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First published online June 18, 2004; 10.1105/tpc.021642 © 2004 American Society of Plant Biologists Overexpression of GLUTAMINE DUMPER1 Leads to Hypersecretion of Glutamine from Hydathodes of Arabidopsis Leaves
a Zentrum für Molekularbiologie der Pflanzen, Pflanzenphysiologie, Universität Tübingen, D-72076 Germany 3 To whom correspondence should be addressed. E-mail wfrommer{at}stanford.edu; fax 650-325-6857.
Secretion is a fundamental process providing plants with the means for disposal of solutes, improvement of nutrient acquisition, and attraction of other organisms. Specific secretory organs, such as nectaries, hydathodes, and trichomes, use a combination of secretory and retrieval mechanisms, which are poorly understood at present. To study the mechanisms involved, an Arabidopsis thaliana activation tagged mutant, glutamine dumper1 (gdu1), was identified that accumulates salt crystals at the hydathodes. Chemical analysis demonstrated that, in contrast with the amino acid mixture normally present in guttation droplets, the crystals mainly contain Gln. GDU1 was cloned and found to encode a novel 17-kD protein containing a single putative transmembrane span. GDU1 is expressed in the vascular tissues and in hydathodes. Gln content is specifically increased in xylem sap and leaf apoplasm, whereas the content of several amino acids is increased in leaves and phloem sap. Selective secretion of Gln by the leaves may be explained by an enhanced release of this amino acid from cells. GDU1 study may help to shed light on the secretory mechanisms for amino acids in plants.
Although plants store a variety of compounds in the vacuole, which are not currently needed or which may even be potentially harmful, they also excrete substances from roots into the soil and release compounds from the aerial parts. In leaves, excretion, which serves a variety of purposes, is mediated by highly specialized structures called hydathodes. Hydathodes are positioned at the leaf margins, close to the endings of conducting vessels. Hydathodes mediate guttation (i.e., secretion of a sap containing ions, metabolites, and proteins) (Komarnytsky et al., 2000  ; Mizuno et al., 2002; Grunwald et al., 2003). The guttation process seems to be related to the water status of the plant (i.e., stomata opening and leaf water potential) (Takeda and Glenn, 1989; Enns et al., 2000).
The structure of the hydathodes suggests an involvement not only in the active secretion of solutes but also in the selective absorption and retrieval of both inorganic and organic solutes (Esau, 1977
To gain insight into the molecular process of guttation and the specific recovery or secretion of compounds, we searched for mutants with altered hydathode activity. Because no such mutants have been reported by screening conventional ethyl methanesulfonate–mutagenized or knockout libraries of Arabidopsis thaliana, a collection of activation tagged lines was screened. Activation tagging enables unbiased upregulation on a genome-wide scale by the insertion of a T-DNA containing endogenous enhancers or promoters (Weigel et al., 2000
Amino Acids Are Present in the Guttation Fluid Wild-type Arabidopsis plants were grown in growth chambers and exposed to high humidity for one night. Guttation droplets were collected the next morning, lyophilized, and analyzed for amino acid content by HPLC. The guttation contained significant amounts of Asp, Gln, and His (Table 1). For comparison, root xylem exudate from Arabidopsis mainly contained Gln, whereas leaf exudate (phloem sap) composition was more complex (Table 1), consistent with previous reports (Shelp, 1987 ; Schobert and Komor, 1989, 1990; Lam et al., 1995). Thus, the amino acid composition of the guttation fluid in wild-type plants is different from that of xylem and phloem saps.
Identification of Two Salt Secreting Mutants To study the secretion mechanism in Arabidopsis, two independent lines (named gdu1-1D and gdu1-2D, see below) that had white salty deposits at the hydathodes (Figures 1A and 1B) were identified by screening  10,000 independent T2 activation tagged lines from Agrinomics (Portland, OR). The white flecks reappeared locally a few days after leaves were washed, showing that the salt deposit is indeed secreted by hydathodes. In young seedlings grown under long-day conditions, deposits often appeared at the tips of cotyledons. Mutant plants were smaller than the wild type when grown in soil in both short- or long-day conditions (Figures 1C to 1E) or in axenic culture (data not shown). Mutant leaves were curled and darker green compared with the wild type. The mutants flowered at the same time as the wild type. All phenotypic alterations observed in gdu1-2D were weaker than those in gdu1-1D: specifically, gdu1-2D secreted less than gdu1-1D and had an intermediate size between gdu1-1D and the wild type (Figures 1D and 1E).
The Secretion Is Composed of Gln Several samples of the salty deposit were collected and analyzed. Crystals contained few inorganic cations: 18.5% Na+, 0.16% Ca2+, and <0.1% K+ and Mg2+ (w/w) as assessed by flame spectrometry. Combustion analysis revealed the presence of a large proportion of organic matter, with 35.6% carbon, 6.7% hydrogen, and 15.6% nitrogen (w/w). The liquid chromatography–mass spectrometry analysis identified a single compound, which produced a main peak with a mass of 146 atomic units and fragmenting in daughter ions, as detected by the tandem mass spectrometry analysis (data not shown). NMR spectra were identical to those produced by analysis of pure Gln (Figure 2A). The carbon, hydrogen, and nitrogen proportions of the secretion were similar to the expected values for Gln as the sole organic compound (34.4, 5.6, and 15.7%, respectively). HPLC assessed purity of Gln. Gln represented 95% of the detected amino acids ( 90% of secretion weight). Apart from Gln, <2% (1% of secretion weight) each of Asp, Thr, Ser, and Arg were present (Figure 2B). Inorganic anions (NO3–, SO42–, and Cl–) were not detected in the secretion. HPLC cation analysis detected Gln and Na+ but not K+ or NH4+ (Figure 2C). The secretion was thus composed of Gln (>80% [w/w]) and Na+ (between 2 and 20% [w/w]). Based on the composition of the secretion, the mutants were called glutamine dumper. The amino acid composition of the solution produced by the hydathodes is different in gdu1-1D than that of the wild type (Figure 2B). Interestingly, the amino acid composition of the xylem and the secretion of gdu1-1D are very similar: both contain >90% Gln, each of the other amino acids representing <2% of the amino acid content (cf. Table 1 and Figure 2B).
Free Amino Acid Content of the Leaf Is Increased in gdu1-1D, and Gln Synthetase Activity Is Not Augmented To assess the origin of the Gln secreted by the hydathodes in gdu1-1D, free and total amino acid contents of the leaf were analyzed. Whereas Ala, Gly, and Asp contents were poorly affected, Ser, Gln, Thr, Asn, and Pro levels increased 2, 2.1, 2.5, 3.1, and 12.8 times, respectively (Figure 3A). Interestingly, the leaf content of basic amino acids, such as Arg, Lys, and His, increased 2.3, 2.6, and 3.7 times, respectively. The total free amino acid content of the leaf was increased by 100%. At the same time, the total amino acid content (free plus protein amino acids) of leaf was increased by 15% (data not shown). These data show that the content of several amino acids, and not only Gln, is increased in the leaf, showing that the composition of the secretion at the hydathodes is not correlated to amino acid content of the leaf.
The effects of gdu1-1D mutation on amino acid accumulation are different from those reported for Gln synthetase (GS) overexpression in tobacco (Nicotiana tabacum) plants. In these tobaccos, only Gln levels were significantly augmented (Fuentes et al., 2001 ). Consistently, total GS activity in leaves of gdu1-1D was not higher than in wild-type plants: when grown in short days, gdu1-1D plants had a 30% lower GS activity than the wild type, and when grown in long days, GS activity was identical in gdu1-1D and the wild type (Table 2). When Gln synthesis was artificially upregulated in wild-type plants by growth on ammonium-rich medium, Gln content increased sixfold, whereas the total amount of the other amino acids was only elevated by 25% (data not shown), but no secretion was observed at the hydathodes. In another experiment, gdu1-1D and wild-type plants were grown on media containing variable amounts of ammonium nitrate (0 to 10 mM). The size of the plants increased with the quantity of available nitrogen, but the ratio between the dry weight of the mutant and that of the wild type remained constant (around 0.6; data not shown). On the contrary, it has been shown that transgenic Lotus corniculatus plants overexpressing soybean (Glycine max) GS1 displayed growth enhancement on ammonium-rich media (Vincent et al., 1997). In another report, growth of GS1 overexpressing tobacco showed improvements under nitrogen limiting conditions but was indistinguishable from the wild type in nitrogen-rich conditions (Fuentes et al., 2001).
These results suggest that the excretion of Gln from the leaves is not the result of an overproduction of Gln or an increased GS activity in the leaves.
Root Xylem Exudates and Apoplasm Wash Fluid of gdu1-1D Contain More Gln
In the mutant and in the wild type, the free amino acid composition of leaf exudate was comparable to leaf composition, as previously described (Winter et al., 1992
In leaves, an apoplasmic-loading step for amino acids requires a cellular export process (Lohaus et al., 1995 The selective increase in Gln levels in hydathode secretion, xylem sap, and AWF in gdu1-1D suggests that a common export mechanism for Gln at these different sites in the plant is affected in the mutant.
The gdu1D Mutations Are T-DNA Linked and Lead to a Gain of Function
Interestingly, the progeny of several heterozygous gdu1-1D plants contained a small number of dwarfed plants (groups B and C, Table 3). These plants were significantly smaller than gdu1-1D (Figure 1E) but showed similar leaf shape and color as gdu1-1D, and all of them were BASTAR, suggesting that the mutation is also T-DNA linked. These plants were called dwarf gdu1 (dwg). Group C contained a single line, which segregated for Dwg– with a higher proportion compared with group B lines and for BASTAR in a non-Mendelian way (Table 3). Moreover, some of the Gdu1– progeny of the group C line were also BASTAS, an attribute never seen in the lines of groups A, B, and D. The abnormal segregation and the generation of a variable percentage of Dwg– plants is likely to come from a genome rearrangement caused by the T-DNA insertion (see below; Nacry et al., 1998 ; Tax and Vernon, 2001). The Dwg– phenotype segregated in a complex way in the next generation (data not shown) and was not further investigated.
Genetic analysis of gdu1-2D mutant showed that, similar to gdu1-1D, the gdu1-2D mutation is dominant (
GDU1 Overexpression Is the Cause of the Phenotype
In the 8-kb region flanking the T-DNAs, a segment of BAC F28M20 containing three genes, namely loci At4g31730, At4g31740, and At4g31750, was common between the two mutants (Figure 4A). Real-time RT-PCR showed that the accumulation of At4g31730 mRNA (named thereafter GDU1) increased by 20 and 40 times in gdu1-2D and gdu1-1D, respectively, whereas expression levels of the other genes remained unaffected (data not shown). To address whether overexpression of GDU1 is the cause for the Gdu1– phenotype, three DNA fragments derived from gdu1-1D, containing the CaMV 35S enhancer and various lengths of genomic DNA (constructs E1, E2, and E3, Figure 4B; see Methods) and a construction composed of the cassava vein mosaic virus promoter (CsVMV; Verdaguer et al., 1996, 1998) fused to GDU1 coding sequence (construct C, Figure 4B) were introduced into wild-type Arabidopsis. E3 produced transformants indistinguishable from the wild type (60 lines analyzed), whereas more than half of the E1 and E2 transformants showed secretion and reduced size. To test the relation between the presence of secretion and overaccumulation of GDU1 mRNA, 21 E1, 12 E2, and 9 C transformants with a single kanamycin resistance locus, irrespective of the phenotype, were selected. Leaf tissues from 8 to 10 plants per line were analyzed for GDU1 mRNA content by RNA gel blot analysis (Figure 4C) and by real-time RT-PCR (data not shown). Twenty-six of these lines displayed the Gdu1– phenotype. In these 26 lines, GDU1 mRNA accumulation was increased more than 10 times compared with the wild type. Secretion was more abundant in the highly expressing plants. In the remaining 16 lines, which did not present the Gdu1– phenotype, GDU1 mRNA accumulated at lower levels only (Figure 4C). Overaccumulation of GDU1 mRNA was thus sufficient and necessary to generate the Gdu1– phenotype.
GDU1 Is a Member of a Novel Multigenic Family of Putative Membrane Proteins
To assess the subcellular localization of GDU1, the green fluorescent protein was fused to the C terminus of GDU1 and expressed transiently in Arabidopsis protoplasts and tobacco epidermis cells. In most of the cases, fluorescence localized only at the plasma membrane in protoplasts (see Supplemental Figure 2 online), whereas several protoplasts also showed staining in endomembranes (data not shown). In tobacco leaves transiently transformed by Agrobacterium tumefaciens, bright green fluorescent protein fluorescence was localized in endomembranes, whereas little fluorescence was detected at the plasma membrane (data not shown). Additional experiments will be required to assess unequivocally the localization of GDU1.
GDU1 Is Expressed in the Vasculature of the Plant and in Hydathodes
Immunolocalization experiments were performed to assess the localization of GDU1 protein in the plant. Because of the low expression levels of GDU1, a transgenic approach was chosen. Wild-type plants were transformed with a construct in which a triple c-Myc tag was fused to the GDU1 open reading frame, placed under the control of its own promoter (Figure 7A). Localization of the c-Myc tag was determined in roots and stems, where background fluorescence was found to be low. The immunochemical reaction was performed in parallel on wild-type and transformed plants using an anti-c-Myc antibody and a Cy3-labeled secondary antibody. No Cy3 fluorescence was detected in wild-type roots or stems, whereas a strong fluorescence was visible in the transformed plants. In roots, only the stele cells were labeled (Figure 7B), with stronger labeling in pericycle cells (data not shown). In stems, the c-Myc tag was detected in phloem cells, the parenchyma cells of the protoxylem, as well as the cells surrounding xylem vessels (Figure 7C). These results confirm the GUS analysis and show that GDU1 is transcribed in the vascular tissues and that GDU1 protein is present in the phloem and xylem. Root pericycle cells are supposed to play a role in xylem loading because they express transporters known to be involved in xylem loading, namely SKOR (Gaymard et al., 1998 ), BOR1 (Takano et al., 2002), and SOS1 (Shi et al., 2002). GDU1 expression in the pericycle would be in agreement with a role of this protein in Gln secretion into the xylem.
Quantitative Relationship between Strength of Phenotype and Accumulation of GDU1 mRNA Together with the secretion of Gln, it was noticed that the mutant lines gdu1-1D and gdu1-2D were smaller compared with the wild type (Figure 1). To further characterize the relationship between overexpression of GDU1 and the phenotype, each of the previously selected 42 recapitulation lines, gdu1-1D, gdu1-2D, and wild-type plants, were studied. The size (i.e., diameter of the rosette) of the plants was recorded. A clear correlation existed between size and leaf accumulation of GDU1 mRNA (Figure 8A). The data point distribution could be fitted by a nonlinear, logarithmic, curve (r2 = 0.89; Figure 8A). Although a 10-fold overaccumulation of GDU1 mRNA led to a strong decrease (40%) in the size of the plants, an increase in GDU1 overexpression by 30 and 60 times led to a further decrease in size of 10 and 20%, respectively. The relationship between GDU1 expression and leaf amino acid content was studied on the 42 recapitulation lines, the gdu1 mutants, and wild-type plants. The free amino acid content increased linearly (r2 = 0.81) with GDU1 mRNA accumulation, reaching a threefold increase when the mRNA quantity was augmented 60 times compared with the wild type (from 15 ± 5 to 46 ± 5 nmol·mg–1 fresh weight; Figure 8B). The content of each of the amino acids was either unchanged (Asp, Glu, Ala, citrulline, and  -amino-butyric acid) or increased (all other amino acids) upon GDU1 overexpression (see Supplemental Figures 3 and 4 online).
The whole set of data regarding size, mRNA overaccumulation, and amino acid content were grouped in a single graph (Figure 8C). For most of the 45 lines, there is a clear correlation between all three parameters. The correlation between GDU1 mRNA accumulation and size of the plant is thus clearly established, but the reason for this relation is at present unknown.
Arabidopsis wild-type plants secrete small amounts of three amino acids from the hydathodes in guttation droplets, namely Gln, Asp, and His. A mutant was identified that deposits salt crystals at the hydathodes. The deposit consists of almost pure Gln (Figure 2). In the mutant, the xylem sap and the AWF also contain elevated levels of Gln. In leaves, Gln content is increased as well, but the content of other amino acids also increases, especially Pro (Figure 2A). The overall elevation of amino acid pools in the whole leaf does not seem to be because of an increase in GS activity and, thus, Gln synthesis. There are three main sites of amino acid export in the plant: at hydathodes, which play a crucial role in solute absorption and possibly excretion; at xylem parenchyma into xylem sap; and from mesophyll into leaf apoplasm. The presented results show that Gln content is specifically increased at these three sites of amino acid export. These data are consistent with a specific increase in net Gln export from hydathodes, export into the xylem, and also potentially export from leaf mesophyll cells. These effects are because of activation tagging of a novel gene, GDU1, encoding a protein with a single putative TM span and a cytosolic domain. GDU1 may thus represent a component of a mechanism responsible for secretion of Gln.
Mechanisms for the Export of Amino Acids
Putative Role of GDU1 in Gln Export
GDU1 was found to be located at the plasma membrane and in intracellular compartments and contains a putative TM domain with a short C terminus probably located in the cytosol. The only homo-oligomeric transporters known so far that are formed from subunits with a single TM are viral channels (Fischer et al., 2000
Conclusion
Plant Growth and Transformation Arabidopsis thaliana plants (ecotype Columbia, Col-7) were grown in soil (Metromix 350) either (1) in a greenhouse (25°C, 75% relative humidity, 16-h light) and watered automatically from below twice a week or (2) in growth chambers (22°C, 50% relative humidity, 16-h light, 120 µmol/m2/s) and watered manually from below twice a week. BASTAR segregation was analyzed in vitro on MS medium (Sigma M5524; St. Louis, MO) containing 15 µg/mL of glufosinate-ammonium (BASTA; Bayer CropScience, Frankfurt, Germany), 0.7% agar, and 1% sucrose (16-h light, 22°C, and 100 µmol/m/s).
Binary plasmids were introduced into plants using heat shock–transformed GV3101 (pMP90) Agrobacterium tumefaciens strain (Van Larebeke et al., 1974
Xylem Exudate, AWF, and Leaf Exudate Collection
AWF was collected using the infiltration-centrifugation method (Lohaus et al., 2001
Leaf phloem exudate was collected in 5 mM EDTA, pH 7, solution (Corbesier et al., 2001
Analytical Methods Crystals secreted by gdu1-1D leaves were solubilized in water with a micropipette, lyophilized, and weighed. A few milligrams were subjected to flame spectrometry for cation analysis and combustion analysis to determine carbon, hydrogen, and nitrogen content (Galbraith Laboratories, Knoxville, TN). Liquid chromatography–mass spectrometry analysis was performed on a Perkin-Elmer Sciex 3000 LC/MS/MS system (Foster City, CA) with an Ion Spray Electrospray Interface to a Shimadzu LC-10AD VP HPLC pumps (Columbia, MD) with 250 µL of high-pressure mixer and a SCL-10A VP pump controller. The columns on the HPLC were two YMC ODS AQ, 2 x 50-mm, 3-µm particle size, 120-Å pore size columns closely coupled in series with a mobile phase of 68% acetonitrile/32% (1.5% acetic acid in water). NMR analyses were performed on a Varian 300 MHz model G300B-2974 NMR system (Palo Alto, CA) with a Sun workstation. The solvent was D2O, frequency of 300.092 Hz, sweep width of 4500.5 Hz, and the number of scans was 16. Cation and anion content of the secretion were respectively analyzed by HPLC with a Hamilton PRP-X200 column (Reno, NV; eluant: 2 mM HNO3 and 15% methanol) and a standard anion chromatography column from Wescan (Gamma Analysen Technik, Bremerhaven, Germany; eluant: 2 mM phtalic acid and 10% methanol, pH 5), and detected by conductivity on a Wescan ion analyzer. Cation content of phloem was analyzed by HPLC (DX120 ion chromatography; Dionex, Idstein, Germany) with an IonPac CS12A column (eluant: 20 mM methanesulfonic acid) and an AS40 automated sampler (Dionex).
Total GS activity was determined using the transferase assay (Shapiro and Stadtman, 1971
Plasmid Rescue Experiments
Recapitulation Constructs
Expression Analyses
The GDU1 promoter region (3 kb) was amplified by PCR from the PstI insert obtained by plasmid rescue (see above) with Pfu Turbo using the primers proF 5'-TTTCTGCAGTTCGTTGGTGATTGGTCGCCTC-3' and proR 5'-TTTTCTAGATTTTGTTGTTGTTGTGTGTAATCTC-3'. The PCR fragment was digested by PstI and XbaI and cloned into the pPZP212 vector (Hajdukiewicz et al., 1994
GDU1 gene (3-kb promoter and coding sequence) was amplified by PCR with Pfu Turbo and the primer proF and 5'-TTCTCGAGGTGACTTGTAGTAGTTGTCTCGC-3'. The PCR fragment was digested by PstI and XhoI and cloned in frame with a tripled c-Myc tag, previously cloned in pBluescript KS+. The construct was cut out using KpnI and SacI and inserted in front of the 3' region of the P. sativum Rbcs gene of a modified pPZP212 binary vector, where the 35S-nptII cassette was replaced by a mannopine (mas) promoter-nptII-mas terminator cassette (provided by Ji Hoon Ahn, Seoul National University). Approximately 15 transformed plants were assayed for GDU1-c-Myc expression by immunolocalization. For stems, stalk of 5- to 6-week-old Arabidopsis plants were cut in 5-mm pieces and fixed overnight at 4°C in 0.1% glutaraldehyde, 4% paraformaldehyde in MTSB (50 mM Pipes, 5 mM EGTA, and 5 mM MgSO4, pH 7). The pieces were embedded in 4% agarose and cut in 50-µm sections using a Leica vibratome VT 1000S in PBS, pH 7.2. The sections were blocked for 2 h at 37°C in 3% BSA in PBS. The reaction with the anti-c-Myc antibody (1:600; Santa Cruz Biotechnology, Heidelberg, Germany) was performed overnight at 4°C and 2 h at 37°C in 3% BSA in PBS. Sections were washed six times for 10 min in PBS. Detection was performed using Cy3-conjugated goat anti-mouse antibody (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA) for 4 h at 37°C in 3% BSA in PBS. Sections were washed six times for 10 min in PBS and observed by confocal microscopy (Leica DMRE with TCS SP). Root immunolocalization was performed as described (Friml et al., 2003
Phenotypic Analysis of Recapitulation Lines
We would like to thank B. Stadelhofer for all HPLC analyses, C. Brancato and P. Neumann for the perfect technical help, A. Martone for the NMR analyses, R. Seymour for the liquid chromatography tandem mass spectrometry experiments, J. Pilot for the helpful discussions on the real-time PCR experiments, and F. de Courcy for the critical reading of the manuscript. G.P. was supported by an EMBO long-term fellowship. This work was supported by the Koerber Foundation and by the Deutsche Forschungsgemeinschaft (Leibniz-Award to W.B.F.).
1 Current address: Vector Research, 710 West Main Street, Durham, NC 27701. 
2 Current address: Carnegie Institution, 260 Panama Street, Stanford, CA 94305.
Online version contains Web-only data. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Wolf B. Frommer (wfrommer{at}stanford.edu). Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.021642. Received February 8, 2004; accepted March 30, 2004.
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ABSTRACT
INTRODUCTION
-CH, respectively.
2 test, P = 0.90), suggesting a single insertion locus. Groups A and D plants did not segregate for BASTAR: progeny of group A plants was BASTAR, whereas progeny of group D plants was BASTA susceptible (BASTAS). At the same time, group A plants displayed the Gdu1– phenotype as did their progeny, whereas group D plants and their offspring were indistinguishable from the wild type. Thus, group A plants were homozygous for gdu1-1D and BASTAR, and group D plants were homozygous for the corresponding wild-type alleles. Segregation analysis of group B plants showed that they also segregate for Gdu1– phenotype with a 3:1 ratio (
Ct; 
