TY - JOUR
AU - Dubois, Frédéric
AB - Abstract Although the physiological role of the enzyme glutamate dehydrogenase which catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate remains to be elucidated, it is now well established that in higher plants the enzyme preferentially occurs in the mitochondria of phloem companion cells. The Nicotiana plumbaginifolia and Arabidopis thaliana enzyme is encoded by two distinct genes encoding either an α- or a β-subunit. Using antisense plants and mutants impaired in the expression of either of the two genes, we showed that in leaves and stems both the α- and β-subunits are targeted to the mitochondria of the companion cells. In addition, we found in both species that there is a compensatory mechanism up-regulating the expression of the α-subunit in the stems when the expression of the β-subunit is impaired in the leaves, and of the β-subunit in the leaves when the expression of the α-subunit is impaired in the stems. When one of the two genes encoding glutamate dehydrogenase is ectopically expressed, the corresponding protein is targeted to the mitochondria of both leaf and stem parenchyma cells and its production is increased in the companion cells. These results are discussed in relation to the possible signalling and/or physiological function of the enzyme which appears to be coordinated in leaves and stems. Introduction Recently, we showed that in tobacco the production of glutamate dehydrogenase (GDH; EC 1.4.1.2) and its activity were strongly induced when ammonia is applied externally or is released during photorespiration. GDH was increased in the mitochondria and appeared in the cytosol of companion cells (CCs) of vascularized organs such as stems, petioles and midribs. It was therefore hypothesized that the enzyme plays a dual role in CCs, either in the mitochondria when mineral nitrogen availability is low or in the cytosol when the ammonium concentration increases above a certain threshold (Tercé-Laforgue et al. 2004). This novel finding revealed that the role of the enzyme in the whole plant context is more complex than previously proposed. In particular, a number of authors had controversial commentaries about its physiological function (Pahlich 1996) because GDH catalyses the reversible amination of 2-oxoglutarate for the synthesis of glutamate using ammonium as a substrate. It was therefore proposed that the enzyme could operate in the direction of either ammonia assimilation (Yamaya and Oaks 1987, Oaks 1995, Melo-Oliveira et al. 1996) or glutamate deamination (Robinson et al. 1992, Fox et al. 1995, Stewart et al. 1995) depending on cellular carbon (C) and nitrogen (N) availability. However, in these investigations, both the organ and the subcellular localization of the protein were not taken into account, since the catalytic function of the enzyme was studied in vivo by providing 15N-labelled ammonium or 15N-labelled glutamate to cells in suspension culture or isolated mitochondria. The recent finding that GDH is mostly located in the vascular tissue led some authors to propose that the enzyme could also play a sensing role, in agreement with the current view that the continuity between CCs and sieve tubes is one of the key elements for C and N metabolite translocation and signalling (Tercé-Laforgue et al. 2004). This hypothesis was also supported by the fact that the enzyme is strongly induced during various stresses and physiological conditions involving a rapid translocation of metabolic signals (Loulakakis and Roubelakis-Angelakis 2001, Chaffei et al. 2004). In most plant species, GDH is encoded by two distinct nuclear genes (Melo-Oliveira et al. 1996, Pavesi et al. 2000, Restivo 2004). Each gene encodes a different subunit termed α- and β-polypeptides, which can be assembled as homo- or heterohexamers composed of a different ratio of α- and β-polypeptides, thus leading to the formation of seven active isoenzymes. These seven isoenzymes can be distinguished using native PAGE followed by in-gel NAD-dependent activity (Turano et al. 1996) or NADH-dependent activity staining (Loulakakis and Roubelakis-Angelakis 1996). Variations of the GDH isoenzyme pattern were observed according to both the organ examined and the N source (Loulakakis and Roubelakis-Angelakis 1992, Turano et al. 1997). However, the physiological function of the seven isoenzymes and the significance of the variations in their relative proportions remain unknown. In order to investigate further the relationship that may exist between the biochemical properties of the enzyme and its function in the vascular tissue, the subcellular localization of the enzyme was studied in Nicotiana plumbaginifolia plants in which the production of either the α- or the β-polypeptide was impaired using an RNA antisense strategy. A similar investigation was performed in the Arabidopsis mutants gdh1 and gdh2 defective in the production of the α- and β-subunit, respectively. We showed that in both species the two GDH subunits are targeted to the mitochondria of the CCs. To determine whether the protein can be produced in other tissues, cytoimmunochemical experiments were also conducted in Arabidopsis plants ectopically expressing the N. plumbaginifolia GDHB gene. In parallel, the changes in the GDH isoenzyme profile and in vitro activity in floral stems and leaves of transgenic plants and mutants modified for GDH gene(s) expression were examined. Results Analysis of GDH isoenzyme activity and protein content in N. plumbaginifolia GHDA and GDHB antisense plants The binary vectors 35Sas-NpGDHA and 35Sas-NpGDHB containing the cauliflower mosaic virus (CaMV) 35S promoter upstream of the N. plumbaginifoliaGDHA and GDHB cDNA placed in the antisense orientation were introduced into N. plumbaginifolia via Agrobacterium tumefaciens-mediated transformation. Following plant transformation and regeneration, PCR analysis allowed the screening and selection of 12 35Sas-NpGDHA and 10 35Sas-NpGDHB primary transformants (data not shown). In-gel activity staining was used to detect the GDH isoenzyme composition in mature leaves of transformed plants. In three 35Sas-NpGDHA transformants (A1, A6 and A14), only the most cathodal isoenzyme (GDH1) was present in comparison with the untransformed wild-type (WT) plant, in which the seven isoenzymes (GDH1-7) were detected. In leaves of the WT, GDH1, 2 and 3 were the most active isoenzymes (Fig. 1A). Fig. 1B shows the results obtained with in-gel GDH activity staining in the 35Sas-NpGDHB primary transformants. In the three transgenic plants B9, B49 and B56, only the two most anodal isoenzymes (GDH6 and 7) were visible, isoenzyme GDH7 being the most active. In the other lines containing the 35Sas-NpGDHA and 35Sas-NpGDHB constructs, the isoenzyme profile was either similar to the WT or exhibited a reduced number of bands of GDH activity. This observation indicates that the synthesis of GDHA and GDHB mRNA was either not modified or partially inhibited. We observed that in leaves of transgenic lines A1, A6 and A14, the activity of GDH1 was higher than in the WT, whereas in transgenic lines B9, B49 and B56, it was GDH7 which was highest (Fig. 1). The three lines displaying the most cathodal (A1, A6 and A14) and the most anodal (B9, B49 and B56) GDH isoenzyme pattern were selected and self-pollinated to obtain seeds for analysis of the T1 generation. For each construct, the resulting seeds from the three independent transformed lines were further collected and germinated on kanamycin-containing media to eliminate untransformed lines and to select for homozygous T3 plants. Since similar results were obtained with each of the three A and B transgenic lines, only homozygous lines A1 and B56 were used for further biochemical and cytoimmunochemical analyses. In these two lines, the leaf GDH isoenzyme pattern was similar compared with primary transformants, indicating that the expression of both antisense constructs was stable in their progeny. In the floral stems of the WT plants, we observed that the most anodal isoenzymes (GDH5, 6 and 7) were more active compared with the leaves in which these three isoenzymes were barely detectable (Fig. 2). In both leaves and stems of the A1 antisense plants, only GDH1 (homohexamer composed of six β-subunits) remained active. In the B56 antisense plants, GDH7 and GDH6 were detected, the former being the most active (Fig. 2). As in the primary transformants, we confirmed that in the leaves of the B56 transgenic line, GDH7 (homohexamer composed of six α-subunits) was more active compared with the WT in which this isoenzyme was barely detectable. In the leaves of the A1 transgenic line, GDH1 activity was higher compared with that of the WT. In the stems of the A1 transgenic plants, we observed a slight increase in the activity of GDH1 in comparison with the WT (Fig. 2). NAD(H)-GDH specific activity was measured in mature leaves and in stems of 35Sas-NpGDHA and 35Sas-NpGDHB antisense plants. In floral stems, GDH activity was 65 and 55% lower in line B56 and line A1, respectively. Compared with the WT, GDH activity was 89 and 52% lower in leaves of line B56 and line A1, respectively (Table 1). The enzyme activity measured in vitro matched that detected after in-gel staining (Fig. 2). Protein gel blot analysis showed that as for the enzyme activity, the reduction in the amount of GDH protein was more important in leaves (Fig. 3A) and stems (Fig. 3B) of line B56. Analysis of GDH isoenzyme activity and protein content in gdh1- and gdh2-deficient mutants of Arabidopsis Like in N. plumbaginifolia, GDH in Arabidopsis is represented by seven isoenzymes. In both species, their relative activity is similar since the most cathodal isoforms (GDH1-4) are more active in leaves whereas the most anodal isoforms (GDH4-7) are more active in floral stems (Fig. 4). In both stems and leaves of the gdh1-deficient mutant, only GDH7 (homohexamer composed of six α-subunits) was detected. Only GDH1 (homohexamer composed of six β-subunits) was detected in the gdh2-deficient mutant (Fig. 4). In a similar manner to that observed in the N. plumbaginifolia B56 antisense plants, we found that in leaves of the Arabidopsisgdh1 mutant, GDH7 is more active compared with the WT. In stems of the Arabidopsisgdh2 mutant, GDH1 is induced in comparison with the WT. In the N. plumbaginifolia A1 antisense line, we found that the activity of the cognate isoenzyme was also enhanced (Fig. 2). In the stems of the gdh1 mutant, GDH activity measured in vitro was 34% higher compared with that of the WT, whereas in leaves it was 70% lower. In the gdh2 mutant, we found a 34% decrease in stem GDH activity, whereas in leaves the enzyme was 19% higher in the WT (Table 1). This compensatory balance for enzyme activity in the stem and leaves in the two mutants was already visible following in-gel staining, as shown in Fig. 4. The protein gel blot presented in Fig. 3C showed that the amount of GDH protein was decreased in the leaves of gdh1 and gdh2 mutants. This decrease was lower in the gdh2 mutant although the corresponding enzyme activity was higher than in the WT (Table 1). In stems, a reduction in the amount of GDH protein was only visible in the gdh2 mutant (Fig. 3D), in agreement with the enzyme activity data (Table 1). In the gdh1 mutant, although the amount of GDH protein was similar to that of the wild type, the corresponding enzyme activity was higher (Table 1). Overexpression of GDH in Arabidopsis The N. plumbaginifolia cDNA encoding GDHB (GDHB) was made constitutive by fusing it with the CaMV 35S promoter. After selection and regeneration on kanamycin, transgenic plants overexpressing GDHB were selected by in-gel enzyme activity staining. Out of several T2 transformants, only two lines (B7-4 and B6-5) showed increased GDH activity in the leaves (Fig. 5). In these two lines, the GDH isoenzyme pattern appeared rather complex compared with the WT. Thirteen bands of NAD-GDH activity were detected, likely to be the result of the assembly of the GDH β-subunit from N. plumbaginifolia and two GDH α- and β-subunits of Arabidopsis producing heterospecific hexamers (Fig. 5). In stems of the two transgenic lines, an increase in the enzyme activity was only visible in line B7-4, but was much lower compared with that detected in leaves (Fig. 5). The seeds of the two T2 independent transformed lines were germinated on kanamycin-containing media to eliminate untransformed lines and to select for homozygous T3 plants. The GDH isoenzyme pattern of the T3 homozygous lines was similar to that of the T2 progeny (data not shown). These B7-4 and B6-5 homozygous lines were used for further biochemical and cytoimmunochemical analysis. In the two transgenic lines B7-4 and B6-5, leaf GDH activity was increased at least 2-fold (Table 1). In floral stems, the increase in GDH activity was only significantly higher in line B7-4 (Table 1). The increase in the enzyme activity measured in vitro was qualitatively similar to that detected by in-gel staining (Fig. 5) and corresponded to the amount of GDH protein detected following protein gel blot analysis (Fig. 3C, D). Subcellular localization of GDH by immuno-gold transmission electron microscopy In order to determine the localization of GDHA and GDHB gene products of N. plumbaginifolia, immuno-gold transmission electron microscopy experiments were conducted on leaf and floral stem sections of 35Sas-NpGDHA and 35Sas-NpGDHB antisense plants. Fig. 6A shows a partial view of a mature leaf mesophyll cell of WT plants. The section was devoid of gold particles, indicating the absence of GDH protein. Similar results were obtained when leaf mesophyll cells sections of 35Sas-GDHA and 35Sas-GDHB antisense plants were examined (data not shown). We did not observe any labelling above the background level when a similar section was treated with pre-immune serum (Fig. 6D). Quantification of gold particles confirmed the absence of GDH in the leaf mesophyll, considering that the background level is around four particles per µm2 (Table 2). In the leaf veins (Fig. 6B) and floral stems (Fig. 6C) of WT plants, gold particles were only detected in the mitochondria of the CCs. Following quantification, we confirmed that gold particles corresponding to GDH protein were only detectable in the mitochondria of CCs found in leaf veins and floral stems (Table 2). In both leaf vein (Fig. 6E) and floral stem (Fig. 6F) sections of 35Sas-NpGDHA antisense plants, it was difficult to visualize a reduction in the amount of gold particles present in the mitochondria. However, following quantification of gold particles, we found that there was a low (30%) but significant reduction in the amount of GDH protein in the mitochondria of both leaf veins and floral stem CCs (Table 2). In 35Sas-NpGDHB antisense plants, the reduction in the amount of gold particles was much stronger (around 85%, as shown in Table 2) and clearly visible in both leaf vein and floral stem sections (Fig. 6G, H). A similar investigation was conducted in gdh1 and gdh2 mutants to verify if in another plant species the corresponding gene products are targeted to the mitochondria of CCs and to estimate the relative amounts of GDH1 and GDH2 proteins in leaves and floral stems. In the floral stem (Fig. 7A, B) of WT plants, GDH protein was localized in the mitochondria of the CCs. A reduction of 52 and 78% in the number of gold particles was observed for the gdh1 mutant (Fig. 7C) and the gdh2 mutant (Fig. 7D), respectively (Table 2). When leaf sections of mature leaves were examined, a reduction in the amount of gold particles was also observed in the veins of the two mutants (data not shown). Quantification of the gold particles (Table 2) revealed that the decrease in the amount of GDH protein was lower in the gdh2 mutant (23%) compared with that of the gdh1 mutant (54%). In the mesophyll parenchyma cells of WT plants, we did not observe any labelling above the background level (Fig. 7E). Similar results were obtained when leaf sections of the two mutants were treated with the GDH antiserum (data not shown). In transgenic Arabidopsis plants overexpressing GDHB from N. plumbaginifolia, gold particles were detected in the mitochondria of leaf mesophyll parenchyma cells (Fig. 7F, G). When floral stem sections of Arabidopsis 35S-NpGDHB plants were treated with the GDH antiserum, gold particles were detected not only in the mitochondria of the CCs, but also in the neighbouring parenchyma cells (Fig. 7H). In WT floral stems, no labelling was observed in the phloem parenchyma cells of the floral stem (Fig. 7E). Compared with the WT, a 40% increase (Table 2) in the amount of GDH protein was observed in the floral stem CCs (Fig. 7I). When substituting the anti-GDH serum with pre-immune serum, no gold particles were visible (data not shown). Although the quantification of gold particles only gives a rough estimation of the amount of protein present in a given organ or tissue due to the relatively small surface observed in a section, the general trend was to observe similar differences in the enzyme activity whatever the type of plant (antisense, mutant or overexpressors) or the organ (leaves and stems) examined. However, we found that GDH activity in the stems of the gdh1 mutant and in the leaves of the gdh2 mutant was higher than in the WT, although the number of gold particles was lower in both organs (see Table 1 and 2 for comparison). This observation suggests that there is a post-translational control of the enzyme activity in the stems of the gdh2 mutant and in the leaves of the gdh1 mutant. Discussion Phylogenic analysis identified two separated groups of GDH genes (Pavesi et al. 2000, Purnell et al. 2005). The genes GDHB from N. plumbaginifolia and GDH1 from Arabidopsis encoding the β-subunit belonged to the first group, whereas the genes GDHA from N. plumbaginifolia and GDH2 from Arabidopsis encoding the α-subunit were clustered in the second group. By examining the GDH isoenzyme profile in transgenic N. plumbaginifoliaGDHA and GDHB antisense lines and Arabidopsisgdh1- and gdh2- deficient mutants, we confirmed that in both leaves and stems the most anodal isoenzyme corresponded to the α-subunit whereas the most cathodal isoenzyme corresponded to the β-subunit. Although the relative activity of GDH differs between stems and leaves of the two species examined in this study, we found that the most anodal isoenzymes predominate in the rosette leaves while the most cathodal isoenzymes are more active in floral stems. However, the significance of these organ-specific differences has never clearly been interpreted (Turano et al. 1997) mostly because the physiological role of the seven GDH isoenzymes is still a matter of speculation (Dubois et al. 2003). In the Arabidopsisgdh1-deficient mutants, Melo-Oliveira et al. (1996) showed that GDH1 transcripts were not expressed and that the gene encoding GDH2 was still transcribed, leading to the production of active α-subunits in the rosette leaves. In a more recent work performed on tobacco expressing a tomato GDHB1 gene in the antisense orientation, only a 50% decrease in the amount of α-subunit was observed using Western blot analysis (Purnell et al. 2005). In this work, we showed that in two different species such as N. plumbaginifolia and Arabidopsis, there is a compensatory mechanism up-regulating the expression of the α-subunit in the stems when the expression of the β-subunit is impaired in the leaves, and of the β-subunit in the leaves when the expression of the α-subunit is impaired in the stems. However, the occurrence of such a compensatory mechanism was not so obvious in N. plumbaginifolia, particularly in line B56, because a cross-inhibition of the expression of the two genes probably occurred when using full-length antisense RNAs. It is also possible that this compensatory mechanism occurs within the same organ when one of the subunits is lacking. Although the nature of this compensatory mechanism remains to be determined, this result suggests that there is a signal possibly circulating via the phloem that controls the balance between the leaf and stem GDH isoenzyme composition, or within the same organ. It has been shown previously that in all the plant species examined so far, including grapevine (Paczek et al. 2002), maize (Dubois et al. 2003), tobacco (Tercé-Laforgue et al. 2004) and wheat (Kichey et al. 2005), GDH is mainly if not exclusively localized in CCs of shoot tissues. Using immuno-gold labelling, we showed that in both N. plumbaginfolia and GDHBArabidopsisgdh1 and gdh2 mutants, the products of the two different genes encoding GDH are targeted to the mitochondria of the CCs. Making the N. plumbaginifolia GDHB gene expression constitutive led to the expression of an active protein localized in the mitochondria of the leaf mesophyll parenchyma cells and of the stem parenchyma cells neighbouring the CCs of transgenic Arabidopsis plants. This observation demonstrates that the production of GDH can be forced in a cell type where the enzyme is not normally expressed and that the targeting of the protein to the mitochondria is conserved although not occurring in its native cellular environment. An increase in the amount of GDH was also observed in the mitochondria of the leaf and stem CCs where the native enzyme is localized, thus indicating that the organelles have the capacity to import more protein (Tercé-Laforgue et al. 2004) even if the transgene comes from a heterologous plant system. Experiments are currently in progress to determine whether a shift in the tissue location of GDH in transgenic plants alters plant metabolism and gene expression profiling in different organs and under different growth conditions for which the nitrogen source and intensity of photosynthesis are varied, as well as stress conditions. The physiological impact of the lack of the α- and/or the β-subunits in Arabidopsis will be examined in stems and leaves using single and double mutants. Materials and Methods Plant material and growth Nicotiana plumbaginifolia (cv. Viviani 2n = 20) and Arabidopsisthaliana (ecotype Columbia) seeds were surface sterilized for 20 min in a 0.25% NaClO solution containing 0.1% Tween-20, washed six times in sterile water and placed on Petri dishes containing MS (Murashige and Skoog 1962) medium (Duchefa, Harlem, The Netherlands) solidified with 0.8% agar, and supplemented with 2% sucrose. After 48–72 h of incubation at 4°C in the dark, the plates were transferred to a controlled environment growth chamber (16 h light, 8 h dark at 25°C). After 2–3 weeks, seedlings were removed from the plates and aseptically transferred to plastic pots (10 and 5 cm diameter for N. plumbaginifolia and Arabidopsis, respectively) containing a soil : vermiculite mix (3 : 1). Both substrates were purchased from a local dealer (Consorzio Agrario, Parma, Italy). Plants were placed in a controlled environment growth chamber (16 h light, 350–400 µmol photons m–2 s–1, 25°C; 8 h dark, 18°C) and watered with diluted (1 : 10) MS solution containing 4 mM NO3– and 2 mM NH4+ as the nitrogen source. N. plumbaginifolia transgenic plants were either sexually propagated or micropropagated using in vitro stem cuttings. Arabidopsis gdh1 (SALK_042736) and gdh2 mutants (SALK_102711) were obtained from the Nottingham Arabidopsis Stock Centre (NASC). The growth conditions were similar to those used for N. plumbaginifolia and Arabidopsis WT and transgenic plants. Production of N. plumbaginofolia GDH antisense plants SacI and BamHI cloning sites were added by PCR to the 3′ and 5′ ends of the GDHA and GDHB cDNAs (NpGDHA and NpGDHB), respectively, from N. plumbaginifolia (Ficarelli et al. 1999). The two amplification products were cloned into the pGEM-T easy vector (Promega, Madison, WI, USA), sequenced and subcloned in the antisense orientation downstream of the 35S CaMV promoter and upstream of the nopaline synthetase terminator (NOS) using the SacI and BamHI sites of the binary vector pBI121 (Clontech, Palo Alto, CA, USA). The resulting constructs were called 35Sas-NpGDHA and 35Sas-NpGDHB. The pBI vector containing the constructs 35Sas-NpGDHA and 35Sas-NpGDHB was placed into Escherichia coli DH5α from where it was subsequently transferred to A. tumefaciens (strain EHA105) by chemically based direct transformation (Walkerpeach and Velten 1994). Kanamycin-resistant N.plumbaginifolia plants were regenerated following leaf disk transformation (Horsch et al. 1985) and transferred to a soil and vermiculite (3 : 1) mixture and watered daily with diluted 1 : 10 MS solution for 6–8 weeks before harvesting for protein extraction. In independent primary transformants (T0), tissue of a mature rosette leaf (leaf 4–5 of the rosette from a plant with 8–10 leaves) was analysed for GDH in-gel activity staining. The plants were self-fertilized and allowed to go to seed to select for homozygous T3 transformants. Further analysis of the GDH isoenzyme composition and immunolocalization experiments were performed using the homozygous T3 plants utilizing the same growth conditions used to analyse the primary transformants. Production of A. thaliana plants overexpresing GDHB from N. plumbaginifolia A fragment containing the GDHB cDNA from N. plumbaginifolia (NpGDHB) was subcloned in the sense orientation into a the binary vector pBI121 to obtain the 35S-NpGDHB construct using the same procedure already described to produce the antisense construct 35Sas-NpGDHB. Transgenic A. thaliana (accession Columbia) plants were produced by the floral dip method (Clough and Bent 1998) infiltrating 4-week-old plants in A. tumefaciens culture containing the 35S-NpGDHB construct. Seeds from treated plants were collected and screened for kanamycin resistance, and transgenic plants identified in this generation were classified as T1 plants. Homozygous T3 progeny were then selected for further biochemical and cytoimmunochemical studies. Enzymatic in vitro and in-gel assay, determination of total soluble protein and protein gel blot analysis For soluble protein extraction, mature leaves (middle leaves of the rosette) and floral stems of 6- to 8-week-old N. plumbaginifolia (having 15–18 leaves in total) and 4-week-old Arabidopsis plants (having 10 visible open flowers) were harvested. Soluble proteins were extracted from frozen leaf and stem material stored at –80°C. All extractions were performed at 4°C. Glutamate dehydrogenase [NAD(H)-GDH] was measured as described by Turano et al. (1996). In-gel detection of GDH-NAD-dependent activity was performed as described by Restivo (2004). As previously shown by Loulakakis and Roubelakis-Angelakis (1996), staining of NADH-GDH activity revealed the same isoenzyme profile (data not shown). However, in the present study, NAD-GDH in-gel detection was used because of its higher sensitivity. Soluble protein was determined using a commercially available kit (Coomassie Protein assay reagent, Biorad, München, Germany) with bovine serum albumin (BSA) as a standard. Leaf and stem soluble proteins were extracted from frozen leaf or stem material in cold extraction buffer containing 50 mM Tris–HCl pH 7.5, 1 mM EDTA, 1 mM MgCl2, PVP 0.5% (w/v), 2-mercaptoethanol 0.1% (v/v) and leupeptin 4 µM, and separated by SDS–PAGE (Laemmli 1970). Equal amounts of protein (10 µg) were loaded in each track. The percentage of polyacrylamide in the running gels was 10%. After SDS–PAGE separation, proteins were transferred to nitrocellulose membranes for Western blot analysis. Antibodies against GDH protein were obtained by immunization of rabbits (Eurogentec X2 protocol, Liege, Belgium) with two synthetic peptides (NH2-VQHDNARGPMKGGIR+C-CONH2 and NH2-PIDLGGSLGRDAATGR-CONH2) corresponding to a highly conserved motif in Arabidopsis GDH1 and GDH2 proteins. The specificity of the antibodies raised against the synthetic peptides was tested using Western blot analysis (Fig. 3) according to the protocol described by Tercé-Laforgue et al. (2004). Statistics For measurement of enzymes activities, results are presented as mean values for four plants with SEs. Cytoimmunochemical studies Leaf and floral stem fragments (2–3 mm2) were fixed in freshly prepared 1.5% (w/v) paraformaldehyde in phosphate buffer 0.1 M, pH 7.4 for 4 h at 4°C. For immunolocalization, material was dehydrated in an ethanol series [final concentration 90% (v/v) ethanol] then embedded in London Resin white resin (Polysciences, Warrington, PA, USA). Polymerization was carried out in gelatin capsules during 10 h at 54°C. For immunotransmission electron microscopy studies, ultrathin sections were mounted on 400 µm mesh nickel grids and allowed to dry at 37°C. Sections were first incubated with 5% (v/v) normal goat serum in T1 buffer [0.05 M Tris–HCl buffer containing 2.5% (w/v) NaCl, 0.1% (w/v) BSA and 0.05% (v/v) Tween-20, pH 7.4] for 1 h at room temperature and then for an additional 6 h at room temperature with the GDH antiserum diluted 70 times in T1 buffer. Sections were then washed five times with T1 buffer, twice with T2 buffer [0.02 M Tris–HCl buffer containing 2% (w/v) NaCl, 0.1% (w/v) BSA and 0.05% (v/v) Tween-20, pH 8] and incubated for 2 h at room temperature with a 10 nm colloidal gold–goat anti-rabbit immunoglobulin complex (Sigma) diluted 50 times in T2 buffer. After several washes, grids were treated with 5% (w/v) uranyl acetate in water and observed with a Philips CM12 electron microscope (Philips, Eindhoven, The Netherlands) at 80 kV. Negative controls were conducted by substituting the serum containing GDH antibodies with pre-immune rabbit serum. Acknowledgments The authors thank Dr. C. Tonelli and Dr. M. Galbiati for their kind help with Arabidopsis transformation, and Dr. Judith Harrison for critically reading the manuscript. This work was supported by the Conseil Régional de Picardie and the Fonds Social Européen, and by a grant from the Italian Ministry of University and Scientific and Technological Research (MIUR). 5 These authors contributed equally to this work. View largeDownload slide Fig. 1 NAD-GDH isoenzyme patterns in leaves of N. plumbaginifolia primary transformants expressing a GDHA and GDHB antisense RNA under the control of the 35S CaMV promoter. Leaf extracts of transformed 35Sas-GDHA (A) and 35Sas-GDHB (B) and untransformed wild-type (WT) plants were subjected to native PAGE followed by NAD-GDH in-gel activity staining. Following Agrobacterium-mediated transformation, plants were grown for 6–8 weeks as described in Materials and Methods. Proteins were extracted from a mature rosette leaf (leaf 4–5 starting from the base). Arrowheads indicate the 35Sas-GDHA (lines A1, A6 and A14) and 35Sas-GDHB (B9, B49 and B56) transgenic lines displaying the strongest alteration of the GDH isoenzymatic pattern compared with the untransformed WT control plant. The positions of the seven isoenzymes are indicated on the left side of the panels and numbered from 1 (proximal to the cathode, –) to 7 (proximal to the anode, +). View largeDownload slide Fig. 1 NAD-GDH isoenzyme patterns in leaves of N. plumbaginifolia primary transformants expressing a GDHA and GDHB antisense RNA under the control of the 35S CaMV promoter. Leaf extracts of transformed 35Sas-GDHA (A) and 35Sas-GDHB (B) and untransformed wild-type (WT) plants were subjected to native PAGE followed by NAD-GDH in-gel activity staining. Following Agrobacterium-mediated transformation, plants were grown for 6–8 weeks as described in Materials and Methods. Proteins were extracted from a mature rosette leaf (leaf 4–5 starting from the base). Arrowheads indicate the 35Sas-GDHA (lines A1, A6 and A14) and 35Sas-GDHB (B9, B49 and B56) transgenic lines displaying the strongest alteration of the GDH isoenzymatic pattern compared with the untransformed WT control plant. The positions of the seven isoenzymes are indicated on the left side of the panels and numbered from 1 (proximal to the cathode, –) to 7 (proximal to the anode, +). View largeDownload slide Fig. 2 NAD-GDH isoenzyme patterns in N. plumbaginifolia35Sas-GDHA and 35Sas-GDHB transformants. Stem (S) and mature leaf (L) extracts of homozygous T3 transformed 35Sas-GDHA (line A1) and 35Sas-GDHB (line B56) and untransformed wild-type (WT) plants were subjected to native PAGE followed by NAD-GDH in-gel activity staining. The positions of the seven GDH isoenzymes are indicated on the left side of the panel and numbered from 1 to 7. View largeDownload slide Fig. 2 NAD-GDH isoenzyme patterns in N. plumbaginifolia35Sas-GDHA and 35Sas-GDHB transformants. Stem (S) and mature leaf (L) extracts of homozygous T3 transformed 35Sas-GDHA (line A1) and 35Sas-GDHB (line B56) and untransformed wild-type (WT) plants were subjected to native PAGE followed by NAD-GDH in-gel activity staining. The positions of the seven GDH isoenzymes are indicated on the left side of the panel and numbered from 1 to 7. View largeDownload slide Fig. 3 GDH protein content in N. plumbaginifolia GDH antisense plants, Arabidopsis gdh mutants and GDH overexpressors. Western blot analysis of GDH in mature leaves (A) and stems (B) of N. plumbaginifolia transgenic plants expressing the GDHA (A1) and GDHB (B56) cDNAs in the antisense orientation in comparison with the wild type (WT). Western blot analysis of GDH in leaves (C) and stems (D) of Arabidopsis gdh1 and gdh2 homozygous mutants and in two Arabidopsis transgenic lines (B6-5 and B7-4) overexpressing GDHB from N. plumbaginifolia under the control of the 35S CaMV promoter. WT is a wild-type control plant. Soluble proteins were extracted from a mature leaf of the rosette. A 10 µg aliquot of soluble protein was loaded onto each lane. View largeDownload slide Fig. 3 GDH protein content in N. plumbaginifolia GDH antisense plants, Arabidopsis gdh mutants and GDH overexpressors. Western blot analysis of GDH in mature leaves (A) and stems (B) of N. plumbaginifolia transgenic plants expressing the GDHA (A1) and GDHB (B56) cDNAs in the antisense orientation in comparison with the wild type (WT). Western blot analysis of GDH in leaves (C) and stems (D) of Arabidopsis gdh1 and gdh2 homozygous mutants and in two Arabidopsis transgenic lines (B6-5 and B7-4) overexpressing GDHB from N. plumbaginifolia under the control of the 35S CaMV promoter. WT is a wild-type control plant. Soluble proteins were extracted from a mature leaf of the rosette. A 10 µg aliquot of soluble protein was loaded onto each lane. View largeDownload slide Fig. 4 NAD-GDH isoenzyme patterns in leaves of Arabidopsisgdh1- and gdh2-deficient mutants. Stem (S) and mature leaf (L) extracts of homozygous gdh1 and gdh2 mutants and of wild-type (WT) plants were subjected to native PAGE followed by NAD-GDH in-gel activity staining. The positions of the seven isoenzymes are indicated on the left side of the panels and numbered from 1 to 7. View largeDownload slide Fig. 4 NAD-GDH isoenzyme patterns in leaves of Arabidopsisgdh1- and gdh2-deficient mutants. Stem (S) and mature leaf (L) extracts of homozygous gdh1 and gdh2 mutants and of wild-type (WT) plants were subjected to native PAGE followed by NAD-GDH in-gel activity staining. The positions of the seven isoenzymes are indicated on the left side of the panels and numbered from 1 to 7. View largeDownload slide Fig. 5 NAD-GDH isoenzyme patterns in leaves and stems of Arabidopsis T3 homozygous transformants overexpressing the GDHB cDNA from N. plumbaginifolia under the control of the 35S CaMV promoter. Leaf and stem extracts of untransformed wild-type (WT) plants and two independent transgenic plants (B7-4 and B6-5) were subjected to native PAGE followed by NAD-GDH in-gel activity staining. Proteins were extracted from a mature leaf and from stems of 30-day-old plants. The positions of the seven isoenzymes in the WT is indicated on the left side of the panels and numbered from 1 to 7. In the centre of the panel is shown the position of the GDH homohexamers 6β from N. plumbaginifolia (protein extract from antisense line A1) and 6α and 6β from Arabidopsis (protein extract from gdh1 and gdh2 mutants, respectively). The three protein extracts were mixed and assayed for in-gel GDH activity staining. View largeDownload slide Fig. 5 NAD-GDH isoenzyme patterns in leaves and stems of Arabidopsis T3 homozygous transformants overexpressing the GDHB cDNA from N. plumbaginifolia under the control of the 35S CaMV promoter. Leaf and stem extracts of untransformed wild-type (WT) plants and two independent transgenic plants (B7-4 and B6-5) were subjected to native PAGE followed by NAD-GDH in-gel activity staining. Proteins were extracted from a mature leaf and from stems of 30-day-old plants. The positions of the seven isoenzymes in the WT is indicated on the left side of the panels and numbered from 1 to 7. In the centre of the panel is shown the position of the GDH homohexamers 6β from N. plumbaginifolia (protein extract from antisense line A1) and 6α and 6β from Arabidopsis (protein extract from gdh1 and gdh2 mutants, respectively). The three protein extracts were mixed and assayed for in-gel GDH activity staining. View largeDownload slide Fig. 6 Immunolocalization of GDH in leaf and floral stem of N. plumbaginifolia GDH antisense plants. (A) Mesophyll cell of a mature leaf of wild-type (WT) plants. (B) Companion cell (CC) of a mature leaf of WT plants. (C) A CC of a basal floral stem of WT plants. (D) Control section showing a CC of a basal floral stem of WT plants treated with pre-immune serum. (E) CC of a leaf of 35Sas-GDHA antisense plants. (F) CC of a floral stem of 35Sas-GDHA antisense plants. (G) CC of a leaf of 35Sas-GDHB antisense plants. (H) CC of a floral stem of 35Sas-GDHB antisense plants. Cy, cytosol; m, mitochondrion; P, plastids; N, nucleus; V, vacuole. Bars = 1 µm. View largeDownload slide Fig. 6 Immunolocalization of GDH in leaf and floral stem of N. plumbaginifolia GDH antisense plants. (A) Mesophyll cell of a mature leaf of wild-type (WT) plants. (B) Companion cell (CC) of a mature leaf of WT plants. (C) A CC of a basal floral stem of WT plants. (D) Control section showing a CC of a basal floral stem of WT plants treated with pre-immune serum. (E) CC of a leaf of 35Sas-GDHA antisense plants. (F) CC of a floral stem of 35Sas-GDHA antisense plants. (G) CC of a leaf of 35Sas-GDHB antisense plants. (H) CC of a floral stem of 35Sas-GDHB antisense plants. Cy, cytosol; m, mitochondrion; P, plastids; N, nucleus; V, vacuole. Bars = 1 µm. View largeDownload slide Fig. 7 Immunolocalization of GDH in Arabidopsis GDH-deficient mutants and GDH overexpressors. (A) Partial view of a basal floral stem section of wild-type (WT) plants. (B) Detail of a floral stem companion cell (CC) of WT plants. (C) A CC in a floral stem of the gdh1 mutant. (D) A CC in a floral stem of the gdh2 mutant. (E) A mesophyll cell of a mature leaf of WT plants. (F) A mesophyll parenchyma cell of Arabidopsis transgenic plants overexpressing GDHB from N. plumbaginifolia. (G) Detail of a mesophyll parenchyma cell of Arabidopsis transgenic plants overexpressing GDHB from N. plumbaginifolia. (H) A CC and neighbouring phloem parenchyma cell in a floral stem of Arabidopsis transgenic plants overexpressing GDHB from N. plumbaginifolia. (I) Detail of a CC in a floral stem of Arabidopsis transgenic plants overexpressing GDHB from N. plumbaginifolia. CC, companion cells; Cy, cytosol; m, mitochondria; P, plastids; PP, phloem parenchyma cell; S, starch granule; V, vacuole. A, bar = 5 µm and B–I, bars = 1 µm. View largeDownload slide Fig. 7 Immunolocalization of GDH in Arabidopsis GDH-deficient mutants and GDH overexpressors. (A) Partial view of a basal floral stem section of wild-type (WT) plants. (B) Detail of a floral stem companion cell (CC) of WT plants. (C) A CC in a floral stem of the gdh1 mutant. (D) A CC in a floral stem of the gdh2 mutant. (E) A mesophyll cell of a mature leaf of WT plants. (F) A mesophyll parenchyma cell of Arabidopsis transgenic plants overexpressing GDHB from N. plumbaginifolia. (G) Detail of a mesophyll parenchyma cell of Arabidopsis transgenic plants overexpressing GDHB from N. plumbaginifolia. (H) A CC and neighbouring phloem parenchyma cell in a floral stem of Arabidopsis transgenic plants overexpressing GDHB from N. plumbaginifolia. (I) Detail of a CC in a floral stem of Arabidopsis transgenic plants overexpressing GDHB from N. plumbaginifolia. CC, companion cells; Cy, cytosol; m, mitochondria; P, plastids; PP, phloem parenchyma cell; S, starch granule; V, vacuole. A, bar = 5 µm and B–I, bars = 1 µm. Table 1 NADH-GDH activity in N. plumbaginifolia GDH antisense plants and in A. thaliana GDH-deficient mutants and GDH overexpressors   Stems  Leaves  Stems  Leaves  Stems  Leaves  N. plumbaginifolia antisense                WT    B56    A1      84.4 ± 7.7  106.2 ± 13.5  29.4 ± 3.0  11.5 ± 2.4  37.5 ± 11.6  51.3 ± 11.6  A. thaliana mutants                WT    gdh1    gdh2      84.6 ± 11.1  39.5 ± 6.3  113.1 ± 17.6  11.8 ± 1.6  56.2 ± 12.2  47.2 ± 10.3  A. thaliana overexpressors                    B7-4    B6-5          125.1 ± 12.2  113.7 ± 45.2  87.5 ± 17.9  79.2 ± 2.5    Stems  Leaves  Stems  Leaves  Stems  Leaves  N. plumbaginifolia antisense                WT    B56    A1      84.4 ± 7.7  106.2 ± 13.5  29.4 ± 3.0  11.5 ± 2.4  37.5 ± 11.6  51.3 ± 11.6  A. thaliana mutants                WT    gdh1    gdh2      84.6 ± 11.1  39.5 ± 6.3  113.1 ± 17.6  11.8 ± 1.6  56.2 ± 12.2  47.2 ± 10.3  A. thaliana overexpressors                    B7-4    B6-5          125.1 ± 12.2  113.7 ± 45.2  87.5 ± 17.9  79.2 ± 2.5  NADH-GDH activity is expressed in nmol min–1 mg protein–1. Values are the means ± SD of three independent experiments. In each experiment, GDH activity was measured on three individual plants. View Large Table 2 Quantification of GDH protein in different tissue sections of N. plumbaginifolia and Arabidopsis   No. of gold particles µm–2 in the mitochondria        Leaf parenchyma cell  Stem CCs  Leaf CCs  N. plumbaginifolia WT  3.8 ± 3.5  200 ± 35  180 ± 40  N. plumbaginifolia 35Sas-NpGDHB  5.0 ± 2.0  25 ± 6*  30 ± 6*  N. plumbaginifolia 35Sas-NpGDHA  3.5 ± 2.0  145 ± 33*  120 ± 45*  Arabidopsis WT  4.5 ± 2.0  193 ± 23  130 ± 20  Arabidopsis gdh1 mutant  5.0 ± 3.0  92 ± 11*  60 ± 15*  Arabidopsisgdh2 mutant  4.0 ± 2.0  42 ± 5*  100 ± 22*  Arabidopsis 35S-NpGDHB  160 ± 20*  280 ± 45*  215 ± 36*    No. of gold particles µm–2 in the mitochondria        Leaf parenchyma cell  Stem CCs  Leaf CCs  N. plumbaginifolia WT  3.8 ± 3.5  200 ± 35  180 ± 40  N. plumbaginifolia 35Sas-NpGDHB  5.0 ± 2.0  25 ± 6*  30 ± 6*  N. plumbaginifolia 35Sas-NpGDHA  3.5 ± 2.0  145 ± 33*  120 ± 45*  Arabidopsis WT  4.5 ± 2.0  193 ± 23  130 ± 20  Arabidopsis gdh1 mutant  5.0 ± 3.0  92 ± 11*  60 ± 15*  Arabidopsisgdh2 mutant  4.0 ± 2.0  42 ± 5*  100 ± 22*  Arabidopsis 35S-NpGDHB  160 ± 20*  280 ± 45*  215 ± 36*  Immunolocalization of the enzyme was performed using transmission electron microscopy. Values are the mean ± SD of gold particles counted on 5–10 different sections. *Significant difference from the corresponding N.plumbaginifolia or Arabidopsis WT (0.05 probality level). View Large Abbreviations BSA bovine serum albumin CaMV cauliflower mosaic virus C carbon CC companion cell GDH glutamate dehydrogenase N nitrogen WT wild type References Chaffei, C., Pageau, K., Suzuki, A., Gouia, H., Ghorbel, M.H. and Masclaux-Daubresse, C. ( 2004) Cadmium toxicity induces changes in nitrogen management in Lycopersicon esculentum leading to metabolic safeguard through an amino acid storage strategy. Plant Cell Environ.  45: 1681–1693. Google Scholar Clough, S.J. and Bent, A.F. ( 1998) Floral dip: a simplified method for Agrobacterium- mediated transformation of Arabidposis thaliana. Plant J.  16: 735–743. Google Scholar Dubois, F., Tercé-Laforgue, T., Gonzalez-Moro, M.B., Estavillo, M.B., Sangwan, R., Gallais, A. and Hirel, B. ( 2003) Glutamate dehydrogenase in plants: is there a new story for an old enzyme? Plant Physiol. Biochem.  41: 565–576. Google Scholar Ficarelli, A., Tassi, F. and Restivo, F.M. ( 1999) Isolation and characterization of two cDNA clones encoding for glutamate dehydrogenase in Nicotiana plumbaginifolia. Plant Cell Physiol.  40: 339–342. Google Scholar Fox, G.G., Ratcliffe, R.G., Robinson, S.A. and Stewart, G.R. ( 1995) Evidence for deamination by glutamate dehydrogenase in higher plants: commentary. Can. J. Bot.  73: 1112–1115. Google Scholar Horsch, R.B., Fry, J.E., Hoffmann, N.L., Eichholtz, D., Rogers, S.G., and Fraley, R.T. ( 1985) A Simple and General Method for Transferring Genes into Plants. Science  227: 1229–1231. Google Scholar Kichey, T., Le Gouis, J., Hirel, B. and Dubois, F. ( 2005) Evolution of the cellular and subcellular localization of glutamine synthetase and glutamate dehydrogenase during flag leaf senescence in wheat (Triticum aestivum L.). Plant Cell Physiol.  46: 964–974. Google Scholar Laemmli, U.K. ( 1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature  227: 680–685. Google Scholar Loulakakis, K.A. and Roubelakis-Angelakis, K.A. ( 1992) Ammonium-induced increase in NADH-glutamate dehydrogenase activity is caused by de-novo synthesis of the α-subunit. Planta  187: 322–327. Google Scholar Loulakakis, K.A. and Roubelakis-Angelakis, K.A. ( 1996) The seven NAD(H)-glutamate dehydrogenase isoenzymes exhibit similar anabolic and catabolic activities. Physiol. Plant.  96: 29–35. Google Scholar Loulakakis, K.A. and Roubelakis-Angelakis, K.A. ( 2001) Ammonium assimilating genes in Vitis vinifera L. In Molecular Biology and Biotechnology of the Grapevine. Edited by Roubelakis-Angelakis, K.A. pp. 59–108 Kluwer Academic Publishers, The Netherlands. Google Scholar Melo-Oliveira, R., Cinha-Oliveria, I. and Coruzzi, G.M. ( 1996) Arabidopsis mutant analysis and gene regulation define a non-redundant role for glutamate dehydrogenase in nitrogen assimilation. Proc. Natl Acad. Sci. USA  96: 4718–4723. Google Scholar Murashige, J. and Skoog, F. ( 1962) A revised medium for rapid growth and bio assay with tobacco tissue culture. Physiol. Plant.  115: 473–497. Google Scholar Oaks, A. ( 1995) Evidence for deamination by glutamate dehydrogenase in higher plants: reply. Can. J. Bot.  73: 1116–1117. Google Scholar Paczek, V., Dubois, F., Sangwan, R., Morot-Gaudry, J.F., Roubelakis-Angelakis, K.A. and Hirel, B. ( 2002) Cellular and subcellular localisation of glutamine synthetase and glutamate dehydrogenase in grapes give new insights on the regulation of C and N metabolism. Planta  216: 245–254. Google Scholar Pahlich, E. ( 1996) Remarks concerning the dispute related to the function of plant glutamate dehydrogenase: commentary. Can. J. Bot.  74: 512–515. Google Scholar Pavesi, A., Ficarelli, A., Tassi, F. and Restivo, F.M. ( 2000) Cloning of two glutamate dehydrogenase cDNAs from Asparagus officinalis: sequenze analysis and evolutionary implications. Genome  4: 306–316 Google Scholar Purnell, M.P., Skopelitis, D.S., Roubelakis-Angelakis, K.A. and Botella, J.R. ( 2005) Modulation of higher-plant NAD(H)-dependent glutamate dehydrogenase activity in transgenic tobacco via alteration of beta subunit levels. Planta  222: 167–180. Google Scholar Restivo, F.M. ( 2004) Molecular cloning of glutamate dehydrogenase genes of Nicotiana plumbaginifolia: structure and regulation of their expression by physiological and stress conditions. Plant Sci.  166: 971–982. Google Scholar Robinson, S.A., Stewart, G.R. and Phillips, R. ( 1992) Regulation of glutamate dehydrogenase activity in relation to carbon limitation and protein catabolism in carrot cell suspension cultures. Plant Physiol.  98: 1190–1195. Google Scholar Stewart, G.R., Shatilov, V.R., Turnbull, M.H., Robinson, S.A. and Goodall, R. ( 1995) Evidence that glutamate dehydrogenase plays a role in oxidative deamination of glutamate in seedlings of Zea mays. Aust. J. Plant Physiol.  22: 805–809. Google Scholar Tercé-Laforgue, T., Dubois, F., Ferrario-Mery, S., Pou de Crecenzo, M.A., Sangwan, R. and Hirel, B. ( 2004) Glutamate dehydrogenase of tobacco (Nicotiana tabacum L.) is mainly induced in the cytosol of phloem companion cells when ammonia is provided either externally or released during photorespiration. Plant Physiol.  136: 4308–4317. Google Scholar Turano, F.J., Dashner, R., Upadhyaya, A. and Caldwell, C.R. ( 1996) Purification of mitochondrial glutamate dehydrogenase from dark-grown soybean seedlings. Plant Physiol.  112: 1357–1364. Google Scholar Turano, F.J., Thakkar, S.S., Fang, T. and Weisemann, J.M. ( 1997) Characterisation and expression of NAD(H) dependent glutamate dehydrogenase genes in Arabidopsis. Plant Physiol.  113: 1329–1341. Google Scholar Walkerpeach, C.R. and Velten, J. ( 1994) Agrobacterium-mediated gene transfer to plant cells: cointegrate and binary vectors. In Plant Molecular Biology Manual, 2nd edn. Edited by Gelvin, S.B. and Schilperoort, R.A. pp. B1/11–B1/12. Kluwer Academic Publishers, Dordrecht, The Netherlands. Google Scholar Yamaya, T. and Oaks, A. ( 1987) Synthesis of glutamate by mitochondria—an anaplerotic function for glutamate dehydrogenase. Physiol. Plant.  70: 749–756. Google Scholar
TI - Control of the Synthesis and Subcellular Targeting of the Two GDH Genes Products in Leaves and Stems of Nicotiana plumbaginifolia and Arabidopsis thaliana
JO - Plant and Cell Physiology
DO - 10.1093/pcp/pcj008
DA - 2006-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/control-of-the-synthesis-and-subcellular-targeting-of-the-two-gdh-ANDEpLg509
SP - 410
EP - 418
VL - 47
IS - 3
DP - DeepDyve
ER -