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Two Cytosolic Glutamine Synthetase Isoforms of Maize Are Specifically Involved in the Control of Grain Production^[W],[OA]

^a Unité de Nutrition Azotée des Plantes UR511, Institut National de la Recherche Agronomique, F-78026 Versailles Cedex, France
^b School of Biological Sciences, University of Bristol, Bristol, BS8 1UG, United Kingdom
^c Laboratoire d'Androgenèse et Biotechnologie, Université de Picardie Jules Verne, Ilot des Poulies, F-80039 Amiens Cedex, France
^d Biogemma, Campus Universitaire des Cézaux, F-63170 Aubière, France
^e Unité Mixte de Recherche de Génétique Végétale, Institut National de la Recherche Agronomique, Centre National de la Recherche Scientifique, Université de Paris-Sud, Institut National Agronomique Louis Grignon, F-91190 Gif sur Yvette Cedex, France
^f Departamento Biología Vegetal y Ecología, Universidad del País Vasco, 48080 Bilbao, Spain
^g Crop Performance and Improvement Division, Rothamsted Research, Harpenden, AL5 2JQ, United Kingdom
^h Department of Biological Sciences, Lancaster University, Lancaster, LA1 4YQ, United Kingdom
ⁱ Biodynamics Research Team, Riken Plant Science Center, Tsurumi, Yokohama 230-0045, Japan

² To whom correspondence should be addressed. E-mail hirel{at}versailles.inra.fr; fax 33-1-30-83-30-96.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
The roles of two cytosolic maize glutamine synthetase isoenzymes (GS1), products of the Gln1-3 and Gln1-4 genes, were investigated by examining the impact of knockout mutations on kernel yield. In the gln1-3 and gln1-4 single mutants and the gln1-3 gln1-4 double mutant, GS mRNA expression was impaired, resulting in reduced GS1 protein and activity. The gln1-4 phenotype displayed reduced kernel size and gln1-3 reduced kernel number, with both phenotypes displayed in gln1-3 gln1-4. However, at maturity, shoot biomass production was not modified in either the single mutants or double mutants, suggesting a specific impact on grain production in both mutants. Asn increased in the leaves of the mutants during grain filling, indicating that it probably accumulates to circumvent ammonium buildup resulting from lower GS1 activity. Phloem sap analysis revealed that unlike Gln, Asn is not efficiently transported to developing kernels, apparently causing reduced kernel production. When Gln1-3 was overexpressed constitutively in leaves, kernel number increased by 30%, providing further evidence that GS1-3 plays a major role in kernel yield. Cytoimmunochemistry and in situ hybridization revealed that GS1-3 is present in mesophyll cells, whereas GS1-4 is specifically localized in the bundle sheath cells. The two GS1 isoenzymes play nonredundant roles with respect to their tissue-specific localization.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
The cereals, including maize (Zea mays), wheat (Triticum aestivum), and rice (Oryza sativa), account for 70% of worldwide food production (http://apps.fao.org; Shewry and Jones, 2005). When such crops are grown for protein content, they require large quantities of nitrogenous fertilizers to attain maximal yields. In the past few years, there has been considerable interest in nitrogen use efficiency, which can be defined as the kernel yield per unit of nitrogen (N) in the soil and the N utilization efficiency, which is the yield per N taken up (Good et al., 2004; Hirel and Lemaire, 2005). A number of physiological and agronomic studies have been undertaken to identify which are the limiting steps in the control of N uptake, assimilation, and recycling during plant growth and development (Jeuffroy et al., 2002; Lawlor, 2002), including cereals such as maize (Hirel et al., 2001, 2005b; Gallais and Hirel, 2004), rice (Yamaya et al., 2002), and wheat (Kichey et al., 2006; Lopes et al., 2006). As maize carries out the NAD–malic enzyme type of C₄ photosynthesis, it has a greater capacity to assimilate and metabolize C and N compounds than plants undergoing C₃ photosynthesis and ultimately has a larger sink for starch and protein storage in the seeds (Oaks, 1994). Using maize as a model crop, Hirel et al. (2005a, 2005b) have investigated the changes in metabolite concentration and enzyme activities involved in N metabolism within a single leaf at different stages of leaf growth and at different periods of plant development during the kernel-filling period. It was concluded that total N, chlorophyll, soluble protein content, and Gln synthetase (GS) activity are strongly interrelated and are indicators that mainly reflect the metabolic activity of individual leaves with regard to N assimilation and recycling, whatever the level of N fertilization. In maize, the putative role of GS for kernel productivity has also been highlighted using quantitative genetic approaches, since quantitative trait loci (QTL) for the leaf enzyme activity have been shown to be coincident with QTL for yield. One QTL for a thousand kernel weight was coincident with the Gln3 locus (corresponding to the gene encoding cytosolic GS Gln1-4 studied in this work; Hirel et al., 2001), and two QTL for a thousand kernel weight and yield were coincident with the Gln4 locus (corresponding to the gene encoding cytosolic GS Gln1-3 studied in this work; Hirel et al., 2001). Such strong coincidences are consistent with the positive correlation observed between kernel yield and GS activity (Gallais and Hirel, 2004). Further QTL for GS gene loci have been identified in relation to remobilization of N from the leaf, stem, and whole plant, postanthesis N uptake (Gallais and Hirel, 2004), and germination efficiency (Limami et al., 2002).

Since all of the N in a plant, whether derived initially from nitrate, ammonium ions, N fixation, or generated by other reactions within the plant that release ammonium (decarboxylation of Gly during photorespiration, metabolism of N transport compounds, and the action of Phe ammonia lyase), is channeled through the reactions catalyzed by GS, it is somehow logical to find that in cereals, the enzyme is likely to be a major checkpoint controlling plant growth and productivity (Miflin and Habash, 2002; Hirel et al., 2005b; Tabuchi et al., 2005; Kichey et al., 2006). Thus, an individual N atom can pass many times through the GS reaction (Coque et al., 2006), following uptake from the soil, assimilation, and remobilization (Gallais et al., 2006) to final deposition in a seed storage protein.

GS in higher plants, including maize, can be readily separated by standard chromatographic, localization, and protein gel blotting techniques into cytosolic (GS1) and plastidic (GS2) forms (Hirel and Lea, 2001). However, this distinction is not as simple as was first thought because although only one gene has been shown to encode the plastidic form, a small family of up to five genes is now known to encode the cytosolic form (Cren and Hirel, 1999). Initial experiments indicated that the five cytosolic GS genes were differentially expressed in the roots, stems, and leaves of maize (Sakakibara et al., 1992, 1996; Li et al., 1993). In a more recent study of maize leaves performed by Hirel et al. (2005b), it was shown that two of the five genes encoding GS1 (Gln1-3 and Gln1-4) were highly expressed regardless of the leaf age and the level of N fertilization, although there was an increase in Gln1-4 transcripts in the older leaves. It has been suggested that Gln1-4 encodes a GS isoform that is involved in the reassimilation of ammonium released during the remobilization of leaf proteins, whereas Gln1-3 encodes a GS isoform, which plays a housekeeping role during plant growth (Hirel et al., 2005a). GS1-2, which is an important GS isoenzyme of the developing kernel, is abundant in the pedicel and pericarp but has also been shown to be present in immature tassels, dehiscing anthers, kernel glumes, ear husks, cobs, and stalks of maize plants (Muhitch et al., 2002; Muhitch, 2003). Compared with the four other genes encoding GS1, Gln1-5 is expressed at a very low level in leaves, roots, and stems (Sakakibara et al., 1992; Li et al., 1993). The plastidic GS (GS2) encoded by Gln2 was only expressed in the early stages of plant development, presumably to reassimilate ammonium released during photorespiration, which is at a much lower rate in a C₄ plant compared with a C₃ plant (Ueno et al., 2005).

Despite the information available, as outlined above, concerning the expression of the five cytosolic GS1 genes in maize and the evidence of their importance from the demonstration of QTL, the precise role of the individual GS1 isoenzymes in controlling the flux of ammonium into Gln is not known. Using the maize Mutator (Mu) system, individual maize mutants have been isolated that have insertions in three of the five GS1 genes (Hanley et al., 2000; S. Haines and K.J. Edwards, unpublished results). In all cases, the insertion position of the transposon within the GS1 gene is approximately midpoint and as such is expected to eliminate the production of a functional transcript. The insertion lines of Gln1-3::Mu and Gln1-4::Mu have undergone extensive backcrossing to the wild-type non-Mu line, and homozygous, heterozygous, and null mutant lines have been obtained. In addition, a double mutant (gln1-3 gln1-4) of the Gln1-3 and Gln1-4 genes has been produced. In this article, we have investigated the role of the cytosolic GS isoenzyme products of the Gln1-3 and Gln1-4 genes by studying the properties of the insertion mutants at the molecular, biochemical, and physiological levels and by examining the impact of the mutation on kernel yield and its components.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Isolation and Characterization of gln1-3– and gln1-4–Deficient Mutants and gln1-3 gln1-4 Double Mutant
A Mu insertion event within Gln1-3 (Gln1-3::Mu1a) was first identified using the services of the maize targeted mutagenesis program (http://mtm.cshl.edu/; May et al., 2003), and two further single Mu insertion events (Gln1-3::Mub and Gln1-3::Muc) were identified using the collection of mutants produced at Biogemma. The Mu insertion event within Gln1-4 was identified by the random sequencing of Mu-tagged fragments as described by Hanley et al. (2000). Sequence analysis of both events indicated that in the case of gln1-3, Mu1 (mutant a) had inserted within exon 8, and in the case of gln1-4, Mu5 had inserted within the intron separating exons 7 and 8 (Figure 1 ). In the cases of gln1-3 mutants b and c, Mu had inserted just upstream of the translation initiation codon in the 5'-untranslated region (Figure 1). In both cases, heterozygous plants had similar phenotypes to other randomly chosen Mu-active lines in the genetic background represented by line B73. During the various backcrosses, crosses, and selfings, no distorted segregation patterns were observed as monitored using a PCR assay designed to detect heterozygous and homozygous plants for the Gln1-3 and Gln1-4 insertion events (data not shown). In the case of gln1-3 mutants a and b after three rounds of crosses with the line FV2 and selfing, a segregating very small or aborted kernel phenotype was recorded, thus confirming a similar effect of the mutation on two other allelic mutants in a different genetic background represented by line FV2 (see Supplemental Figure 1 online). In all further experiments, only the homozygous gln1-3a single mutant, which exhibited the strongest phenotype, was studied.

View larger version (21K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Characterization of the Gln1-3::Mu and Gln1-4::Mu Insertion Events. (A) Insertion position of the Mu elements within the Gln1-3 and Gln1-4 genes. For clarity and as the Gln1-3 and Gln1-4 genes show high sequence similarity, they are shown as a single structure. The Gln1-3 and Gln1-4 gene structure and exon sizes were determined by sequencing genomic DNA PCR products using primers designed from the corresponding cDNA sequences (Li et al., 1993): the Mu insertion sequences and the rice genomic clone AC105364. The rice and Gln1-3 and Gln1-4 genes consist of 10 exons (black arrows), ranging in size from 40 to 252 bp, and nine introns (black lines). The size of the last exon was 150 bp in Gln1-3 and 147 bp in Gln1-4. The intron and exon structure is drawn to scale. The gene numbering starts with the ATG codon (1 bp) and continues to the stop codon (position 3857 bp). The four Mu insertion events are indicated by triangles (not to scale). Also shown is the relative position of the GS-NH4+ binding site signature (green rectangle) and the GS-ATP binding region signature (blue rectangle). (B) Sequence characterization of Mu insertions in Gln1-3 and Gln1-4. The nucleotide sequences flanking the 5' and 3' ends of the inserted transposons in Gln1-3::Mu1 and Gln1-4::Mu5, respectively, are presented and in each case show the Mu-specific 9-bp duplication of the target DNA (boxed). Sequence analysis of the flanking regions surrounding the Mu elements indicated that in the case of Gln1-3::Mu1a, the insertion had occurred within exon 8, and in the case of Gln1-4::Mu5, the insertion had occurred within the intron separating exon 7 and exon 8, while Gln1-3::Mub and c are both located within the 5'-untranslated region. Black lettering indicates sequences derived from exons, and blue lettering indicates sequences derived from introns.

View larger version (60K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Steady State Level of Transcripts for GS in GS1-Deficient Mutants. (A) RNA gel blot showing the steady state level of the genes encoding cytosolic GS (Gln1-2, Gln1-3, and Gln1-4) and plastidic GS (Gln2) in leaves of the wild type and in gln1-3, gln1-4, and gln1-3 gln1-4 mutants. (B) RNA gel blot showing the steady state level of the genes encoding cytosolic GS (Gln1-1, Gln1-3, and Gln1-4) in roots of the wild type and in gln1-3, gln1-4, and gln1-3 gln1-4 mutants. The transcripts were detected by hybridization with cDNA 32P-labeled probes corresponding to the 3' noncoding regions specific for each gene (Sakakibara et al., 1992). Each sample contains 10 µg of total RNA. Ethidium bromide–stained gels (rRNA) are presented at the bottom of each figure to show that similar amounts of total RNA were loaded in each track. Experiments were repeated three times using three different plants with similar results.

The GS isoenzyme protein content of roots and leaves of the wild type and three mutants was examined following one-dimensional polyacrylamide gel electrophoresis and protein gel blot analysis using antibodies raised against tobacco (Nicotiana tabacum) GS2. This technique provides a reliable method of estimating the relative amounts of both GS1 and GS2 in a crude protein extract of maize leaves (Becker et al., 2000). In the leaves of wild-type plants at VS, two polypeptides of similar relative abundance were detected. The 44-kD polypeptide corresponds to the plastidic form of GS, whereas the 40-kD polypeptide corresponds to the cytosolic form of GS. In the gln1-3 and gln1-4 mutants, a decrease in the amount of GS1 protein was observed, which was more pronounced in the former. In gln1-3 gln1-4, the GS1 protein was barely detectable. Similar amounts of GS2 protein were visible in the wild type and in all three mutants (Figure 3A ). The decrease in GS1 protein contents of the gln1-3, gln1-4, and gln1-3 gln1-4 mutants at the VS was confirmed using antibodies raised against Phaseolus vulgaris root nodule GS (Cullimore and Miflin, 1984). However, since this antibody preparation is apparently more specific for cytosolic GS, GS2 protein was less readily visible on the protein gel blot (Figure 3B). When the GS proteins were analyzed in the leaves 55 DAS using the tobacco antibody, a similar pattern of decrease in GS1 protein content was obtained in the mutants. However, at this later stage of plant development, the amount of GS2 protein isolated from all four lines was much lower (Figure 3C).

View larger version (37K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. GS Subunit Composition of GS1-Deficient Mutants and Overexpressing Lines. (A) Protein gel blot analysis of the GS subunit composition of leaves of the wild type, gln1-3, gln1-4, and gln1-3 gln1-4 harvested at VS using tobacco GS antibodies (Hirel et al., 1984). The top band (molecular mass of 44 kD) corresponds to the plastidic GS (GS2) subunit, and the bottom band (molecular mass of 39 kD) corresponds to the cytosolic GS (GS1) subunit. On the right side of the panel is the position of the protein molecular mass markers. (B) Protein gel blot analysis of GS subunit composition of leaves of the wild type, gln1-3, gln1-4, and gln1-3 gln1-4 harvested at VS using P. vulgaris GS antibodies (Cullimore and Miflin, 1984). (C) Protein gel blot analysis of GS subunit composition in leaves of the wild type, gln1-3, gln1-4, and gln1-3 gln1-4 harvested at 55 DAS using tobacco GS antibodies. (D) Protein gel blot analysis of GS subunit composition in roots of the wild type, gln1-3, gln1-4, and gln1-3 gln1-4 harvested at VS using tobacco GS antibodies. The two bands (molecular masses of 40 and 38 kD) correspond to the GS1 and GSr forms of cytosolic GS. (E) Protein gel blot analysis of GS subunit composition of leaves of the two null segregants (WT1 and WT9) and the two transgenic lines (Lines 1 and 9) with expression of Gln1-3 cDNA under the control of the CsVMV promoter. Plants were harvested at VS, and tobacco GS antibodies were used in the experiment. The top band (molecular mass of 44 kD) corresponds to the plastidic GS (GS2) subunit, and the bottom band (molecular mass of 39 kD) corresponds to the cytosolic GS (GS1) subunit.

View larger version (12K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. GS Isoenzyme Composition of GS1-Deficient Mutants. Ion exchange chromatography of GS activity in leaf extracts from the wild type (A), gln1-4 (B), gln1-3 (C), and gln1-3 gln1-4 (D). The first peak of GS activity corresponds to cytosolic GS (GS1) and the second peak to plastidic GS (GS2). The relative amounts of GS1 and GS2 activity indicated in parentheses were calculated using as a maximum the value measured for the amount of GS1 and GS2 activities in the wild type. GS activity was measured using the transferase reaction (Lea et al., 1999). Similar results were obtained with three independent extractions. FW, fresh weight.

Identification of GS Protein Sequences
Using plants harvested at the VS, leaf proteins from the wild type, the gln1-3, the gln1-4, and the gln1-3 gln1-4 mutant were separated by two-dimensional gel electrophoresis (Figure 5A ). To unambiguously demonstrate that the corresponding GS protein was lacking in the gln1-3 and gln1-4 mutants, 36 protein spots in the range of the pI and molecular mass of GS were analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Protein identification revealed that out of these 36 spots, four of them (spots 8, 9, 14, and 38) contained a GS protein.

View larger version (84K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Proteomic Analysis of GS1-Deficient Mutants. (A) Two-dimensional gel of proteins extracted from the leaves of the wild type. The rectangle shows the region containing GS spots that is enlarged in (B). (B) Identification of GS1-3, GS1-4, and GS2 proteins in the wild type and in gln1-3, gln1-4, and gln1-3 gln1-4. White crosses with no black arrowheads in the wild type show the spots that were analyzed by LC-MS/MS but did not contain a GS protein. (C) Sequence of the peptides that allowed the distinction between GS1-3 and GS1-4. Entry names of the sequence in the UNIPROT Database are given. Ref 2 of GS1-3 and GS1-4 refers to the allelic form sequenced by Sakakibara et al. (1992). Positions are relative to the first Met for all GS except for GS2, where it is relative to the first amino acid after the signal peptide. Shaded background shows the differences between GS1-3 and GS1-4 and between GS1-3 allelic forms. Differences between GS1-3 and GS1-4 and the rest of GS proteins are underlined.

Two sites of sequence polymorphism were used to distinguish the GS1-3 and GS1-4 proteins from each other: the amino acid at position 41 is a Ser in GS1-3 and a Pro in GS1-4, and the amino acid at position 278 is an Arg in GS1-3 and a Lys in GS1-4. Peptides containing these sites of polymorphism were identified: TLSGPVTDPSK (and the miscleaved TLSGPVTDPSKLPK) allowed the identification of GS1-3, while TLPGPVTDPSK (and TLPGPVTDPSKLPK) allowed the identification of GS1-4 according to the S/P polymorphism at position 41, and HREHIAAYGEGNER and HKEHIAAYGEGNER allowed the identification of GS1-3 and GS1-4 proteins, respectively, according to the R/K polymorphism at position 278. It should be noted that the peptide HREHIAAYGEGNER is similar to the allelic form of GS1-3 sequenced by Sakakibara et al. (1992) but not to that sequenced by Li et al. (1993), in which the two last amino acids of the peptide, ER, are substituted by DG (Figure 5C).

Most of the GS spots were excised twice, and the numbers of peptides indicated below refer to the analyses that identified the largest number of peptides. According to the presence of the discriminating peptides, the excision of spot 8 from gels from the gln1-4 mutant allowed the identification of the GS1-3 protein (eight GS1 peptides from which three were specific to GS1-3), and the excision of spot 9 from the gln1-3 mutant allowed the identification of the GS1-4 protein (12 GS1 peptides from which three were specific to GS1-4). When excised from the wild type, no discrimination between the two proteins was possible because of a partial overlap of the two spots. Spots 14 and 38, which both exhibit a higher mass compared with GS1, were found to contain the plastidic GS2 protein (9 and 23 peptides allowed the identification of GS2 in spots 38 and 14, respectively). Interestingly, spot 14 was not visible in gln1-3, gln1-4, or the gln1-3 gln1-4 mutant (Figure 5B). It is worth noting that the intensity of spot 38 in the three mutants and the wild type was visibly proportional to GS2 activity (Figure 5).

Immunolocalization of GS
Immunocytochemical studies using both light (Figure 6 ) and electron microscopy (Figure 7 ) techniques were employed to determine in which leaf tissue GS was impaired in the three GS mutants. GS was first localized in leaf sections of the wild type and the gln1-3 gln1-4 mutant using immunogold labeling, followed by silver enhancement. A bright-field microscope emitting epipolarized light was used for these experiments, and the silver-enhanced gold particles appeared as a bright yellow color (Figure 6). In the wild-type leaf tissue sections treated with gold-labeled tobacco anti-GS antibodies, the presence of GS both in the bundle sheath cells (BSCs) and mesophyll cells (MCs) was clearly visible. The phloem companion cells (PCCs) located in the central part of the BSC were also faintly stained (Figure 6B). In leaf sections of the gln1-3 gln1-4 mutant, the fluorescent signal was much weaker and apparently restricted to the plastids (arrows) of both BSCs and MCs (Figure 6C). In addition, faint labeling was still detected in the PCC. Similar results were obtained when leaf sections of the gln1-3 and gln1-4 mutants were treated with the gold-labeled GS antiserum (data not shown). However, the resolution of the technique did not allow a clear differentiation of a specific signal in the various cell types and cellular compartments of the three mutants. In a control leaf section treated with ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) antibodies, a fluorescent signal was detected in plastids of the BSC, whereas the MCs were unstained, thus allowing a clear identification of the two cell types (Figure 6A).

View larger version (66K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Histological Immunolocalization of Rubisco and GS in Leaf Transversal Sections of the Wild Type and the gln1-3 gln1-4 Mutant Stained by Fuchsin and Examined under Bright-Field Epipolarized Light. (A) Immunolocalization of Rubisco in the leaf of the wild type. (B) Immunolocalization of GS in the leaf of the wild type. (C) Immunolocalization of GS in the leaf of the gln1-3 gln1-4 mutant. n, nucleus. Bars = 100 µm.

View larger version (124K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Transmission Electron Microscopy Immunolocalization of GS in Maize Leaf Sections at VS. (A) to (C) Localization of GS in parenchyma cells of the wild type (A). Magnification of the zone containing MC (B) and BSC (C). (D) to (F) Localization of GS in parenchyma cells of the gln1-3 gln1-4 mutant (D). Magnification of the zone containing an MC (E) and a BSC (F). (G) and (H) Localization of GS in MCs of the gln1-3 mutant (G) and in the BSC of the gln1-4 mutant (H). (I) and (J) Localization of GS in PCC in the wild type (I) and in the gln1-3 gln1-4 mutant (J). (K) Control section in the wild type treated with preimmune serum. cy, cytosol; p, plastid; m, mitochondrion; s, starch granules. Arrows indicate the presence of GS protein. Bars = 1 µm in (A) and (C) and 0.5 µm in (B) and (D) to (K).

In Situ Hybridization of Cytosolic GS Transcripts
In situ hybridization was performed in leaves of the wild type and the three GS mutants to confirm at the transcript level that Gln1-4 and Gln1-3 were expressed in two different cell types. Tissue sections of the wild type were hybridized with digoxygenin-labeled sense and antisense RNA probes from the coding region of Gln1-3, which shares >85% homology with the four other genes encoding GS1 (Sakakibara et al., 1992). In all tissue sections, hybridization of the probe was observed by the presence of a blue signal corresponding to the reaction catalyzed by the alkaline phosphatase. In transverse leaf sections of the wild type, the antisense probe from the coding region of Gln1-3 hybridized with all the different leaf tissues, including the MC, the BSC, and the two epidermal cell layers (Figures 8A and 8B ). In the gln1-4 mutant, there was no significant hybridization signal in the BSC (Figure 8C). In the gln1-3 mutant, the signal was much stronger in the BSC (Figure 8D). In the gln1-3 gln1-4 double mutant, the GS1 probe hybridized with some of the vascular cells present in the BSC, whereas both the MC and BSC remained unstained (Figures 8E and 8F). In the three mutants, a weak signal was always visible in both the lower and upper epidermis (Figures 8C to 8E).

View larger version (127K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. In Situ Localization of Cytosolic GS Transcripts in Leaf and Root Sections of the Wild Type and the gln1-4, gln1-3, and gln1-3 1-4 Mutants. (A) and (B) Localization of GS1 transcripts in the leaves of the wild type (A). Magnification of the zone containing the MC and BSC cells (B). (C) Localization of GS1 transcrips in the gln1-4 mutant showing the absence of staining in the BSC. (D) Localization of GS1 transcripts in the gnl1-3 mutant showing staining in the BSC. (E) and (F) Localization of GS1 transcripts in the gln1-3 gln1-4 mutant. (F) Magnification of the zone containing the BSC showing the presence of staining in the vascular tissue (arrow). (G) and (H) Localization of GS1 transcripts in the roots of the wild type showing the presence of a strong signal in the epidermis and in the two outermost cortical cell layers. (H) A magnification of the root central cylinder showing the presence of signal in the vascular tissue (arrows). (I) Transverse section of a root tissue hybridized with the digoxygenin-labeled sense probe showing the absence of signal. (J) Transverse section of a leaf tissue hybridized with the digoxygenin-labeled sense probe showing the absence of signal. c, cortex; cc, central cylinder; e, epidermis. Bars = 100 µm.

Phenotype and Kernel Production
To determine the impact of the mutations on plant phenotype and kernel production, plants were grown in a glasshouse until maturity under suboptimal N feeding conditions. No significant differences were seen in the dry matter production of the vegetative parts of the shoot (Figure 9A ), which exhibited a similar phenotype (Figure 10 ). By contrast, a major decrease in the dry matter content of the ear was observed, which when compared with the wild type was 68% for gln1-3, 48% for the gln1-4 mutant, and 84% for the gln1-3 gln1-4 mutant. A reduction of ear size was observed in gln1-3 and gln1-4 mutants and was even more severe in the gln1-3 gln1-4 mutant (Figure 9B). The main agronomic characteristics of the three GS mutants grown under N suboptimal conditions are presented in Table 3 . In both gln1-3 and gln1-4 mutants, a strong reduction in kernel yield was observed, which was more important in the former, thus confirming the phenotype shown in Figure 11A . In the gln1-3 gln1-4 mutant, the kernel yield was reduced to only 12% of the wild type (Table 3). In gln1-4, there was a greater reduction in kernel weight but a larger kernel number when compared with gln1-3. In the gln1-3 gln1-4 mutant, both yield components were strongly reduced compared with the wild type and the two single mutants (Table 3). Compared with the wild type, an 20% increase in N kernel content was observed in the three mutants (Table 3).

View larger version (8K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. Shoot and Ear DW Accumulation in GS1-Deficient Mutants. (A) Total DW of shoot vegetative parts of wild type, gln1-3, gln1-4, and gln1-3 gln1-4. Plants were harvested at maturity and grown under N suboptimal conditions on a nutrient solution containing 10 mM NO3–. (B) Total ear DW of wild type, gln1-3, gln1-4, and gln1-3 gln1-4. Plants were harvested at maturity and grown under N suboptimal conditions on a nutrient solution containing 10 mM NO3–. The plants were grown in the glasshouse on soil and watered daily with the medium described by Coïc and Lesaint (1971). Values are the mean ± SE of three individual plants.

View larger version (144K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 10. Phenotype of the Shoot in GS1-Deficient Mutants. The wild type, gln1-3, gln1-4, and gln1-3 gln1-4 maize plants (line B73) at a developmental stage corresponding to 55 d after flowering. Plants were grown in the glasshouse on soil and watered daily with the medium described by Coïc and Lesaint (1971). Note that both the number of remaining leaves and the position of the ear were similar in the wild type and the three mutants.

View larger version (96K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 11. Phenotype of the Ear in GS1-Deficient Mutants and Overexpressing Lines. (A) Ears of wild type, gln1-3, gln1-4, and gln1-3 gln1-4 of maize plants (line B73) harvested at maturity and grown under N suboptimal conditions on a nutrient solution containing 10 mM NO3–. (B) Ears of wild-type null segregants and T4 transgenic lines (Lines 1 and 9) overexpressing the Gln1-3 cDNA. Maize plants (line FV2) were harvested at maturity and grown under N suboptimal conditions on a nutrient solution containing 10 mM NO3–. The ear in the control null segregants WT1 and WT9 was smaller compared with that of the wild type shown in (A). This is due to the fact that the European line FV2 used for generating the transgenic maize plants produces fewer kernels than the North American line B73.

View larger version (23K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 12. Shoot and Grain Yield in Relation to GS Activity of Plants Overexpressing Gln1-3. (A) Total leaf GS activity in the two null segregants (WT1 and WT2) and the corresponding two transgenic lines (Lines 1 and 9) overexpressing GS1. (B) Kernel yield (DW) of two null segregants (WT1 and WT2) and two transgenic lines (Lines 1 and 9) overexpressing GS1. Plants were harvested at maturity and grown under N suboptimal conditions on a nutrient solution containing 10 mM NO3–. (C) Total DW of shoot vegetative parts of two null segregants (WT1 and WT2) and two transgenic lines (Lines 1 and 9) overexpressing GS1. Plants were harvested at maturity and grown under N suboptimal conditions on a nutrient solution containing 10 mM NO3–. (D) Scatterplots between leaf GS activity and kernel yield in the two null segregants (WT1 and WT2) and the corresponding two transgenic lines (Lines 1 and 9) overexpressing GS1. Each symbol corresponds to a leaf sample harvested at the VS. Values are the mean ± SE of six individual plants.

To determine the impact of Gln1-3 overexpression on plant phenotype and kernel production, plants were grown in a glasshouse until maturity under suboptimal N feeding conditions. A significant increase in grain yield was observed, which when compared with two corresponding wild-type control plants was 30% for both Lines 1 and 9 (Figure 12C), thus confirming the phenotype shown in Figure 11B. Kernel number was the yield component that was mostly responsible for the increase in kernel production (Table 3). Linear regression and the resulting correlation coefficient were calculated for the level of leaf GS activity versus grain yield in the two null segregants and the two transgenic lines. Figure 12D shows that there is a strong relationship between leaf GS activity and grain yield (r² 0.91), thus indicating that the increase in yield is proportional to the increase in enzyme activity. By contrast, no significant differences were seen in shoot dry matter production between the two wild-type control plants and the two transgenic lines (Figure 12C).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
GS and Yield
Using a reverse genetic approach, we have demonstrated the functional importance of cytosolic GS in the control of yield and its components in maize. Under suboptimal N feeding conditions, a reduction in kernel size and kernel number was observed in the gln1-3 and gln1-4 single mutants and was even more severe in the gln1-3 gln1-4 double mutant. In addition, we have shown that in the gln1-3 mutant, kernel number is the yield component that is preferentially affected, whereas in the gln1-4 mutant, it is kernel size. In transgenic plants, in which Gln1-3 was overexpressed in the MC, an increase in kernel number was observed. This finding confirms that the GS1-3 isoenzyme plays a major role in controlling yield through the production of kernels.

This finding is in agreement with the coincidence previously demonstrated with the QTL for the two yield components and the two GS1 structural genes (Hirel et al., 2001). Therefore, these observations suggest that the reduced N channeled through the reaction catalyzed by GS1-3 is more specifically invested in kernel set, whereas the reduced N arising from the reaction catalyzed by GS1-4 is rather used for kernel filling. The expression of Gln1-4 is enhanced during leaf aging (Hirel et al., 2005a). Thus, the isoform GS1-4 is likely to be involved in the reassimilation of NH₄⁺ released during protein breakdown to provide Gln for kernel growth during the filling period. The housekeeping role of the Gln1-3 gene product is in agreement with its role in providing enough N assimilates to the developing ear to avoid kernel abortion (Below, 1987). The increase in kernel number observed in transgenic plants overexpressing Gln1-3 further supports this hypothesis. In the gln1-3 gln1-4 mutant, both kernel size and kernel number were reduced, thus demonstrating the cumulative effect of the two mutations.

Previous studies have shown that the supply of N assimilates facilitates the utilization of carbohydrates by the kernels (Below et al., 2000), thus influencing both kernel weight and yield (Below et al., 1981). The qualitative importance of N assimilate supply to the kernels has also been emphasized by Seebauer et al. (2004), who proposed that Gln and Asn, two molecules used for N transport to the kernel, play an important role during the kernel filling process.

The finding that vegetative biomass production is not affected in the two single GS mutants and the gln1-3 gln1-4 double mutant reinforces the conclusion that the leaf isoforms GS1-3 and GS1-4 are specifically involved in the synthesis of Gln that is further used for kernel set and kernel development. This therefore implies that the root GS (represented by the GSr isoform encoded by Gln1-1), whose activity is not notably affected in the mutants, is probably able to provide the major part of the reduced N that is used for vegetative growth. The contribution of GS2 and GS1-2 in providing Gln to the leaf, at least in the early phase of plant development before the silking period, can also be considered, since both the expression of corresponding genes and the presence of an active protein were found in all three mutants. Although the exact physiological function of GS1-5 remains to be determined, its role may be confined to photosynthetic epidermal cells under certain conditions of illumination (Sakakibara et al., 1992).

The importance of GS in controlling cereal productivity has recently been strengthened by a study performed in rice, in which a strong reduction in both growth rate and kernel yield was observed in a mutant deficient in cytosolic GS (Tabuchi et al., 2005).

Physiological Impact of Decreased GS Activity
The main impact of the reduction in GS activity in the mutants was an increase in the leaf amino acid content at a rather late stage during the kernel-filling period (55 DAS), which was primarily due to an increase in Asn and to a lesser extent Glu, notably in the gln1-3 gln1-4 mutant. The increase in Glu was proportional to the reduction of GS1 activity in the mutants, indicating that there was an accumulation of substrate as a result of lower enzyme activity. No major changes in the profile of N and C metabolites were observed before that period, except an accumulation of NH₄⁺ in vegetative plants, which is likely to be the result of lower GS1 activity during full leaf growth. In legumes, an accumulation of Asn was observed when GS activity was impaired, suggesting that other enzymes (e.g., Asn synthetase) may be important in bypassing the flux of reduced nitrogen to avoid a toxic accumulation of NH₄⁺, thus ensuring plant survival (Carvalho et al., 2003; Harrison et al., 2003; Wong et al., 2004). The question as to whether these alternative metabolic pathways are important in the control of plant productivity has been extensively studied in legumes and nonlegumes (Knight and Langston-Unkefer, 1988; Brears et al., 1993; Lam et al., 2003) but has still not been fully elucidated, particularly in cereals.

According to Seebauer et al. (2004), Gln is the major amino acid entering the cob just after silking and is then further metabolized into Asn, which is used for optimal kernel growth and protein accumulation. Phloem sap analysis confirmed that Gln is a major N transport form in maize (Oaks, 1992) and that the concentration is considerably reduced in the three mutants. This reduction was also observed for most of the main amino acids of the phloem sap, which is a typical symptom of N deficiency in plants (Tercé-Laforgue et al., 2004). Consequently, less Gln and other amino acids were apparently transported to the developing kernels, thus causing a shortage in the amount of N translocated and therefore a reduction in both kernel number and kernel size. The higher leaf N content of the mutants at maturity confirms that N assimilates are not efficiently translocated to the ear. Since Asn accumulated in the leaves of the mutants during the kernel-filling period, it seems likely that Asn is synthesized at the expense of Gln but is not efficiently transported to the developing ear. This hypothesis was further confirmed by the finding that although there was an accumulation of Asn in the leaves of the mutants, the amino acid remained a relatively minor N compound transported in the phloem sap, the concentration in all three mutants being lower than that of the wild type. The lower relative content of soluble Asn in the kernels in the mutants compared with the wild type is also an argument in favor of this hypothesis.

The higher kernel N content of the mutants may be explained by the fact that there are less kernels to fill. Thus, the amount of N available to an individual kernel may be increased, even if the amino acids (mainly represented by Gln) translocated in the phloem are reduced.

The results so far show that at the VS, the impact of altered GS1 expression was minimal or absorbed by the system. This could due to support from the root in producing Gln and/or compensation by other isozymes (GS1-2, GS1-1, and GS2). However, there was a drastic phenotype at the reproductive stage, which implicates the deficient isoenzymes play major roles after VS and/or that the flux of ammonia through those enzymes is of magnitudes higher during the grain-filling period.

Now, the function of GS1-2 and GS1-5 remains to be determined, which may be involved in the control of Gln synthesis in reproductive tissues, such as the cobs (Seebauer et al., 2004), the kernels and other vascular-rich organs (Muhitch, 2003), and the leaf or stem epidermis (Li et al., 1993).

Cell-Specific Expression and Function of GS Isoenzymes in Leaves
Originally, Sakakibara et al. (1992) and Li et al. (1993) showed that there is an organ-specific expression of the different genes encoding GS1 and GS2. Later on, Becker et al. (2000) found that the relative proportions of GS1 and GS2 were different in the MC and BSC. This finding led these authors to propose that the source of Gln in both the MC and in the BSC originated from protein catabolism through the reaction catalyzed by GS1. GS2 in the MC was responsible for ammonia assimilation as the result of NO₃^– reduction occurring specifically in this cell type. In the BSC, GS2 in conjunction with GOGAT was involved in the reassimilation of ammonium arising from photorespiration.

In this work, the analysis of the cellular localization of GS in the wild type and in the three mutants revealed that in leaves, the tissue distribution of the individual cytosolic GS isoenzymes is different. The GS1-3 isoenzyme is localized in the MC, whereas the GS1-4 isoenzyme is restricted to the BSC. The in situ hybridization experiments confirmed the cell-specific expression of the corresponding transcripts.

Since the gene encoding GS1-3 (Gln1-3) is constitutively expressed until a very late stage of leaf development (Hirel et al., 2005a), we propose that GS1-3 in the MC is more specifically involved in the synthesis of Gln following NO₃^– reduction until plant maturity. The finding that GS1 protein is more abundantly expressed in the MC of transgenic plants overexpressing Gln1-3 further supports this hypothesis. This function is in agreement with the finding that postflowering NO₃^– uptake still occurs during the kernel-filling period and represents 50% of the N allocated to the grain (Martin et al., 2005). Therefore the major impact of the lack of GS1-3 activity would be a reduction in grain number due to the lack of N available to induce the setting of more kernels. This hypothesis is in agreement with the well-known phenomenon of kernel abortion as the result of N deficiency during ear development (Below, 1987).

The BS-specific GS1-4 isoform is encoded by a gene induced during leaf aging (Hirel et al., 2005a; Martin et al., 2005). Since Rubisco is located in this cell type and is a major source of N during the transition from sink to source (Esquivel et al., 2000), we hypothesize that GS1-4 has a catabolic function in the reassimilation of ammonium released during protein degradation in senescing leaves. In the gln1-4 mutant, kernel size was reduced, which suggests that the N originating from this catabolic process is more specifically used to fill the kernels.

The finding that only Gln1-2 transcripts were detected in the leaves of the three mutants and that GS is still detected in the PCCs suggests that this gene encodes a phloem-specific GS isoenzyme. We have also shown that in the double mutant, GS1 transcripts were detected in the leaf and root vascular tissue, which strengthens our hypothesis. A phloem-specific GS isoenzyme has only been identified previously in C₃ plant species. Its physiological role appears to be related either to N storage under stress conditions (Brugière et al., 1999) or to N export during plant growth and development (Suarez et al., 2002; Tabuchi et al., 2005). In maize, it is likely that the GS1-2 isoenzyme contributes to N translocation but is not directly involved in supplying N to the kernels.

We have also demonstrated that a high level of GS1 transcripts were expressed in the root cortex and the two outermost cell layers of the cortex, thus confirming that the GS1-1 isoenzyme is likely to be involved in Gln synthesis following NO₃^– uptake and reduction in these two cell types, which have been shown to be directly involved in the process of NO₃^– uptake (Nazoa et al., 2003).

Although the signal was low, transcripts for GS1 were detected in the upper and lower leaf epidermis of the three mutants, thus suggesting that the fifth gene encoding GS1 (Gln1-5) may