein and the accumulation of amino acids. Gln represented the major amino acid transported through xylem sap of Cd-treated and control plants. Cadmium treatment increased the total amino acid content in the phloem, maintaining Gln/Glu ratios. Western and Northern blot analysis of Cd-treated plants showed a decrease in chloroplastic GS protein and mRNA and an increase in cytosolic GS and glutamate dehydrogenase transcripts and proteins. An increase in asparagine synthetase mRNA was observed in roots, in parallel with a strong increase in asparagine. Taken together, these results suggest that the plant response to Cd stress involved newly induced enzymes dedicated to coordinated leaf nitrogen remobilization and root nitrogen storage.

Characterization of nitrogen relationships between Sorghum bicolor and the root-hemiparasitic angiosperm Striga hermonthica (Del.) Benth. using K15 NO3 as isotopic tracer.

The role of the host in the nitrogen nutrition of Striga hermonthica (Del.) Benth. (Scrophulariaceae) parasitic on Sorghum bicolor cv. SH4 Arval has been investigated using (15)N-nitrate as the tracer. It is shown that, when nitrate is absorbed only by the roots of the host plant, a rapid transfer of nitrogen to the parasite can be detected. The xylem sap of S. hermonthica contained approximately equal amounts of nitrate and amino acids, mostly glutamine and asparagine. Infection altered the free amino acid profile of the host tissues, leading notably to a large increase in asparagine and a decrease in glutamine. The haustoria of S. hermonthica, although rich in nitrate, showed a low concentration of free amino acids, particularly lacking in asparagine and glutamine. The roots of S. hermonthica, in contrast, were rich in both asparagine and glutamine while, in the shoots, asparagine constituted 80% of the total FAA pool. Asparagine was also found to be the primary (15)N-enriched amino acid in the shoots of S. hermonthica while, interestingly, it was glutamate that was most strongly enriched in the roots. It is concluded that nitrogen nutrition in S. hermonthica is based on a supply of both nitrate and amino acids from the host. This implies a non-specific transfer in the transpiration stream. Nitrate reduction probably occurs mainly in the leaves of the parasite. Assimilation also occurs in S. hermonthica and excess nitrogen is stored as the non-toxic nitrogen-rich compound, asparagine. This specific trait of nitrogen metabolism of the parasite is discussed in relation to the effect of nitrogen fertilization on reducing infestation.