Implication of the glutamine synthetase ⁄glutamate synthase pathway in conditioning the amino acid metabolism in bundle sheath and mesophyll cells of maize leaves ´ ` Marie-Helene Valadier1, Ayako Yoshida2, Olivier Grandjean3, Halima Morin3, Jocelyne ´ Kronenberger3, Stephanie Boutet1, Adeline Raballand1, Toshiharu Hase2, Tadakatsu Yoneyama4 and Akira Suzuki1 ´ ´ Unite de Nutrition Azotee des Plantes, Institut National de la Recherche Agronomique, Versailles, France Institute for Protein Research, Osaka University, Japan Laboratoire Commun de Cytologie, Institut National de la Recherche Agronomique, Versailles, France Department of Applied Biological Chemistry, University of Tokyo, Japan Keywords amino acid translocation; compartmentation; glutamine and glutamate synthesis; nitrogen assimilation; Zea mays L Correspondence ´ ´ A Suzuki, Unite de Nutrition Azotee des Plantes, Institut National de la Recherche Agronomique, Route de St-Cyr, 78026 Versailles cedex, France Fax: +33 30 83 30 96 Tel: +33 30 83 30 87 E-mail: suzuki@versailles.inra.fr (Received 20 February 2008, revised 16 April 2008, accepted 17 April 2008) doi:10.1111/j.1742-4658.2008.06472.x We investigated the role of glutamine synthetases (cytosolic GS1 and chloroplast GS2) and glutamate synthases (ferredoxin-GOGAT and NADHGOGAT) in the inorganic nitrogen assimilation and reassimilation into amino acids between bundle sheath cells and mesophyll cells for the remobilization of amino acids during the early phase of grain filling in Zea mays L The plants responded to a light ⁄ dark cycle at the level of nitrate, ammonium and amino acids in the second leaf, upward from the primary ear, which acted as the source organ The assimilation of ammonium issued from distinct pathways and amino acid synthesis were evaluated from the diurnal rhythms of the transcripts and the encoded enzyme activities of nitrate reductase, nitrite reductase, GS1, GS2, ferredoxin-GOGAT, NADH-GOGAT, NADH-glutamate dehydrogenase and asparagine synthetase We discerned the specific role of the isoproteins of ferredoxin and ferredoxin:NADP+ oxidoreductase in providing ferredoxin-GOGAT with photoreduced or enzymatically reduced ferredoxin as the electron donor The spatial distribution of ferredoxin-GOGAT supported its role in the nitrogen (re)assimilation and reallocation in bundle sheath cells and mesophyll cells of the source leaf The diurnal nitrogen recycling within the plants took place via the specific amino acids in the phloem and xylem exudates Taken together, we conclude that the GS1 ⁄ ferredoxin-GOGAT cycle is the main pathway of inorganic nitrogen assimilation and recycling into glutamine and glutamate, and preconditions amino acid interconversion and remobilization In the C4 plant maize, inorganic nitrate reduction to ammonium and subsequent ammonium assimilation into amino acids occur in two different photosyn- thetic cells: bundle sheath cells (BSCs) and mesophyll cells (MCs) Nitrate taken up by roots moves in part, via the vascular bundle, to leaves for reduction Abbreviations AS, asparagine synthetase (EC 6.3.5.4); BSC, bundle sheath cells; DIG, digoxigenin; Fd, ferredoxin; Fd-NiR, ferredoxin-nitrite reductase (EC 1.6.6.4); FNR, ferredoxin:NADP+ oxidoreductase (EC 1.18.1.2); GDH, glutamate dehydrogenase; GOGAT, glutamate synthase (Fd-GOGAT, EC 1.4.7.1; GS, glutamine synthetase (EC 6.1.1.3); MC, mesophyll cells; NADH-GOGAT, EC 1.4.1.14); NR, nitrate reductase (EC 1.6.6.1); PS I (II), photosystem I (II) FEBS Journal 275 (2008) 3193–3206 ª 2008 The Authors Journal compilation ª 2008 FEBS 3193 Amino acid metabolism and translocation M.-H Valadier et al In leaves, nitrate is reduced to ammonium by cytosolic nitrate reductase (NR; EC 1.6.6.1), and then by plastidial ferredoxin-nitrite reductase (Fd-NiR, EC 1.6.6.4) [1] Ammonium, also derived from photorespiration, is assimilated first into the glutamineamide group by glutamine synthetase (cytosolic GS1 and plastidial GS2, EC 6.3.1.2) and then into glutamate-amino group by glutamate synthase (FdGOGAT, EC 1.4.7.1; NADH-GOGAT, EC 1.4.1.14) in vegetative organs GS1 is encoded by five genes in maize, and the regulation and function of each gene have been elucidated in part [2–4] Ammonium and glutamine-amide group are also assimilated into asparagine by asparagine synthetases (ASs; ammonia ligase AS, EC 6.3.1.1; glutamine hydrolyase AS, EC 6.3.5.4) Alternatively, mitochondrial NADH-glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2) can incorporate high levels of ammonium into glutamate under stress [5] Nitrogen assimilation and amino acid synthesis require reductants and ATP Fd and Fd:NADP+ oxidoreductase (FNR, EC 1.18.1.2) occupy a central position to mediate chloroplast electron flow to yield reducing equivalents [6] The nitrogen metabolism between BSCs and MCs depends on the efficient distribution of energy between photosystem I (PS I) and photosystem II (PS II) via the electron flow specific to the two cell types As a result, inorganic nitrogen assimilation into amino acids is tightly correlated with photosynthesis Furthermore, light at low fluence entrains circadian rhythms and plays an essential role for molecular signalling in the expression of the genes and encoded enzymes involved in nitrate assimilation and amino acid synthesis [7] The stalk and leaves below and above the ear act as the source organs for nitrogen reallocation in the reproductive stage of maize [8,9] In the source leaves, it has been postulated that the metabolic shift from the GS2 ⁄ GOGAT cycle to the GS1 ⁄ GDH pathway is responsible for ammonium assimilation into glutamine and glutamate, as a result of a decline in GS2 and the induction of the a-GDH subunit (for a review, see [10]) However, the role of GDH is controversial [11–14], and the regulation and function of GOGATs in nitrogen remobilization remain to be evaluated In this study, we examined the diurnal responses of the plants, which provide valuable cues to nitrogen and carbon metabolism [7,15] We assessed the role of the GS ⁄ GOGAT cycle in the nitrogen assimilation between MCs and BSCs in the amino acid synthesis and remobilization during the early phase of grain filling in Zea mays L 3194 Results High levels of glutamate and glutamate derivatives in reproductive leaves Leaves above and below the ear act as sources to export nitrogen resources to sink organs via vascular bundles In order to examine the nitrogen status in the leaves, we determined the inorganic nitrogen and amino acid contents in the second leaf above the ear every h during a 16 h light ⁄ h dark cycle The nitrate content was high during the second half of the light phase and remained at about 16 lmolỈ(g fresh weight))1 (Fig 1A) Ammonium accumulated in the middle of the light phase up to lmolỈ(g fresh weight))1, indicating that a part of the ammonium was not assimilated in the light (Fig 1B) The major amino acids in the leaves were alanine (26–39%), glycine (26–40%), glutamic acid (6–14%), serine (8–12%) and aspartic acid (4–16%) in both the light and dark (Fig 1, Table 1) Following ammonium accumulation, glutamine increased about four-fold in the light (Fig 1C) In contrast, asparagine remained at a fairly constant level in the light and dark (Fig 1E, Table 1) The increase in ammonium was inversely correlated with the decline in glutamic acid and aspartic acid in the light (Fig 1B,D,F) Expression of the genes involved in nitrogen assimilation Light is a signal that regulates nitrogen metabolism, and nitrogen assimilation into amino acids is tightly correlated with the expression of the genes involved [15] Thus, we analysed the diurnal expression of the genes encoding the enzymes of nitrogen assimilation in the second leaf above the ear every h during a 16 h light ⁄ h dark cycle Total mRNAs were isolated and estimated on the basis of equal total amounts of 18S rRNA as the internal standard (data not shown) We measured the NR transcripts as an additional control, as the light regulation of NR expression has been defined in maize and several plant species The NR mRNAs peaked at h during the dark to light transition, and then decreased to undetectable levels (Fig 2) Similar diurnal patterns have been reported for other plant NRs [15–17] Gln1-1, encoding the main form of cytosolic GS1 in leaves [2], was strongly expressed, and slightly smaller signals were detected for the Gln1-2 and Gln1-3 mRNAs In contrast, strong expression of Gln1-4 was observed, as also reported in [3] The FEBS Journal 275 (2008) 3193–3206 ª 2008 The Authors Journal compilation ª 2008 FEBS M.-H Valadier et al Amino acid metabolism and translocation Nitrate B8 Amount µmol·g–1 FW 20 Amount µmol·g–1 FW 10 12 15 18 21 24 Glu D 20 Relative amount % 0 Asn E 2.5 2.0 1.5 1.0 0.5 0.0 12 15 18 21 24 12 15 18 21 24 Asp F 20 0.6 15 15 0.4 10 10 0.2 5 0 0.0 12 15 18 21 24 Ala G H 12 15 18 21 24 Gly 50 Relative amount % C Relative amount % 25 15 Gln Ammonium A 20 10 12 15 18 21 24 12 15 18 Time of day (h) 21 24 Ser 30 20 40 30 I 50 40 10 15 10 0 0 12 15 18 21 24 Time of day (h) 12 15 18 21 24 Time of day (h) Fig Levels of nitrate (A), ammonium (B) and amino acids (C–I) in maize leaves collected every h during a 16 h light ⁄ h dark cycle Nitrate and ammonium contents represent the mean from five independent plants ± standard error Amino acid contents are expressed as a percentage relative to the total free amino acid contents, which represent the mean [lmolỈ(g fresh weight))1] from five independent plants ± standard error as follows: 17.9 ± 1.1 (3 h), 19.8 ± 1.2 (6 h), 25.4 ± 1.6 (9 h), 23.4 ± 1.4 (12 h), 18.9 ± 1.2 (15 h), 29.4 ± 1.9 (18 h), 36.0 ± 2.2 (21 h) and 28.0 ± 1.8 (24 h) The standard errors for individual amino acid contents are of the same order of magnitude as those of the total amino acid contents for glutamine (C), glutamic acid (D), asparagine (E), aspartic acid (F), alanine (G), glycine (H) and serine (I) Grey boxes indicate the dark phase GS1 genes (Gln1-1 to Gln1-4) were expressed in a similar diurnal rhythm with an increase in the dark to a maximum at 12 h, and then barely detectable (Fig 2) The Gln2 mRNAs were low, peaking early in the light phase and persisting longer than the Gln1 mRNAs (Fig 2) The GLU mRNAs for Fd-GOGAT were found at the highest level at h, similar to the Gln2 mRNAs (Fig 2) The NADHGOGAT mRNAs could not be detected in our assay conditions As GDH genes are expressed at high levels in reproductive plant leaves [18], we also measured gdh expression Maize NADH-GDH is encoded by gdh1 and gdh2 for the b-subunit and a-subunit, respectively This gdh1 is phylogenetically related to tomato gdh1, whose b-homohexamer complexes appear to oxidize glutamate in transgenic tobacco [14] The gdh1 mRNAs accumulated on illumination, reaching a maximum level at 12 h, and FEBS Journal 275 (2008) 3193–3206 ª 2008 The Authors Journal compilation ª 2008 FEBS 3195 Amino acid metabolism and translocation M.-H Valadier et al Table Amino acid composition in leaves, and amino acid percentage ratio in the phloem exudates and leaves and xylem bleeding sap and leaves in the light and dark The amino acid composition in leaves is expressed as a percentage relative to the total amino acid contents, which represent the mean [lmolỈ(g fresh weight))1] from five independent plants ± standard error as follows: 25.48 ± 1.58 (light) and 21.90 ± 1.93 (dark) The standard errors for the individual amino acid contents are of the same order of magnitude as those of the total amino acid contents Phloem exudates and xylem sap were collected over a 16 h light ⁄ h dark cycle % in phloem exudates ⁄ % in leaves Leaves (%) % in xylem sap ⁄ % in leaves Light Dark Light Dark Light Dark Glutamine Glutamate Asparagine Aspartate Alanine Glycine Serine 1.1 9.7 0.3 5.9 32.7 32.4 10.4 0.5 13.8 0.3 12.4 28.4 26.0 10.4 15.7 1.7 12.4 1.5 0.7 < 0.1 0.9 24.9 1.7 17.6 1.3 0.3 0.1 1.2 27.8 < 0.1 98.8 < 0.1 0.5 < 0.1 0.7 84.3 0.2 76.1 0.3 0.4 < 0.1 0.7 100 12 15 18 21 24 50 12 15 18 21 24 100 Fd II 50 12 15 18 21 24 6 12 15 18 21 24 Time of day (h) 50 6 12 15 18 21 24 Time of day (h) 12 15 18 21 24 12 15 18 21 24 50 12 15 18 21 24 100 50 50 0 3 50 12 15 18 21 24 100 L-FNR Fd VI 12 15 18 21 24 100 100 50 50 12 15 18 21 24 100 100 100 12 15 18 21 24 50 12 15 18 21 24 100 50 100 GLU Gln2 50 Gln1-3 100 50 0 gdh1 12 15 18 21 24 Fd III 50 L-FNR 3 Fd V relative amount % 50 0 Gln1-2 Gln1-1 50 100 100 100 100 Fd I ASN relative amount % Gln1-4 relative amount % NR relative amount % Amino acid 12 15 18 21 24 Time of day (h) 12 15 18 21 24 Time of day (h) Fig Levels of the transcripts for NR (NR), cytosolic GS1 (Gln1-1, Gln1-2, Gln1-3, Gln1-4), chloroplast GS2 (Gln2), Fd-GOGAT (GLU), GDH (gdh1), AS (ASN), Fd (Fd I, Fd II, Fd III, Fd V, Fd VI) and leaf FNR (L-FNR and L-FNR 2) in maize leaves The mRNAs were estimated by RT-PCR using an equal amount of total RNA from each sample, collected every h during a 16 h light ⁄ h dark cycle The time of day corresponds to the light phase (6–22 h) and dark phase (22–6 h) The values represent the mean from five independent plants mixed together and expressed as a percentage relative to the maximum a second peak appeared at the beginning of the dark phase (Fig 2) The gdh2 mRNAs could not be detected under our assay conditions The ASN gene for maize AS belongs to the light-inducible genes, 3196 such as monocot rice ASN and Arabidopsis ASN2 and ASN3 [19] The level of ASN mRNAs was higher in the dark and decreased to about 70% in the middle of the light phase (Fig 2) FEBS Journal 275 (2008) 3193–3206 ª 2008 The Authors Journal compilation ª 2008 FEBS M.-H Valadier et al Amino acid metabolism and translocation GOGATs in the later light phase This contrasts with the diurnally active Fd-GOGAT and NADH-GOGAT in developing maize seedlings, in which GOGATs cope with large amounts of primary and photorespiratory ammonium [22] Interestingly, there was a substantial increase in NADH-GOGAT activity at the end of the light phase (Fig 3E) Fairly high and constant activities were detected for GDH in both the synthesis and deamination of glutamate (Fig 3F) In vitro activities of nitrogen assimilation into amino acids The in vitro activities of the key enzymes of nitrogen assimilation were determined NR displayed a delayed light-induced activity compared with its mRNA abundance (Fig 3A) We detected a lower activity of NiR than NR, and the primary nitrate reduction to nitrite and then to ammonium took place potentially at rates of 4–9 and 1.5–2 lmolỈh)1Ỉ(g fresh weight))1, respectively (Fig 3A,B) There was a small change in NiR activity, with a 25% decrease early in the light phase (Fig 3B), as observed in other plants [20] The total GS activity remained fairly constant at 30 lmolỈh)1Ỉ(g fresh weight))1 (Fig 3C) Because the activity ratio of GS1 to GS2 reaches 20 in stalks at similar maturity after anthesis [21], it is probably cytosolic GS1, which assimilates ammonium during a day ⁄ night cycle Fd-GOGAT is the primary form in the source leaves, accounting for 90% of total GOGAT activity, with the rest being NADH-GOGAT (Fig 3D,E) Both Fd-GOGAT and NADH-GOGAT were induced at the end of the dark phase, peaked at the dark ⁄ light transition, and then became undetectable The patterns indicate that there was no further nitrogen flux through the NR A NiR EDTA Mg2+ 0 D Fd-GOGAT 1.5 0.5 12 15 18 21 24 40 µmol·h–1·g–1 FW E 12 NADH-GOGAT 6 12 15 18 21 24 Time of day (h) 10 12 15 18 21 24 GDH 25 0.5 0 20 F µmol·h–1·g–1 FW µmol·h–1·g–1 FW 30 12 15 18 21 24 1.5 10 GS C 2.5 µmol·h–1·g–1 FW Activity µmol·h–1·g–1 FW Ammonium assimilation into glutamine by GSs (GS1 and GS2) occurs in the two cell types [23], but the localization of the major Fd-GOGAT between MCs and BSCs remains controversial [24] Therefore, we first determined the cellular and subcellular localization of Fd-GOGAT Fd-GOGAT mRNAs were hybridized in situ with the digoxigenin (DIG)-labelled antisense GLU mRNA probe Staining was found in the cytoplasmic layers of BSCs (Fig 4A) No positive staining was detected in the BSCs using the control sense probe (Fig 4B) The localization of GLU mRNAs in the vicinity of the vascular bundle of BSCs suggests a role of Fd-GOGAT in amino acid translocation Therefore, B 10 Activity µmol·h–1·g–1 FW Localization of Fd-GOGAT 12 15 18 21 24 Time of day (h) 20 15 Amination Deamination 10 0 12 15 18 21 24 Time of day (h) Fig Enzyme activities of NR (A), NiR (B), GS (C), Fd-GOGAT (D), NADH-GOGAT (E) and NADH-GDH and NAD-GDH (F) in maize leaves collected every h during a 16 h light ⁄ h dark cycle The NR assay was carried out in a reaction mixture in the presence of 10 mM MgCl2 (Mg2+) or mM EDTA (EDTA) for the divalent cation-dependent activity and maximum catalytic activity, respectively The GDH activity was assayed for NADH-dependent glutamate synthetic activity (amination) and NAD+-dependent glutamate oxidation activity (deamination) Error bars represent the standard error from five independent plants Grey boxes indicate the dark phase FEBS Journal 275 (2008) 3193–3206 ª 2008 The Authors Journal compilation ª 2008 FEBS 3197 Amino acid metabolism and translocation A M.-H Valadier et al A B BSC B BSC BSC MC MC chl D C MC chl MC F BSC BSC MC MC Fig In situ hybridization of GLU mRNA for Fd-GOGAT and ASN mRNA for AS in thin sections of 21-day-old maize leaves (A) Leaf bundle sheath cell section using an antisense GLU mRNA probe (B) Control leaf bundle sheath cell section using a sense GLU mRNA probe (C) Leaf mesophyll cell section using an antisense GLU mRNA probe (D) Control leaf mesophyll cell section using a sense GLU mRNA probe (E) Leaf section using an antisense ASN mRNA probe (F) Control leaf section using a sense ASN mRNA probe BSC, bundle sheath cell; chl, chloroplast; MC, mesophyll cell Bar: 10 lm in situ mRNA hybridization was carried out for cytosolic AS, which provides asparagine for nitrogen transport The signal was found in the cytoplasm of BSCs in a ring around the vascular bundle with the antisense ASN mRNA probe (Fig 4E) Staining was not detected with the control probe (Fig 4F) Furthermore, the signal was also found on the surface of MC chloroplasts in the cytoplasmic layers (Fig 4C) No positive staining was observed with the control probe in MCs, and chloroplasts appeared to be pink against a pale background (Fig 4D) The cellular and subcellular localization of Fd-GOGAT peptide was determined in leaf sections by the indirect immunofluorescence method, as described in [13] Using a confocal laser scanning microscope, specific immunofluorescence was found in the chloroplasts of BSCs (Fig 5A) No fluorescence was detected using nonimmune serum as a primary antibody (Fig 5B) 3198 chl MC MC E chl D C chl chl BSC Fig Immunocytochemical localization of Fd-GOGAT in thin sections of 21-day-old maize leaves (A) Leaf bundle sheath cell and vascular bundle section using antibody against Fd-GOGAT as the primary antibody (B) Control leaf bundle sheath cell and vascular bundle section using nonimmune serum as the primary antibody (C) Leaf mesophyll cell section using antibody against Fd-GOGAT as the primary antibody (D) Control leaf mesophyll cell section using nonimmune serum as the primary antibody BSC, bundle sheath cell; chl, chloroplast; MC, mesophyll cell Bar: 10 lm Higher magnification of MCs showed that the Fd-GOGAT proteins were localized to the chloroplasts (Fig 5C) No signal was detected with nonimmune serum as the primary antibody (Fig 5D) Expression of the genes involved in chloroplast electron transport Although the GS ⁄ Fd-GOGAT pathway was found to be distributed between BSCs and MCs, the Fd-dependent electron donor to the enzyme in BSC chloroplasts is not well understood Therefore, we determined the transcript levels of Fd and FNR, which are both encoded by a small gene family [25] We found constitutive mRNA levels of Fds and FNRs, except for Fd VI, which gave two peaks at and 15 h (Fig 2) Fd I, Fd II, Fd V, L-FNR and L-FNR are mainly distributed in the leaves, whereas Fd III and Fd VI are found in nonphotosynthetic tissues [26,27] The lowest mRNA level was detected for Fd I at h at 80% of the maximum (Fig 2) Two-phase specific promoters and ⁄ or mRNA stability could entrain two peaks of Fd VI and gdh1 [7] FEBS Journal 275 (2008) 3193–3206 ª 2008 The Authors Journal compilation ª 2008 FEBS M.-H Valadier et al Amino acid metabolism and translocation Glutamate synthesis in the reconstituted system using NADPH, FNRs, Fds and GOGAT Glutamate synthesis depends on a subtle specialization of FNRs and Fds We reconstituted a NADPH-dependent glutamate synthesis system using recombinant FNR, Fd and Fd-GOGAT proteins to assess whether NADPH serves as the initial electron donor for the catalytic reaction of Fd-GOGAT through a redox cascade of Fd and FNR As shown in Fig 6A, rapid NADPH oxidation was observed when all protein components and the substrates glutamine and 2-oxoglutarate were present in the assay mixture In contrast, only a basal level of NADPH oxidation, A B C uncoupled to glutamate formation, was observed without either substrate The N-terminal cysteine of Fd-GOGAT was essential for the amido transfer reaction, and an Fd-GOGAT mutant with this cysteine residue substituted by glycine, Cys1Gly, showed no significant NADPH oxidation (Fig 6B) NADPH oxidation was correlated with glutamate formation measured by HPLC (data not shown), confirming that NADPH supported glutamate synthesis To further investigate the NADPH ⁄ FNR ⁄ Fd-GOGAT electron pathway in glutamate synthesis, NADPH oxidation was assayed using several combinations of photosynthetic isoproteins (L-FNR ⁄ Fd I, L-FNR ⁄ Fd II or L-FNR ⁄ Fd V) or a combination of nonphotosynthetic isoproteins (R-FNR ⁄ Fd III) The rate of NADPH oxidation in the nonphotosynthetic system was most efficient of all combinations of FNRs and Fds (Table 2) The R-FNR ⁄ Fd III combination gave an activity of about three-fold higher than those of L-FNR ⁄ Fd I, L-FNR ⁄ Fd II and L-FNR ⁄ Fd V, all of which showed a similar activity (Table 2) When glutamate formation was determined as a function of Fd concentration, the kinetics of NADPH oxidation in the R-FNR ⁄ Fd III system were high, particularly at lower Fd concentrations, compared with the L-FNR ⁄ Fd I system (Fig 6C) The results indicate that nonphotosynthetic R-FNR and Fd III isoproteins promote efficient glutamate formation using NADPH as reductant Amino acid translocation in the vascular streams In order to monitor the amino acids supplied for allocation by the source tissues, we analysed the amino acid contents in the phloem sap Phloem exudate was promoted and collected as described in [28] As the plants exuded at lower rates in the dark, the amino Fig Assay for Fd-GOGAT activity in the reconstituted electron transfer system The complete reaction mixture contained 50 mM Tris ⁄ HCl, pH 7.5, 100 mM NaCl, 0.2 mM NADPH, mM 2-oxoglutarate, mM glutamine and maize recombinant proteins as follows: 0.2 lM L-FNR 1, 20 lM Fd I and 0.36 lM of either WT (A) or Cys1Gly mutant (B) of Fd-GOGAT As a control, 2-oxoglutarate or glutamine was omitted from the reaction mixture The kinetics of Fd-GOGAT activity were assayed by increasing the concentrations of Fd isoprotein as indicated in the figure (C) Photosynthetic (s) and nonphotosynthetic (d) combinations contained L-FNR ⁄ Fd I and R-FNR ⁄ Fd III, respectively Oxidation of NADPH was followed by monitoring the decrease in A340 nm Table Comparison of Fd-GOGAT activity supported by different combinations of Fds and FNRs The reaction mixture contained 50 mM Tris ⁄ HCl, pH 7.5, 100 mM NaCl, 0.2 mM NADPH, mM 2-oxoglutarate, mM glutamine and maize recombinant proteins as follows: 0.2 lM L-FNR or R-FNR, 20 lM Fd isoprotein and 0.36 lM Fd-GOGAT Fd-GOGAT activity is expressed as the rate of NADPH oxidation [lmolỈmin)1Ỉ(mg Fd-GOGAT protein))1] Reaction Photosynthetic isoproteins L-FNR ⁄ Fd I L-FNR ⁄ Fd II L-FNR ⁄ Fd V Nonphotosynthetic isoproteins R-FNR ⁄ Fd III FEBS Journal 275 (2008) 3193–3206 ª 2008 The Authors Journal compilation ª 2008 FEBS Specific activity 0.508 0.476 0.405 1.28 3199 Amino acid metabolism and translocation M.-H Valadier et al acid analysis was carried out in exudates harvested over a 16 h light phase and h dark phase The amino acid composition in the phloem exudates was very different from that in xylem sap (Fig 7), suggesting that there was little contamination from xylem, and vice versa Glx (glutamine and glutamate: five carbon amide and amino acid) and Asx (asparagine and aspartate: four carbon amide and amino acid) were found to be the main nitrogen compounds in the phloem exudates, amounting to 51% of the total amino acids in the light and 56% in the dark (Fig 7A) The total amount of alanine, serine and glycine was reduced from 33% in the light to 25% in the dark (Fig 7A) Glutamine and asparagine were the major amino acids in the xylem sap in both the light (63% of the total amino acids) and dark (60%) (Fig 7B) The amino acid percentage ratio in the leaves and phloem distinguished three groups First, glutamine and asparagine appeared to be preferentially Phloem exudates Relative amount % A 30 Light Dark 20 10 Gln Glu Asn Asp Ala Gly Ser Xylem saps Relative amount % B 40 Light Dark 30 20 10 Gln Glu Asn Asp Ala Amino acids Gly Ser Fig Amino acid composition in phloem exudates (A) and xylem bleeding sap (B) collected from maize during a 16 h light phase (grey bars, Light) and h dark phase (black bars, Dark) Phloem exudates were collected in tubes filled with 10 mM Hepes buffer, pH 7.5 containing mM EDTA Xylem sap was obtained from cut stumps of decapitated plants The amino acid composition is expressed as a percentage relative to the total amino acid contents, which represent the mean (nmolỈ100 lL)1) from three independent plants ± standard error as follows: 44.3 ± 2.9 (phloem, Light), 19.8 ± 1.2 (phloem, Dark), 34.1 ± 2.0 (xylem, Light) and 24.6 ± 1.5 (xylem, Dark) The standard errors for individual amino acid contents are of the same order of magnitude as those of the total amino acid contents 3200 transported in the phloem, as indicated by high phloem ⁄ leaf ratios (12–25) (Table 1) Translocation of glutamine and asparagine in the phloem seemed to be increased in the dark (Table 1) Second, glutamate, aspartate and serine were similarly distributed in leaves and phloem sap, yielding phloem ⁄ leaf ratios of between 0.9 and 1.7 (Table 1) Third, alanine and glycine were poorly translocated, with phloem ⁄ leaf ratios below 0.7 (Table 1) Finally, amino acids were selectively translocated in the xylem in the form of glutamine and asparagine, which showed a significantly high xylem ⁄ leaf ratio of between 28 and 99 in the light and dark (Table 1) Discussion Glutamine is the main entry point of ammonium, which can be derived from nitrate reduction, protein turnover and, to a lesser extent, photorespiration in the post-flowering maize ear leaf A large accumulation of ammonium in the second half of the light period revealed that ammonium assimilation was substantially inhibited in response to ammonium formation Despite the low abundance of mRNA for four Gln1 genes, active GS1 partially converted a high level of ammonium into glutamine, which transiently increased shortly after the ammonium peak However, glutamine could not be further metabolized because of glutamate deficiency (Fig 1) To obtain an insight into nitrogen assimilation, we showed that Fd-GOGAT was located in the chloroplasts of both BSCs and MCs (Fig 5) To our knowledge, this is the first demonstration of FdGOGAT mRNAs in the cytoplasm on the periphery of chloroplasts and of the enzyme protein in the chloroplasts of the two cell types This spatial distribution of Fd-GOGAT contrasts with its exclusive localization in BSCs of maize [29] BSCs contain most of the photorespiratory enzymes [23] In the post-flowering maize ear leaf, mitochondrial glycine decarboxylase complex (EC 1.4.4.2 ⁄ 2.1.2.10) produces photorespiratory ammonium [30] at rates between 25 and 50% of primary nitrate reduction (Fig 3) As the [15N] label from [15N]glycine, fed to maize leaf, is recovered within 45 exclusively in glutamine and glutamate [31], photorespiratory ammonium is primarily re-fixed via the vascular bundle-located GS1 [32], in concert with BSC-located Fd-GOGAT However, the physiological role of Fd-GOGAT in BSC chloroplasts is a matter of debate, because BSC chloroplasts contain only 20–30% of PS II polypeptides, and most of the capacity for noncyclic electron transport and concomitant Fd reduction is localized to MC chloroplasts [25,33] FEBS Journal 275 (2008) 3193–3206 ª 2008 The Authors Journal compilation ª 2008 FEBS M.-H Valadier et al In vitro Fd-GOGAT assay showed that Fds reduced with NADPH via FNRs display a several-fold higher ability to donate electrons to GOGAT (Table 2) than does photoreduced Fd [34] In spite of the low FNR and Fd concentrations in BSCs (30 and 40 lm) [35,36], a close location of FNRs, Fds and Fd-GOGAT on the thylakoid membrane [25,37] allows protein–protein complex formation essential for the GOGAT reaction [24] To our knowledge, our results provide evidence for the first time that FNR couples Fd reduction with NADPH oxidation in the GOGAT reaction The data indicate that the NADPH ⁄ FNR ⁄ Fd system drives a specific redox reaction in vivo for BSC-located Fd-GOGAT in ammonium assimilation Moreover, the nonphotosynthetic R-FNR ⁄ Fd III system yields a 2.5–3-fold higher GOGAT activity than all photosynthetic FNR ⁄ Fd systems This reflects a higher redox potential of Fd III ()345 mV) than the other Fds [26], leading to rapid thermodynamic electron transfer Without light energy, the NADPH ⁄ R-FNR ⁄ Fd III system presumably substitutes for photoreduced Fd and sustains the GS1 ⁄ Fd-GOGAT cycle to assimilate ammonium in the dark (Fig 3) The reversible electron transfer between NADPH and Fd via FNR has been shown to occur in cyanobacteria and green algae [38,39] However, the contribution of this system in the light and dark to meet the needs for reductant supply to Fd-GOGAT has not been elucidated In addition, the reductant supply system from NADPH to Fd-GOGAT via L-FNR ⁄ Fd II and R-FNR ⁄ Fd III is relevant in BSC chloroplasts (Figs and 5), because of the internal light gradient within the translucent veins of BSCs The light absorption and scattering attenuate the photon fluence rate by 34% at 450 ⁄ 680 nm and 15% at 725 nm by the initial 50 lm across the maize mesocotyl [40] As a result, Fd reduction deprived of sufficient light at the core of vascular bundles depends on the sensitive NADPH ⁄ FNR ⁄ Fd system Glutamate synthesis increases on addition of NADPH at a large excess of stromal concentrations in the dark (0.3– 0.48 mm) [41] (data not shown) This provides evidence that the supply of reduced Fd via NADPH limits nitrogen assimilation and presumably sulfur reduction in the plastids, where the oxidative pentose phosphate pathway produces NADPH [42,43] Large amounts of ammonium are produced in the ear leaf in response to the induction of proteolysis [44], up to several fold higher than primary ammonium (Fig 3) Ammonium incorporation into glutamine and glutamate occurs exclusively by GS, GOGAT and GDH in a broad range of organisms [45] The rapid ammonium accumulation and contrasting shortage of glutamate in the second half of the light phase provide Amino acid metabolism and translocation evidence that the impairment of ammonium assimilation by the GS1 ⁄ GOGAT cycle is caused by the decline in Fd-GOGAT and NADH-GOGAT (Figs and 3) The active GDH does not contribute to alleviate the excess ammonium into glutamate This contrasts with the proposed role of GDH in assimilating excess ammonium in the source leaves in which GDH is induced after pollination (for a review, see [9,10]) Genetic evidence indicates that members of the GDH S_50II class, including plant mitochondrial NADH-GDHs, oxidize glutamate By contrast, members of the GDH S-50I class, such as plastidial NADPH-GDH (EC 1.4.1.4) of Chlorella, assimilate ammonium into glutamate [46] In fact, chloroplast NADPH-GDH is found in higher plants [47], suggesting a possible alternative role of this isoform Therefore, NADH-GDH may provide the anaplerotic pathway with 2-oxoglutarate to regenerate NADH and 2-oxalacetate for further transaminations In addition to GOGATs, glutamate can be produced by the aminotransferases, which, in turn, consume the equivalent amount of glutamate in the reverse reactions to form aspartate and alanine for further amino acid interconversions Therefore, the net synthesis of glutamate through the GS1 ⁄ GOGAT cycle is a prerequisite for grain development This view is supported by the evidence that the overall glutamate level remains constant in the source organs (stalks and cobs) [8,9], and the nitrate supply to roots after pollination reduces the loss of amino acids from these stalks and leaves for use in grain filling [8] The amino acids were selectively remobilized in the phloem in the form of glutamine, asparagine, glutamate and aspartate, which had high phloem ⁄ leaf ratios (Table 1) As a result, these amino acids make up the major components of the seed storage proteins [9] The abundance of glutamine, asparagine and glutamate in the phloem sap correlates with the spatial distribution of GS1 [32], AS and Fd-GOGAT in BSCs, arranged in one or more layers adjacent to the sieve tubes (Figs and 5) The phloem loading of glutamine, asparagine and glutamate from BSCs takes place via H+-coupled amino acid transporters into the vascular parenchyma at the border of BSCs ⁄ vascular parenchyma The amino acids are then apoplastically loaded into the companion cell–sieve element complexes because of the low abundance of plasmodesmata [48] By contrast, the phloem loading from MCs requires additional H+-amino acid transporters across the MC–BSC interface, and depends on the continuity of the electrochemical H+ gradient between the two cell types The location of the GS1 ⁄ Fd-GOGAT cycle in the BSCs, surrounding sieve element, meets the demand of amino FEBS Journal 275 (2008) 3193–3206 ª 2008 The Authors Journal compilation ª 2008 FEBS 3201 Amino acid metabolism and translocation M.-H Valadier et al acid synthesis in these cells, from which the amino acids are loaded to the phloem for the grain In the phloem sap, glutamine is the preferred nitrogen carrier rather than asparagine (Table 1) This can be attributed in part to the amide group on the d-carbon of glutamine, which increases the binding affinity to the transporter (AAP5) by at least three orders of magnitude compared with asparagine [49] In Arabidopsis, dark and sugar induce ASN1 and gln1-1, respectively, and repress gln1-1 and ASN1, respectively These expression patterns correlate with the relative abundance of asparagine and glutamine in the leaves [50] The expression of gln1-3 and ASN in maize is inhibited by light and sugar, respectively [51,52] Therefore, the increased ratios of asparagine to glutamine in the phloem sap in the dark could be attributed partly to the conversion of glutamine to asparagine by AS in the dark Materials and methods Plant growth Seeds of maize (Zea mays L cv DEA) were germinated on sand by supplying a complete nutrient solution, as described in [22] Maize seedlings were grown for 21 days in a controlled growth chamber under a regime of 16 h light (photosynthetic photon flux density of 300 lmol photonsỈm)2Ỉs)1 at 25 °C) and h dark (18 °C) Plants were then grown in a glasshouse for months under natural light with irrigation by complete nutrient solution as described previously [22] Two weeks before harvest, plants were transferred to a controlled chamber and grown in a 16 h light ⁄ h dark cycle under the conditions described above Leaves were numbered from the bottom of the plant, and the second leaves upward from the first ear were harvested for analysis Relative quantitative RT-PCR Total RNA was extracted using a kit according to the manufacturer’s instructions (Qiagen GmbH, Hilden, Germany) Relative RT-PCR was carried out using rRNA as an endogenous standard, and the first cDNA strands were synthesized from lg of RNA using an Omniscript RT kit (Qiagen GmbH) The abundance of initial cDNA strands between samples was corrected using agarose gel electrophoresis and Quantum RNA 18S internal standards (Ambion, Austin, TX, USA) PCR was performed on a LightCycler Instrument (Roche, Basle, Switzerland) For the genes of the multigene family, the specific oligonucleotides were designed along the nonconserved stretches of the genes in the same gene family The following specific primer sets were used for each gene, indicated by the GenBank database accession 3202 number: NR1 (accession number M27821): forward primer, 5¢-CTCAAGCGCATCATCGTCAC-3¢; reverse primer, 5¢-ATGATCTGGTACATGGGCGTG-3¢; GS1-1 (D14576, X65929): forward primer, 5¢-CCCTCCTTCCTCCTTGG GTT-3¢; reverse primer, 5¢-ATGGAATGGAAGTGGTGG GAA-3¢; GS1-2 (D14577, X65928): forward primer, 5¢-TCTCGGACAACACCGAGAAGA-3¢; reverse primer, 5¢-CACAAGTGTGGTACGGCCATT-3¢; GS1-3 (D14578, X65930): forward primer, 5¢-CAGCTCTTCTTGGGTTGC CTA-3¢; reverse primer, 5¢-GTACCCAATAAACGGGA AGCG-3¢; GS1-4 (D14579, X65926): forward primer, 5¢-CTTCTCGTCTGCCCGAGT-3¢; reverse primer, 5¢-CTG GAAGCACAGCCAAACGTA-3¢; GS2 (X65931): forward primer, 5¢-GACGGTTGGTTCGGGAATG-3¢; reverse primer, 5¢-TCCGATGAATCAAAGACAGCC-3¢; Fd-GO GAT (M59190): forward primer, 5¢-GCTGCTATGGGAG CTGATGAA-3¢; reverse primer, 5¢-GCAACGGCCAAG AATCATGTA-3¢; GDH1 (D49475): forward primer, 5¢TTGTTCCTTGGGAGGATAGAAAAA-3¢; reverse primer, 5¢-TTGCTTGCAGACAGCATCTCA-3¢; ASN (X82849): forward primer, 5¢-AAAGCTTCATCGCAGCTCGT-3¢; reverse primer, 5¢-CACGACACACACACACACGT-3¢; Fd I (M73830): forward primer, 5¢-CTACAACGTGAAGCT GATCAC-3¢; reverse primer, 5¢-GATGGGCATGAATGAT TATGCGC-3¢; Fd II (AB016810): forward primer, 5¢-CCTG GCGGTGTATAGCTAAGCAG-3¢; reverse primer, 5¢-CTG AGCATGAGCATCCTCC-3¢; Fd III (M73831): forward primer, 5¢-CGAAGGTTCCAAGCCTGAAGACC-3¢; reverse primer, 5¢-CTAGCAGAACATAGAAGACAGC-3¢; Fd V (M73828): forward primer, 5¢-TCCAGCCATTACCCGCA GCTAGC-3¢; reverse primer, 5¢-GCTTAGGAGATAAG GTCGTCCTCC-3¢; Fd VI (AB001385): forward primer, 5¢-GACGGAGCACGAGTTCGAGGC-3¢; reverse primer, 5¢-CTCATATGCCATGATCTCATCG-3¢, L-FNR (AB035644): forward primer, 5¢-ACAACACAAAATGTCAGCTGC AAAA-3¢; reverse primer, 5¢-AAGGCCAAGAAGGAGTC CAAGAAG-3¢; L-FNR (AB035645): forward primer, 5¢-TTGCTTGAGCTGAACAATACAATGAA-3¢; reverse primer, 5¢-GAGCCGGTCAAGAAGCTGGAG-3¢ PCRs were carried out using : 5, : 10, : 20 and : 40 dilutions of cDNA Reactions were hot started at 95 °C, and carried out for 32 cycles of 94 °C for 30 s, annealing temperature for and 30 s and 72 °C for 30–90 s Products were visualized by ethidium bromide in agarose gels, and bands were quantified by scanning with an FLA-5000 imaging system (Fujifilm SAS, St-Quentin, France) In situ hybridization experiment Tissue inclusion Leaf sections were harvested at 2–3 h into the light phase, and immediately fixed in 4% (v ⁄ v) paraformaldehyde containing 0.1% Triton X-100 in NaCl ⁄ Pi (10 mm sodium phosphate, pH 7.0 and 130 mm NaCl) After dehydration in a FEBS Journal 275 (2008) 3193–3206 ª 2008 The Authors Journal compilation ª 2008 FEBS M.-H Valadier et al Amino acid metabolism and translocation graded ethanol series (10%, 30%, 50%, 70% and 96%), tissues were incubated in an ethanol ⁄ histoclear series (2 : 1, : 1, : 2, v ⁄ v), histoclear and histoclear ⁄ paraffin (1 : 1, v ⁄ v), and embedded in paraffin at 59 °C (75 mgỈmL)1) Slides were sealed with gel mount formol1 (Microm Microtech France, Francheville, France), and fluorescence was observed using a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany) Hybridization probe preparation Indirect immunofluorescence analysis Total RNA was extracted from maize leaves using an RNA isolation kit (Qiagen GmbH) First cDNA strands were synthesized from lg of RNA using an Omniscript RT kit (Qiagen GmbH) Partial DNAs of Fd-GOGAT and AS were amplified by PCR using the following primers FdGOGAT: sense probe: forward primer, 5¢-TGTAATTCGA CTCACTATAGGGTACGCAGCCACCAGTCATGTA-3¢; reverse primer, 5¢-TACGCAGCCACCAGTCATGTA-3¢; antisense probe: forward primer, 5¢-CTTAGGGTGGACG GTGGATTC-3¢; reverse primer, 5¢-TGTAATTCGACTC ACTATAGGGTACGCAGCCACCAGTCATGTA-3¢; AS: sense probe: forward primer, 5¢-TGTAATTCGACTCACT ATAGGGCCTCCCTGCTAGCTTCTACCG-3¢; reverse primer, 5¢-TCCAGACATACAGACACGGGC-3¢; antisense probe: forward primer, 5¢-CCTCCCTGCTAGCTTC TACCG-3¢; reverse primer, 5¢-TGTAATTCGACTCACTA TAGGGCTCCAGACATACAGACACGGGC-3¢ Sense and antisense DNAs (400 ng each) were labelled with DIGUTP using a transcription kit (Promega, Madison, WI, USA) After DNase digestion (1 unit per reaction), RNA probes were hydrolysed in a carbonate solution (120 mm Na2CO3 and 80 mm NaHCO3, pH 10.2), and controlled by anti-DIG IgG conjugated with alkaline phosphatase (Roche Diagnostics GmbH, Penzberg, Germany) on GeneScreen membrane Leaf sections were fixed in 3.7% (w ⁄ v) formaldehyde in 50 mm PIPES buffer, pH 6.9, mm MgSO4 and mm EGTA (MTSB), and then in NaCl ⁄ Pi (6.5 mm Na2HPO4, 1.5 mm KH2PO4, pH 7.3, 14 mm NaCl and 2.7 mm KCl) Tissues were dehydrated in a graded ethanol series (30%, 50%, 70%, 90% and 97%) Samples were incubated in a mixture of wax and ethanol (1 : 1, v ⁄ v), and then embedded in wax at 40 °C Sections (10 lm) were prepared using a microtome, and slides were dewaxed and rehydrated through a degraded ethanol series (97%, 90% and 50%) Antigen unmasking was carried out in 10 mm citrate buffer, pH 6.0 at 95 °C for min, and blocked with 1% (w ⁄ v) BSA in NaCl ⁄ Pi (blocking solution) Antibody hybridization was carried out with the primary rabbit antibody against tobacco Fd-GOGAT, and then with goat antirabbit IgG labelled with Alexa 405 (Molecular Probes, Carlsbad, CA, USA) in blocking solution As a control, preimmune serum was used as the primary antibody Immunofluorescence was observed using a spectral confocal laser scanning microscope (Leica TCS SP2 AOBS) (Leica Microsystems) Immunofluorescence was observed with a laser diode (25 mW, 405 nm) using a Leica HC PL APO 63· ⁄ 1.20 Water Corr ⁄ 0.17 Lbd.BL objective Low-speed scan (200 lines per second) images (512 · 512 pixels) were generated, and Alexa 405 fluorescence was collected with a specific bandwidth (407–427 nm) after spectral adjustment to eliminate background noise The red autofluorescence of tissues was observed between 509 and 628 nm In situ hybridization Tissue sections (8 lm) were prepared using a microtome, and samples on slides (DAKO 2024, Dako, Basingstoke, UK) were deparaffinized in histoclear and hydrated by a degraded ethanol series (96%, 85%, 50% and 30%) After proteinase K digestion (4 lgỈmL)1) in TE buffer (10 mm Tris ⁄ HCl, pH 7.5 and 50 mm EDTA), samples were treated with 0.5% (v ⁄ v) acetic anhydride in 1.3 m triethanolamine, pH 7.0, and dehydrated in a graded ethanol series (30%, 50%, 70%, 85%, 96% and 100%) Sense and antisense probes were denatured, dissolved and hybridized in situ in mRNA hybridization solution (Dako) at 45 °C overnight Slides were washed in 0.2 · SSC (1 · SSC: 150 mm NaCl and 15 mm sodium citrate, pH 7.0) at 45 °C, T2 solution (100 mm Tris ⁄ HCl, pH 7.5 and 150 mm NaCl containing 0.5% blocking reagent) (Roche Diagnostics GmbH) and T3 solution (T1 including 1% BSA and 0.5% Triton X-100) at room temperature Slides were incubated with alkaline phosphatase-conjugated anti-DIG IgG in T3 Alkaline phosphatase activity was developed with 5-bromo-4-chloro-3indolyl-phosphate (50 mgỈmL)1) and nitroblue tetrazolium Enzyme preparation and assays NR was extracted as described previously [17] The activation state of NR was determined by the activity ratio in the presence of 10 mm MgCl2 or mm EDTA for the divalent cation-dependent activity and maximum catalytic activity, respectively NiR and GS were extracted and assayed according to [17] Fd-GOGAT and NADH-GOGAT were extracted and assayed by measuring glutamate formation by HPLC as described in [22] GDH was extracted and assayed for reductive glutamate synthetic activity and glutamate oxidation activity according to [17] Reconstituted electron transfer system to Fd-GOGAT Fd-GOGAT was assayed by reconstituting the electron transfer pathway from NADPH to Fd via FNR as FEBS Journal 275 (2008) 3193–3206 ª 2008 The Authors Journal compilation ª 2008 FEBS 3203 Amino acid metabolism and translocation M.-H Valadier et al described essentially in [36] The recombinant maize proteins of FNRs and Fds (L-FNR, R-FNR, Fd I, Fd II, Fd III and Fd V) were prepared in the Escherichia coli expression system [25,35] Maize Fd-GOGAT was also prepared in a similar system (T Hase, unpublished work) The complete reaction mixture contained 50 mm Tris ⁄ HCl, pH 7.5, 100 mm NaCl, 0.2 mm NADPH, mm 2-oxoglutarate, mm glutamine and maize recombinant proteins as follows: 0.2 lm L-FNR or R-FNR, 20 lm of Fd I, Fd II, Fd III or Fd V and 0.36 lm of Fd-GOGAT The oxidation of NADPH was followed by monitoring the decrease in A340 nm The formation of glutamate was also analysed with an equivalent assay system The reaction mixture contained 25 mm sodium phosphate buffer, pH 7.3, 0.14 mm NADPH, 0.1 lm L-FNR, 20 lm Fd I, mm glutamine, mm 2-oxoglutarate and 0.73 lm of either wild-type or mutant Fd-GOGAT The reaction was carried out at 30 °C, and glutamate formation was measured by HPLC as described above Amino acid analysis Samples were freeze-dried and amino acids were extracted from 20 mg samples at °C with mL of 2% (w ⁄ v) sulfosalicylic acid After centrifugation at 17 500 g for 15 min, supernatants were adjusted to pH 2.1 with LiOH and stored at )70 °C prior to analysis Total amino acid contents were estimated according to the method of Rosen [53] Amino acids were separated by ion-exchange chromatography on a JLC-500 ⁄ V amino acid analyser (JEOL Ltd, Tokyo, Japan) Collection of phloem exudates and xylem bleeding sap Phloem exudates and xylem bleeding sap were collected during a 16 h light ⁄ h dark cycle Shoots were cut off and rapidly immersed in tubes filled with 5–10 mL of collection buffer consisting of 10 mm Hepes, pH 7.5 and mm EDTA, as described previously [54] Xylem sap was collected from the cut stumps of decapitated plants using a micropipette [28] Phloem exudates were adjusted to pH 2.1, and both phloem exudates and xylem sap were concentrated by speedvac and stored at )70 °C prior to amino acid analysis Determination of metabolites, total soluble proteins and chlorophylls Metabolites were extracted from lyophilized materials with 2% 5-sulfosalicylic acid Nitrate contents were analysed as described in [17] Free ammonium contents were determined by the phenol hypochlorite assay (Berthelot assay) Soluble protein contents were determined by the Coomassie blue 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Planta 222, 667–677 FEBS Journal 275 (2008) 3193–3206 ª 2008 The Authors Journal compilation ª 2008 FEBS ... serine and glycine was reduced from 33% in the light to 25% in the dark (Fig 7A) Glutamine and asparagine were the major amino acids in the xylem sap in both the light (63% of the total amino acids)... 3195 Amino acid metabolism and translocation M.-H Valadier et al Table Amino acid composition in leaves, and amino acid percentage ratio in the phloem exudates and leaves and xylem bleeding sap and. .. molecular signalling in the expression of the genes and encoded enzymes involved in nitrate assimilation and amino acid synthesis [7] The stalk and leaves below and above the ear act as the source