Báo cáo khoa học: Assimilation of excess ammonium into amino acids and nitrogen translocation in Arabidopsis thaliana – roles of glutamate synthases and carbamoylphosphate synthetase in leaves ppt

Pheno-typic analysis revealed a high level of photorespiratory ammonium, gluta-mine⁄ glutamate and asparagine ⁄ aspartate in the GLU1 mutant lacking the major ferredoxin-glutamate syntha

nitrogen translocation in Arabidopsis thaliana – roles of glutamate synthases and carbamoylphosphate synthetase

in leaves

Fabien Potel1, Marie-He´le`ne Valadier1, Sylvie Ferrario-Me´ry1, Olivier Grandjean2, Halima Morin2, Laure Gaufichon1, Ste´phanie Boutet-Mercey3, Je´re´my Lothier1, Steven J Rothstein4, Naoya Hirose1 and Akira Suzuki1

1 Unite´ de Nutrition Azote´e des Plantes, Institut National de la Recherche Agronomique, Versailles, France

2 Plateforme de Cytologie et Imagerie Ve´ge´tale, Institut National de la Recherche Agronomique, Versailles, France

3 Plateforme Chimie du Ve´ge´tal, Institut National de la Recherche Agronomique, Versailles, France

4 Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Ontario, Canada

Keywords

amino acid translocation; Arabidopsis thaliana;

carbamoylphosphate synthetase; glutamate

synthase; nitrogen assimilation

Correspondence

A Suzuki, Unite´ de Nutrition Azote´e des

Plantes, Institut National de la Recherche

Agronomique, Route de St-Cyr, 78026

Versailles cedex, France

Fax: +33 1 30 83 30 96

Tel: +33 1 30 83 30 87

E-mail: suzuki@versailles.inra.fr

(Received 27 March 2009, revised 22 May

2009, accepted 27 May 2009)

doi:10.1111/j.1742-4658.2009.07114.x

This study was aimed at investigating the physiological role of ferredoxin-glutamate synthases (EC 1.4.1.7), NADH-ferredoxin-glutamate synthase (EC 1.4.1.14) and carbamoylphosphate synthetase (EC 6.3.5.5) in Arabidopsis Pheno-typic analysis revealed a high level of photorespiratory ammonium, gluta-mine⁄ glutamate and asparagine ⁄ aspartate in the GLU1 mutant lacking the major ferredoxin-glutamate synthase, indicating that excess photorespiratory ammonium was detoxified into amino acids for transport out of the veins Consistent with these results, promoter analysis and in situ hybridization demonstrated that GLU1 and GLU2 were expressed in the mesophyll and phloem companion cell–sieve element complex However, these phenotypic changes were not detected in the GLU2 mutant defective in the second ferredoxin-glutamate synthase gene The impairment in primary ammonium assimilation in the GLT mutant under nonphotorespiratory high-CO2 con-ditions underlined the importance of NADH-glutamate synthase for amino acid trafficking, given that this gene only accounted for 3% of total gluta-mate synthase activity The excess ammonium from either endogenous pho-torespiration or the exogenous medium was shifted to arginine The promoter analysis and slight effects on overall arginine synthesis in the T-DNA insertion mutant in the single carbamoylphosphate synthetase large subunit gene indicated that carbamoylphosphate synthetase located in the chloroplasts was not limiting for ammonium assimilation into arginine The data provided evidence that ferredoxin-glutamate synthases, NADH-glutamate synthase and carbamoylphosphate synthetase play specific physi-ological roles in ammonium assimilation in the mesophyll and phloem for the synthesis and transport of glutamine, glutamate, arginine, and derived amino acids

Abbreviations

AS, asparagine synthetase; CP, carbamoylphosphate; CPSase, carbamoylphosphate synthetase (EC 6.3.5.5); Fd, ferredoxin; Fd-GOGAT, ferredoxin-glutamate synthase (EC 1.4.1.7); Fd-NiR, ferredoxin-dependent nitrite reductase; GDC, glycine decarboxylase complex; GDH, glutamate dehydrogenase; GOGAT, glutamate synthase; GS, glutamine synthetase (EC 6.1.1.3); NADH-GOGAT, NADH-glutamate synthase (EC 1.4.1.14); NAGK, N-acetyl-glutamate kinase; NR, nitrate reductase.

Inorganic nitrogen in the form of nitrate and

ammo-nium in the soil is absorbed by roots across the

plasma membrane, and it is in part transported via

the xylem to leaves prior to incorporation into amino

acids in Arabidopsis [1] Primary nitrogen reduction

from nitrate to ammonium is catalyzed by cytosolic

nitrate reductase (NR; EC 1.6.6.1), and then by

plas-tidial ferredoxin (Fd)-dependent nitrite reductase

(Fd-NiR; EC 1.6.6.4) Photorespiratory glycine oxidation

in the mesophyll mitochondria releases the bulk of

ammonium at high rates of as much as 10–20-fold

those of primary nitrate reduction in leaves [2]

Pri-mary and photorespiratory ammonium assimilation

into amino acids could take place by four distinct

pathways in Arabidopsis, to meet the needs of protein

synthesis, the maintenance of amino acid pool levels

within the leaves, and nitrogen transport to the

grow-ing apical sinks and roots via the phloem First, the

glutamine synthetase (GS)–glutamate synthase

(GO-GAT) cycle is the major assimilatory pathway

Gluta-mine is generated from ammonium and glutamate by

cytosolic GS1 and plastidial GS2 (EC 6.3.1.2) Then,

GOGAT transfers the glutamine amide group to the

2-position of 2-oxoglutarate to yield two molecules of

glutamate, one of which is cycled to GS The

Arabid-opsis nuclear genome carries multiple genes for many

of the nitrogen assimilatory enzymes, and GOGAT

exists as Fd-GOGAT (EC 1.4.7.1), encoded by GLU1

and GLU2, and as NADH-GOGAT (EC 1.4.1.14),

encoded by GLT [3] Second, either ammonium or a

glutamine amide group is integrated into asparagine

by cytosolic asparagine synthetase (AS)

[ammonia-ligasing AS (EC 6.3.1.1) or glutamine-hydrolyzing AS

(EC 6.3.5.4)] [4] Third, carbamoylphosphate

synthe-tase (CPSase) forms carbamoylphosphate (CP) using

bicarbonate (HCO3), ATP and ammonium

(ammonia-ligasing CPSase; EC 6.3.4.16), or the glutamine amide

group (glutamine-hydrolyzing CPSase; EC 6.3.5.5) [5]

In Arabidopsis, a single copy each of carA and of

carB encode the small subunit and large subunit,

respectively The small and large subunits form a

single heterodimeric enzyme that supplies CP as a

precursor for arginine and pyrimidine synthesis [6]

Finally, mitochondrial NADH-glutamate

dehydro-genase (EC 1.4.1.2) could alternatively incorporate

ammonium into glutamate in response to high levels

of ammonium under stress [7]

GOGATs are involved in the major synthetic

path-way of glutamate from primary and photorespiratory

nitrogen [8], and CPSase seems to catalyze a

commit-ted step to recover photorespiratory nitrogen in amino

acid synthesis in Arabidopsis [9] The amino acids are then translocated in the apoplasm and in the phloem via the plasma membrane-located amino acid trans-porters [10] Glutamine and asparagine, and to a lesser extent arginine, glutamate, and aspartate, are trans-ported in Arabidopsis phloem sap for use in sink cell development [11] Therefore, we investigated whether two Fd-GOGAT isoenzymes and NADH-GOGAT play overlapping or distinct roles in nitrogen assimila-tion into amino acids for transport in planta using mutants deficient in GLU1, GLU2, or GLT Despite the in silico data of the Arabidopsis databases, experi-ments on the in vivo function of CPSase remain largely unaddressed Inasmuch as arginine synthesis from CP relies on the regulation of glutamate conversion to ornithine [6], we studied the impact of CPSase on overall arginine synthesis in the carB mutant In fact, amino acid synthesis is tightly correlated with amino acid transport under the fine control of the cellular and subcellular expression of the nitrogen assimilatory genes and of the encoded enzymes [12] Despite their primary importance, the spatial location and expres-sion patterns have not been investigated for Fd-GO-GAT isoenzymes, NADH-GOFd-GO-GAT and CPSase in Arabidopsis Thus, we defined their subcellular localiza-tion and cell type-specific and tissue-specific expression patterns by promoter::GUS fusion expression in trans-genic Arabidopsis, in situ mRNA hybridization, and immunohistochemical localization The results showed that each isoenzyme of Fd-GOGAT, NADH-GOGAT and CPSase had distinct physiological relevance in the mesophyll and in the phloem for the biosynthesis and transport of amino acids under photorespiratory and nonphotorespiratory conditions

Results

Expression of the genes for GOGATs and CPSase

In order to understand the physiological role of GO-GATs and CPSase, we first examined the expression pattern of the genes encoding these enzymes in leaves and roots from 42-day-old Arabidopsis plants A search of the Arabidopsis genome database [13] revealed that there are two genes for Fd-GOGAT [GLU1 (AGI: At5g04140)] and GLU2 (At2g41220)], and one gene for NADH-GOGAT [GLT (At5g53460)] GLU1and GLU2 are composed of 33 exons coding for

a protein of 165 kDa, containing a class II (purF)-type glutaminase domain and short regions for binding to FMN and iron sulfur center GLT is composed of 20

exons encoding a large protein of 240 kDa CPSase is

encoded by two genes: carA (At3g27740) and carB

(At1g29900) carA is composed of 10 exons encoding

the 40 kDa small subunit The small subunit contains

a class I (trpG)-type glutaminase domain to hydrolyze

glutamine to ammonia carB is composed of three

exons encoding a 120 kDa large subunit, consisting of

the duplicated synthetase regions and the ATP-binding

domains to synthesize CP GLU1 was mainly expressed

in the leaves, at significantly higher level than GLT

and GLU2 (Fig 1A) Although GLT and GLU2 were

expressed in the leaves and in the roots, GLT mRNAs

were at least seven-fold more abundant than GLU2

mRNAs (Fig 1A) carA and carB were more highly

expressed in the leaves than in the roots, and both

leaves and roots contained slightly more abundant

carB mRNAs (Fig 1B) Among the cytosolic GS1

genes, higher mRNA levels were found for Gln12,

Gln13 and Gln14 than for Gln11 in the leaves

(Fig 1C) The highest mRNA level was also found for

Gln12 in the roots (Fig 1C) As compared with gln12,

the chloroplastic GS2 mRNAs were more abundant

than gln12 mRNAs in the leaves and in the roots (about two-fold and 1.5-fold, respectively) (data not shown)

Characterization of the T-DNA mutants for GOGATs and CPSase

With a reverse genetic screen, individual plants with homozygous mutant alleles were identified for GLU2, GLT and carB by PCR in combination with the prim-ers specific for the T-DNA left and right bordprim-ers The GLU2mutant was truncated by a T-DNA insertion in intron 9 (Fig 2A) With the use of primers down-stream of the insertion site, the GLU2 mRNA level was approximately 10% of the wild-type level in leaves (Fig 2D) The GLT mutant was characterized by a T-DNA insertion in exon 13 about 50 amino acids upstream of the FMN-binding domain (Fig 2B) The GLT T-DNA mutant contained about 20% of the wild-type level of GLT mRNA (Fig 2D) The carB mutant was disrupted by a T-DNA insertion in the promoter close to 600 nucleotides upstream of the

A

C

B

Fig 1 Transcript levels of the genes for

GOGATs, CPSase and GSs in leaves and

roots of Arabidopsis Arabidopsis plants

were grown for 42 days by hydroponic

cul-ture using 5 m M nitrate [37] in air

supple-mented with 3000 p.p.m CO 2 Transcript

levels were determined by quantitative

real-time RT-PCR (A) GOGAT genes: GLU1,

GLU2, and GLT (B) CPSase genes: carA

and carB (C) GS1 genes: Gln11, Gln12,

Gln13, and Gln14 The values are expressed

as percentage ± standard error relative to

the marker EF1a gene.

initial ATG codon (Fig 2C) The carB mutant

expressed about 10% of the wild-type level of carB

mRNA (Fig 2D) To evaluate whether the decrease in

the GOGAT transcripts correlates with a functional deficiency, we assayed GOGAT activities in leaves from plants grown in air or in high CO2(3000 p.p.m.), where photorespiration is repressed The Fd-GOGAT activity, encoded by GLU1 and GLU2, was reduced to less than 3% in the GLU1 mutant (ethylmethanesulfo-nate-mutagenized CS254 line) [2], whereas almost wild-type activity was recovered in the GLU2 mutant in air and in high CO2 (Table 1), indicating that GLU1 encodes the major Fd-GOGAT isoenzyme The NADH-GOGAT activity, encoded by GLT and repre-senting only 3% of the total GOGAT activity, was reduced to approximately one-fourth in the GLT mutant, whereas NADH-GOGAT activity was less affected in the GLU1 and GLU2 mutants, irrespective

of the photorespiratory conditions (Table 1) We also assayed GS and glutamate dehydrogenase (GDH), as these enzyme activities are closely related to ammo-nium assimilation The GS activity was not affected in the mutants, except for a slight reduction in the GLT mutant in high CO2 (Table 1) The GDH activity var-ied between 75% and 135% of the wild-type activity for glutamate synthesis and between 45% and 65% for glutamate oxidation in the three mutants (Table 1)

Phenotypic changes in the GOGAT and CPSase mutants

As our target was to evaluate the impact of gene func-tion on ammonium assimilafunc-tion and amino acid

1 kb

A

B

C

D

3′

5′

Fig 2 Schematic presentation of the gene structure with the

T-DNA insertion site, and RT-PCR analysis of transcript levels in the

Arabidopsis T-DNA insertion mutants (A) GLU2 with T-DNA

inser-tion in intron 9 (B) GLT with T-DNA inserinser-tion in exon 13 (C) carB

with T-DNA insertion in the promoter Gray triangles correspond to

T-DNA, which is not to scale Boxes indicate exons, and lines

indi-cate 5¢-flanking regions and introns The nucleotide sequences at

the gene–insertion junction are shown The number of the first

nucleotide refers to the position relative to A of the initial

transla-tion initiatransla-tion ATG codon for methionine (D) Transcripts estimated

by RT-PCR for GLU1, GLU2, GLT, carB and 25S ribosomal RNA

(rRNA) in the T-DNA mutants for GLU2, GLT and carB and the

wild-type Arabidopsis (WT).

Table 1 Activities of Fd-GOGAT, NADH-GOGAT, GS and GDH in the mutants and wild-type (WT) leaves of Arabidopsis under

3000 p.p.m CO2or atmospheric air The enzyme assay conditions are described in Experimental procedures GDH was assayed for NADH-dependent reductive amination (NADH-GDH) and oxidative deamination (NAD-GDH) of glutamate The activity is expressed as lmol of glutamate formed (GOGATs), lmol of hydroxylamine formed (GS), or lmol of NADH oxidized (or of NAD reduced) (GDH)

h)1Æg)1fresh weight.

Arabidopsis

CO 2 Fd-GOGAT 0.5 ± 0.1 26.4 ± 2.4 27.3 ± 2.12 8.7 ± 2.2 NADH-GOGAT 0.8 ± 0.1 0.6 ± 0.1 0.2 ± 0.1 0.8 ± 0.1

GS 115.5 ± 10.3 115.0 ± 12.3 87.0 ± 8.1 114.0 ± 10.5 NADH-GDH 28.6 ± 2.4 37.7 ± 4.2 26.8 ± 2.5 36.5 ± 3.2 NAD-GDH 5.5 ± 0.6 13.4 ± 1.5 7.7 ± 0.6 12.3 ± 1.9 Air

Fd-GOGAT 0.5 ± 0.1 28.5 ± 2.3 29.2 ± 2.7 30.2 ± 3.3 NADH-GOGAT 0.8 ± 0.1 0.7 ± 0.1 0.2 ± 0.1 0.9 ± 0.1

GS 84.1 ± 8.9 76.8 ± 7.1 76.6 ± 6.7 75.0 ± 7.0 NADH-GDH 45.7 ± 5.9 32.6 ± 2.8 44.2 ± 3.9 38.2 ± 3.9 NAD-GDH 5.9 ± 0.7 13.4 ± 1.1 11.1 ± 1.0 9.9 ± 0.7

metabolism, we determined the levels of ammonium

and free amino acids in leaves and compared them to

the levels in the control wild-type lines The GLU1

mutant accumulated a large amount of ammonium

48 h after transfer from high CO2 to air, owing to

photorespiratory ammonium release (Fig 3A) A slight

accumulation of photorespiratory and

nonphotorespi-ratory ammonium was detected in the GLT mutant

(Fig 3A) By contrast, the GLU2 and carB mutants contained a wild-type level of ammonium (Fig 3A,B) The ammonium level of the control wild-type line of the GLU1 mutant 48 h after transfer from high CO2to air (0.66 lmolÆg)1 fresh weight) (Fig 3A) was higher than that of the control wild-type line of the carB mutant in air (0.5 lmolÆg)1 fresh weight) (Fig 3B) This may be explained in part by the two experiments

Fig 3 Ammonium and amino acid contents in leaves of the GLU1 (ethylmethanesulfonate-mutagenized CS254 line), GLU2, GLT and carB mutants and the wild-type Arabidopsis (WT) Arabidopsis plants were grown for 42 days by hydroponic culture using 5 m M nitrate [37] in air supplemented with 3000 p.p.m CO2, and then in air for 48 h (A) Ammonium contents under high-CO2conditions and in air (B) Ammonium contents in air (C) Glutamine (GLN), glutamate (GLU), asparagine (ASN) and aspartate (ASP) contents under high-CO 2 conditions (D) Gluta-mine, glutamate, asparagine and aspartate contents in air (E) Ornithine (ORN), citrulline (CIT) and arginine (ARG) contents in leaves of Arabidopsis plants cultured with 5 m M nitrate in air (F) Ornithine, citrulline and arginine contents in leaves of Arabidopsis plants cultured with 2 m M ammonium in air (G) Ornithine, citrulline and arginine contents under high-CO 2 conditions (H) Ornithine, citrulline and arginine contents in air Arabidopsis lines represent the EMS mutant for GLU1 (GLU1), T-DNA mutants for GLU2 (GLU2), GLT (GLT), and carB (carB), and wild-type Arabidopsis The amino acid contents represent means of analysis on leaves from five independent plants.

being performed independently However, many of the

reactions of nitrogen assimilation and amino acid

syn-thesis depend on ATP, reduced Fd, and NAD(P)H,

and take place in the chloroplast Elevated CO2causes

an imbalance of energy and electron transport because

of the lack of photorespiration, which dissipates excess

photochemical energy and reducing equivalents [14]

This increases the number of chloroplasts and starch

grains per mesophyll cell [15], and higher ammonium

accumulation suggests that the control wild-type line

did not completely recover the nitrogen assimilatory

capacity damaged in high CO2 In high CO2, the

GLU1and GLT mutants had reduced glutamate levels

and increased glutamine levels (Fig 3C) The

gluta-mate and glutamine levels were unaffected in the

GLU2 mutant (Fig 3C) These observations indicate

that the GS⁄ GLU1 Fd-GOGAT and GS⁄ GLT

NADH-GOGAT cycles are involved in

nonphotorespi-ratory ammonium assimilation In air, the highest

glu-tamine⁄ glutamate ratio of 13.3 was obtained for the

GLU1mutant, confirming that the GS⁄ GLU1

Fd-GO-GAT cycle is the main pathway of photorespiratory

ammonium reassimilation (Fig 3D) No impairment in

glutamine to glutamate conversion was observed in the

GLT and GLU2 mutants, whereas the GLT mutant

accumulated asparagine (Fig 3C,D) As CPSase

sup-plies CP for arginine synthesis, the amino acid levels

of the urea cycle were determined Despite a tight

link-age of CPSase to arginine synthesis, the carB mutant

showed negligible effects on overall arginine levels

The levels of ornithine, citrulline and arginine

remained low, at between 0.01 and 0.04 lmolÆg)1fresh

weight (Fig 3E) However, arginine accumulated up to

70-fold and 80-fold in the carB mutant and in the

wild-type plants on 2 mm ammonium medium as

com-pared with nitrate medium (Fig 3E,F) The results

suggest that excess ammonium was incorporated into

arginine as a nitrogen storage compound The GLU1

mutant showed a 5.8-fold increase in arginine relative

to the wild-type plants in air, whereas in high CO2,

arginine remained at a wild-type level, indicating that

the high level of photorespiratory ammonium was in

part refixed into arginine as a detoxification molecule

(Fig 3H)

Changes in gene expression patterns caused by

exogenous ammonium

As the endogenous photorespiratory ammonium

affected the levels of ammonium and amino acids in

the GLU1 and GLT mutants (Fig 3), we investigated

whether expression of the ammonium assimilatory

genes of GOGAT, CPSase and GS1 is modified in

response to exogenous excess ammonium (10 mm), provided as a supplement to the culture medium Both GLU1 and GLT were expressed at higher levels than GLU2(Fig 4A) The ammonium caused up to 4.7-fold induction of GLT mRNAs; the GLU1 mRNA was induced to a lesser extent (Fig 4A) The level of carA mRNA was unaffected and that of carB mRNA was lowered by the ammonium treatment (Fig 4B) The GS1 genes exhibited the contrasting patterns in response to excess ammonium: a decrease in the Gln12 mRNA and increases in the Gln11 and Gln13 mRNAs (Fig 4C)

Expression of promoter::GUS fusions

To investigate the tissue-specific expression of the genes for GOGATs and CPSase, transgenic lines expressing an N-terminal translational construct fused

to a GUS reporter gene were generated The promoter region upstream of ATG, including a partial coding sequence, was isolated by PCR from GLU1 (2385 bp) ()1931 ⁄ 454), GLU2 (1501 bp) ()1089 ⁄ 412), carA (1121 bp) ()1021 ⁄ 100), and carB (992 bp) ()922 ⁄ 70) The translational fusions to the uidA gene under the control of the gene promoter were constructed by inserting the PCR product in-frame to the 5¢-end of the GUS reporter gene In the leaf sections of the transformed Arabidopsis lines, the GLU1::GUS fusion was expressed in chloroplasts of the mesophyll (Fig 5A) Furthermore, a high level of expression was detected in the vascular cells of minor veins (Fig 5B) GUS activity was detected in a layer of cells composed

of the companion cell–sieve element complex close to the several xylem tracheary elements (Fig 5B) A low level of GLU2::GUS expression was found not only in the mesophyll chloroplasts, but also in the phloem of minor veins (Fig 5C) A high level of carA::GUS expression was found in a cell layer close to the trache-ary elements of the vascular bundle, together with its neighboring mesophyll cells (Fig 5D) The expression

of carB::GUS was associated with the mesophyll chlo-roplasts and the companion cell–sieve element complex

in the phloem of minor veins (Fig 5E) In the leaf sec-tions from the plant transformed with empty vector,

no staining was detected (Fig 5F)

In situ hybridization of the transcripts

In situ hybridization analysis was carried out to deter-mine the tissue-specific expression pattern of GLU1, GLU2 and GLT in the leaf sections After hybridiza-tion to the antisense RNA probe, the GLU1 mRNAs were found on the periphery of the mesophyll

chloro-plasts (Fig 6A) In addition, specific staining appeared

in the phloem against a pale background (Fig 6B),

consistent with the GLU1 promoter expression patterns

(Fig 5) The GLU2 mRNAs were found around the

mesophyll chloroplasts (Fig 6C) Furthermore, strong

GLU2 mRNA staining was detected in the phloem

adjacent to the mesophyll (Fig 6E) The sense GLU2

mRNA probe gave no specific signal in the mesophyll

or in the vascular cells (Fig 6D) The GLT mRNAs

were strongly expressed in the phloem, whereas a weak

GLTmRNA signal was associated with the mesophyll

(Fig 6F), indicating that GLT was mainly expressed in

the vascular cells

Immunohistochemical localization

As the GLU1::GUS fusion and the GLU1 mRNAs

were expressed both in the mesophyll cells and in the

vascular cells, we examined the localization of

Fd-GO-GAT by the indirect immunofluorescence method,

using a specific antibody against tobacco Fd-GOGAT

as the primary antibody [4] With the use of confocal

laser-scanning microscopy, the Alexa 405 fluorochrome

signal was detected in the mesophyll cells and in the vascular cells of minor veins bordering the mesophyll cells (Fig 7A) With higher-magnification resolution, the specific fluorescence of Fd-GOGAT was found to

be located in the mesophyll chloroplasts (Fig 7C) The immunofluorescent signal and the corresponding trans-mission microscopy of the magnified vascular section showed that the specific signal was associated with the clustered oval companion cells, which flanked the sieve elements in close vicinity to the phloem parenchyma (Fig 7E,F) With nonimmune serum as the first anti-body, no signal was found in the leaf sections (Fig 7B,D)

Discussion

Recovery of excess ammonium into amino acids

in the mesophyll The expression analysis showed that the GLU1 mRNAs were mainly expressed in leaves, in which the GS1 and GS2 genes were coexpressed (Fig 1) The GLU1 mRNAs were found around the mesophyll

A B

C

Fig 4 Regulation of transcript levels of the

genes for GOGATs, CPSase and GS1 in

Ara-bidopsis leaves in response to exogenous

ammonium Arabidopsis seedlings were

grown for 12 days on Petri dishes with

5 m M nitrate, and then for 48 h in the

absence or in the presence of 10 m M

ammonium Transcript levels were

deter-mined by real-time RT-PCR (A) GOGAT

genes: GLU1, GLU2, and GLT (B) CPSase

genes: carA and carB (C) GS1 genes:

Gln11, Gln12, Gln13, and Gln14 The values

are expressed as percentage ± standard

error relative to the marker EF1a gene.

chloroplasts, where Fd-GOGAT protein was

immuno-histochemically located (Figs 5–7) GLU2, the other

Fd-GOGAT gene, was also expressed in the mesophyll

cells, albeit at lower levels than GLU1 (Figs 5 and 6)

The high level of expression of GLU1 in comparison

with that of GLU2 and the conditional lethal

pheno-type of the GLU1 mutant confirm that the defect in

the GLU1 Fd-GOGAT cycle caused the inhibition of

photosynthesis, owing to the extensive release of

pho-torespiratory ammonium (up to 5–20 lmolÆh)1Æg)1

fresh weight) [2,16,17] The high levels of glutamine

and glutamate (nitrogen-rich five-carbon amino acids)

and asparagine and aspartate (four-carbon amino

acids) (up to 80% of the total amino acids) (Fig 3)

suggest that excess photorespiratory ammonium was detoxified, in part, in the form of amino acids for export out of parenchyma cells of the veins The high glutamine⁄ glutamate ratio in the GLU1 mutant (13.3)

as compared with the wild type in air (1.4) (Fig 3) reflects the inability of mitochondrial GDH to act as

an alternative ammonium assimilatory pathway in the leaves, as GDH is a vascular-located enzyme [18] As demonstrated here, the minor effects on ammonium accumulation in the GLU2 mutant in air (Fig 3) pro-vide epro-vidence that the GS⁄ GLU2 Fd-GOGAT cycle does not contribute to photorespiratory ammonium reassimilation The low GLU2 mRNA levels in the

chl

mc

mc

se

te cc

cc

bs

cc

se

te

se

chl

cc

mc

mc

se

se cc

te

cc te

chl se

Fig 5 Histochemical analysis of promoter::GUS expression for

GLU1, GLU2, carA and carB in Arabidopsis leaves (A) Mesophyll

section for GLU1 (B) Mesophyll and vascular section for GLU1 (C)

Mesophyll and vascular section for GLU2 (D) Mesophyll and

vascu-lar section for carA (E) Mesophyll and vascuvascu-lar section for carB (F)

Control mesophyll and vascular section from Arabidopsis

trans-formed with an empty vector bs, bundle sheath; cc, companion

cell; chl, chloroplast; mc, mesophyll cell; se, sieve element; te,

tracheary element Bar: 10 lm.

chl mc

cc

te cc cc

pp

bs mc

chl

cc

se

te cc

mc chl

se

chl cc

se bs cc

te cc

te

bs

mc

mc mc

Fig 6 In situ hybridization of the transcripts of GLU1, GLU2 and GLT in Arabidopsis leaves (A) Mesophyll section hybridized with the antisense GLU1 mRNA probe (B) Vascular section hybridized with the antisense GLU1 mRNA probe (C) Mesophyll section hybridized with the antisense GLU2 mRNA probe (D) Mesophyll and vascular section hybridized with the sense GLU2 mRNA probe (E) Vascular section hybridized with the antisense GLU2 mRNA probe (F) Mesophyll and vascular section hybridized with the anti-sense GLT mRNA probe bs, bundle sheath; cc, companion cell; chl, chloroplast; mc, mesophyll cell; pp, phloem parenchyma cell; se, sieve element; te, tracheary element Bar: 10 lm.

leaves (Figs 1 and 4) suggest that GLU2 Fd-GOGAT

supplies a constitutive level of glutamate to maintain a

basal level of protein synthesis

The high levels of photorespiratory ammonium in

the GLU1 mutant seem to be shifted in part to the

CPSase pathway, resulting in substantial accumulation

of arginine (Fig 3) Arginine synthesis involves

orni-thine formation from glutamate [6] Carbamoylation

of the ornithine d-amino group with CP leads to the

formation of citrulline as a precursor of arginine

syn-thesis (see Fig 8 for a diagram of arginine synsyn-thesis)

It has been proposed that photorespiratory ammonium

released by mitochondrial glycine decarboxylase com-plex (GDC; EC 1.4.4.2⁄ 2.1.2.10) is reassimilated into glutamine by GS, and then into CP by CPSase in the mitochondria [19] However, the subcellular compart-mentation of CPSase has been unclear We showed that the promoter from either carA or carB directed the GUS signal to the mesophyll chloroplasts (Fig 5), indicating that photorespiratory ammonium is shuttled via glutamine to CP in the chloroplasts Glutamine is hydrolyzed via the class I or trpG-type glutaminase of the CPSase small subunit The carB domain of the CPSase large subunit forms the Cys-NH2 intermediate

by the conserved triad (Cys293-His377-Glu379) to acti-vate HCO3-dependent ATP cleavage prior to release

of CP [20] The databases also predict importation of the large subunit (cleavage at Cys62) and small subunit (cleavage at Val33) to the chloroplast stroma [21,22]

In addition, plastid-located carbonic anhydrase 1 (At1g58180, cleavage at Ala113) and cytosolic carbonic anhydrase 2 (At5g14740) can increase the HCO3 sup-ply via CO2⁄ HCO3 interconversion [23,24] Consis-tently, mitochondria have been shown to be unable to use ammonium, and only 0.2% of [15N]ammonium from [15N]glycine was metabolized to [15N]glutamate,

at a rate of 2.64 nmolÆh)1Æmg)1 protein [25] However,

it has been shown that GS is localized to the mito-chondria and that the mitomito-chondria are highly capable

of using glycine to convert ornithine to citrulline (up

to 126 lmolÆh)1Æmg)1protein) [9] Because of a lack of bioinformatic tools to predict to what extent the large and small precursors are seemingly dual-targeted, a dual organelle location of the CPSase in the chloro-plasts and mitochondria cannot not be excluded Excess ammonium from either endogenous photores-piration or exogenous medium appears to be, in part, shuttled to arginine (Fig 3) The fact that there were only slight effects of the carB mutation on overall argi-nine synthesis, either with excess ammonium or under standard nitrate conditions, suggests that CPSase is not the limiting enzyme for arginine biosynthesis However, the GLU1 mutant accumulated arginine at a higher level than the wild-type plants under photore-spiratory conditions (Fig 3) It can thus be assumed that photorespiratory ammonium was shuttled to argi-nine under the control of N-acetyl-glutamate kinase (NAGK; EC 2.7.2.8), a key regulatory enzyme in the arginine synthetic pathway [6]

Nitrogen entry into amino acids and translocation in the vascular tissue Under high-CO2 conditions, when photorespiration is suppressed, leaf cells depend on the importation of

cc cc bs

cc

cc

se

mc

8.00 µm

mc

phl

phl

mc

mc

phl

A B

C D

E F

Fig 7 Immunohistochemical localization of Fd-GOGAT in

Arabidop-sis leaves (A) Mesophyll and vascular section hybridized with the

antibody against Fd-GOGAT as the primary antibody (B) Control

mesophyll and vascular section hybridized with nonimmune serum

as the primary antibody (C) Mesophyll section hybridized with the

antibody against Fd-GOGAT as the primary antibody (D) Control

mesophyll section hybridized with nonimmune serum as the

pri-mary antibody (E) Vascular section hybridized with the antibody

against Fd-GOGAT as the primary antibody (F) Transmission of

vascular section corresponding to (E) bs, bundle sheath; cc,

com-panion cell; chl, chloroplast; mc, mesophyll cell; phl, phloem;

pp, phloem parenchyma cell; se, sieve element Bar: 8 lm.

nitrogen via the tracheary elements for amino acid

syn-thesis and subsequent export of the derived amino

acids via phloem sieve elements for use by sink cells

(Fig 8) Cellular localization of GOGATs and CPSase

in the vascular tissue has been unknown in

Arabidop-sis To dissect the regulation of amino acid

transloca-tion, we determined whether GOGATs and CPSase

were localized in the phloem companion cell–sieve

ele-ment complex Cis-acting regulatory eleele-ments upstream

of ATG were examined in silico, using the place

data-base [26] The TATA or TATA-like boxes were

identi-fied for GLU1 ()61TTATTT)56 and )37TTATTT)32),

GLU2[)506TTATTT)501and )90TTATTT)85()strand)],

GLT ()311TATAAAT)305), carA ()277TATATAA)271

and)188TTATTT)183), and carB [)361TTATTT)356and

)143TTATTT)138()strand)] Consistent with the

meso-phyll localization, cis-elements active in mesomeso-phyll

expression were found: Mem1 motif (CACT) [27];

GLU1 (at positions )258 ⁄ )255, )220 ⁄ )217, and )129 ⁄ )216), GLU2 ()223 ⁄ )220, )218 ⁄ )215, and )139 ⁄ )216), GLT ()310 ⁄ )307, )28 ⁄ )25, and )24 ⁄ )21), carA ()237 ⁄ )234, )151 ⁄ )148, and )115 ⁄ )112), and carB ()405 ⁄ )402 and )79 ⁄ )76) The Mem1 sequence

is supposed to direct mesophyll expression as a result

of transcription repression in the vascular bundle [27]

In addition, the strong cis-elements that determine vascular patterning were identified: the BS1 motif [28] [carA ()875AGCGGG)869), )strand] and the NtBBF1 motif (ACTTTA) [GLU1 ()1180 ⁄ )1175), GLU2 ()381 ⁄ )376), GLT ()1499 ⁄ )1494), carA ()237 ⁄ )232), and carB ()526 ⁄ )521)] The NtBBF1 motif directs expression of the oncogene rolB in phloem and xylem parenchyma [29] By in situ hybridization, the GLT mRNAs were found to be confined to the phloem companion cell–sieve element complex (Fig 6) The GLT mutant showed strong inhibition of primary

Fig 8 Proposed diagram for the role of GOGATs and CPSase in primary nitrogen assimilation, the photorespiratory nitrogen cycle, and nitrogen translocation The organelle localizations and stoichiometries of the interconnected enzymatic reactions are not included CH2-THF,

N 5 ,N 10 -methylene tetrahydrofolate; FdH, reduced ferredoxin; glycolate-P, 2-phosphoglycolate; N-acetylglutamate-5-P, N-acetyl-glutamate 5-phosphate; OH-pyruvate, hydroxypyruvate; OTC, ornithine transcarbamoylase (EC 2.1.3.3); PGA, 3-phosphoglycerate; RuBP, ribulose 1,5-bisphosphate.