Planta DOI 10.1007/s00425-014-2214-z

ORIGINAL ARTICLE

Over-expression of a glutamate dehydrogenase gene, MgGDH, from Magnaporthe grisea confers tolerance to dehydration stress in transgenic rice Yanbiao Zhou • Caisheng Zhang • Jianzhong Lin • Yuanzhu Yang • Yuchong Peng • Dongying Tang • Xiaoying Zhao • Yonghua Zhu • Xuanming Liu

Received: 24 September 2014 / Accepted: 24 November 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Main conclusion Heterologous expression of a fungal NADP(H)-GDH gene (MgGDH) from Magnaporthe grisea can improve dehydration stress tolerance in rice by preventing toxic accumulation of ammonium. Glutamate dehydrogenase (GDH; EC 1.4.1.2 and EC 1.4.1.4) may act as a stress-responsive enzyme in detoxification of high intracellular ammonia and production of glutamate for proline synthesis under stress conditions. In present study, a fungal NADP(H)-GDH gene (MgGDH) from Magnaporthe grisea was over-expressed in rice (Oryza sativa L. cv. ‘kitaake’), and the transgenic plants showed the improvement of tolerance to dehydration stress. The kinetic analysis showed that His-TF-MgGDH preferentially utilizes ammonium to produce L-glutamate. Moreover, the affinity of His-TF-MgGDH for ammonium Y. Zhou, C. Zhang and J. Lin contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s00425-014-2214-z) contains supplementary material, which is available to authorized users. Y. Zhou  C. Zhang  J. Lin  Y. Peng  D. Tang  X. Zhao  Y. Zhu  X. Liu (&) Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, Hunan University, Changsha 410082, China e-mail: [email protected] Y. Zhou  C. Zhang  J. Lin  Y. Peng  D. Tang  X. Zhao  Y. Zhu  X. Liu State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha 410082, China Y. Yang Academy of Seed Industry of Hunan Yahua, Changsha 410001, Hunan, China

was dramatically higher than that of His-TF-OsGDH for ammonium. Over-expressing MgGDH transgenic rice plants showed lower water-loss rate and higher completely close stomata than the wild-type plants under dehydration stress conditions. In transgenic plants, the NADP(H)-GDH activities were markedly higher than those in wild-type plants and the amination activity was significantly higher than the deamination activity. Compared with wild-type plants, the transgenic plants accumulated much less NH4? but higher amounts of glutamate, proline and soluble sugar under dehydration stress conditions. These results indicate that heterologous expression of MgGDH can prevent toxic accumulation of ammonium and in return improve dehydration stress tolerance in rice. Keywords Rice  Magnaporthe grisea  NADP(H)dependent glutamate dehydrogenase  Dehydration stress Abbreviations Mg Magnaporthe grisea GDH Glutamate dehydrogenase GOGAT Glutamate synthase GS Glutamine synthetase 2-OG 2-oxoglutarate TF Trigger factor P5CS 41-Pyrroline-5-carboxylate synthetase P5CR 41-Pyrroline-5-carboxylic acid reductase GSA Glutamic c-semialdehyde PCR Polymerase chain reaction

Introduction Plants are subjected to abiotic stresses such as drought and salinity during their life cycles (Xiang et al. 2007). The

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stresses can negatively affect plant growth, development and productivity. In response to environmental cues, a variety of physiological and biochemical mechanisms have evolved in plants to adapt to adverse conditions during their growth and development (Boyer 1982). Under stress conditions, enhanced proteolytic activity results in increased intracellular hyperammonia and toxicity, if ammonia is not removed efficiently (Lutts et al. 1999). Assimilation of ammonium into amino acids is performed mainly through the glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (GOGAT; EC1.4.7.1 and EC 1.4.1.14) pathway (Lea and Miflin 1974). In addition, ammonium ions are incorporated into glutamate by glutamate dehydrogenase (GDH; EC 1.4.1.2 and EC 1.4.1.4) in the presence of NADP(H) as a cofactor (Wootton 1983). Since the discovery of the GS/GOGAT pathway in plants in the 1970s, the role of GDH in ammonium assimilation remains controversial. However, in microorganisms, such as bacteria and fungi, NADP(H)GDH and GS both play important roles in ammonium assimilation (Kinghorn and Pateman 1973; Kanamori et al. 1987). GDH may act as a stress-responsive enzyme (Skopelitis et al. 2006) and play a complementary role to the usual GS/GOGAT pathway in the re-assimilation of excess ammonia released during stress (Srivastava and Singh 1987) or intracellular hyperammonia conditions (Skopelitis et al. 2006). The capacity to assimilate ammonium may be an important factor in the alleviation of consequence of stress, because ammonium can be toxic at high concentrations (Hoai et al. 2003). Therefore, the introduction of NADP(H)-GDH from microorganisms could improve plant tolerance under stress conditions. For instance, over-expression of a bacterial gdh (the a-subunit of GDH) increased tolerance to toxic amount of ammonia and to herbicides and produced higher biomass in tobacco (Ameziane et al. 2000). Similarly, Nolte et al. (2004) found that the introduction of an Escherichia coli gdhA gene encoding NADPH-dependent GDH into tobacco increased the resistance to glufosinate. In Zea mays, over-expression of the glutamate dehydrogenase gene (gdhA) of E. coli improved drought tolerance (Lightfoot et al. 2007). More recently, Du et al. (2014) found that, although grain yields decreased dramatically, over-expression of SsGDH isolated from the fungus Sclerotinia sclerotiorum significantly enhanced the tolerance to herbicide in rice. Although GDH mediates the amination of 2-oxoglutarate (2-OG) and the deamination of glutamate, its amination activity role is more prominent under adverse environmental conditions (Robinson et al. 1991; Gulati and Jaiwal 1996). In senescing leaves, GS and GOGAT decreased while GDH protein accumulated and the in vitro amination activity of GDH increased in tissues of senescing leaves (Masclaux et al. 2000; Loulakakis et al. 2002; Terce´-

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Laforgue et al. 2004a, b). The amination activity of GDH in salt-sensitive rice cultivars was less compared with salt tolerance ones in the face of increased salinity concentration (Kumar et al. 2000). Similar results were found in ammonium-tolerant pea (Pisum sativum) plants, where the amination activity of GDH was high in roots (Lasa et al. 2002). From these studies, it appears that the amination activity of GDH increases in plant cells deals with stress and detoxifies the high intracellular ammonia concentrations generated by proteolytic activities (Skopelitis et al. 2006). The results suggest that the amination activity of GDH might play an important role in the improvement of plant tolerance to environmental stress. Proline biosynthesis occupies a critical intersection between carbon and nitrogen assimilation pathways (Lutts et al. 1999). Glutamate is the main precursor of proline in the biosynthetic pathway mediated by 41-pyrroline-5-carboxylate synthetase (P5CS; EC 1.5.1.12) and 41-pyrroline-5carboxylic acid reductase (P5CR; EC 1.5.1.2; Yoshiba et al. 1997). Glutamate is reduced to glutamic c-semialdehyde (GSA) catalyzed by P5CS, and GSA is non-enzymatically converted into 41-pyrroline-5-carboxylic acid (P5C), which is reduced to proline by P5CR (Lutts et al. 1999). Under stress conditions, GDH catalyzes the amination of 2-OG to form glutamate, which drives proline synthesis (Skopelitis et al. 2006). Proline accumulation plays a critical protective role in plants that are subjected to abiotic stress, conferred osmotic adjustment (Valliyodan and Nguyen 2006). Overexpression of the P5CS gene in tobacco increased proline production and improved tolerance to osmotic stress (Kishor et al. 1995). Additionally, P5CR-suppressed transgenic soybean plants showed suppressed proline synthesis and increased sensitivity to water deficit (de Ronde et al. 2000). In this study, a fungal NADP(H)-GDH gene (MgGDH) was isolated from Magnaporthe grisea for ectopic expression in the cytosol of rice plants. The NADP(H)-GDH activities of transgenic plants were significantly higher than those of wild-type plants. Moreover, the amination activity was markedly higher than the deamination activity in shoots of transgenic plants. Compared with wild-type plants, MgGDH transgenic plants reduced intracellular toxic amount of ammonium and increased the concentration of glutamate and proline under dehydration stress conditions. The results demonstrate that the heterologous expression of MgGDH can improve the tolerance to dehydration in rice.

Materials and methods Construction and transformation of MgGDH To generate the MgGDH over-expression constructs, the full-length cDNA of MgGDH was obtained from the

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Magnaporthe grisea (from Fujian Agriculture and Forestry University) by reverse transcription (RT)-PCR with the primers 50 -ATGACTGTCCTCCCCCTCGAG-30 (forward) and 50 -CCCCACCAGTCACCCTGGGC-30 (reverse). The PCR product was cloned into a GATEWAY donor vector pGWC, according to the method described by Chen et al. (2006) and sequenced. To achieve better translation in rice, the code of MgGDH was optimized (Supplementary Fig. S1), according to the Codon Usage Database (http://www. kazusa.or.jp/codon/). Overlap PCR was used to generate the point mutations for codon optimization, and the primers are shown in the Supplementary Table S1. To enhance the translation level of eukaryotic genes in the transgenic rice plants, a Kozak sequence (GCCACC) was added before the initiation codon of MgGDH as described by Li et al. (2006). The optimized sequence of MgGDH was introduced in the destination vector pCAMBIA1301GW (under the control of the ubiquitin promoter) by LR reaction following the manufacturer’s instructions (Invitrogen, Carlsbad, USA). The pCAMBIA1301GW vector, a modified pCAMBIA1301 vector (Ohta et al. 1990), contained the HPT gene for hygromycin selection and AsRed gene for red fluorescence selection in plants (Fig. 2a). The gateway cassette sequence was also added to the multiple cloning sites of pCAMBIA1301GW vector. The construct was introduced into rice (Oryza sativa L. cv. ‘kitaake’; from Hokkaido of Japan) via Agrobacterium-mediated transformation as described by Toki et al. (2006). Plant material and stress treatment Rice seeds were germinated following the method of Lin et al. (2009). Seedlings of rice were grown in the greenhouse with light provided at a photon flux density of 300–350 lmol m-2 s-1 and with a 16 h light/8 h dark cycle at 28 °C. The relative humidity in the greenhouse was maintained at 60–70 %. The T2 generation of transgenic rice plants was used to investigate the stress response. For polyethylene glycol (PEG) treatment, 15-day-old seedlings including transgenic and wild-type plants grown in culture dish under normal conditions were treated with 1/2 MS (Murashige and Skoog 1962) solution contained 20 % (w/v) PEG4000 for 15 days. After 15 days of treatment, the plants were subjected to regular 1/2 MS for recovery. For drought stress, each chamber contained the same weight of soil. After growing for 3 weeks under normal conditions, the chamber-grown plants were subjected to drought conditions by withholding water for 9 days before re-watering. Each data point represents the average of three replicates. Each stress experiment was repeated at least three times, and the results were consistent. The result from one set of experiments is presented here.

Expression and purification of the His-TF-MgGDH and His-TF-OsGDH proteins and enzyme assays in vitro The cDNA fragments encoding the MgGDH and OsGDH proteins were amplified by PCR from the optimized pGWC-MgGDH vector and the rice (Oryza sativa L. cv. ‘kitaake’) cDNA, respectively, and then cloned into the pCold-TF vector (TaKaRa, Shiga, Japan) with a Trigger Factor (TF) and a His-tag sequence in 50 -end. The fusion proteins were induced and expressed in E. coli BL21 (DE3). His-TF-OsGDH and His-TF-MgGDH fusion proteins were affinity purified using a nickel–nitrilotriacetic acid (Ni–NTA) resin (Invitrogen, Carlsbad, USA), according to the manufacturer’s instructions. The His-TF protein itself was also expressed, so it could be used as a control. Both purified His-TF-OsGDH and His-TFMgGDH fusion proteins were assayed for enzymatic activity in vitro. The NADP(H)-GDH activities of His-TFOsGDH and His-TF-MgGDH were determined as described previously (Zhou et al. 2014b). One unit of enzyme activity is defined as the reduction or oxidation of 1 lM of NADP(H) per minute per microgram His-TF-OsGDH and His-TF-MgGDH at 25 °C. Soluble proteins were extracted from the shoots and roots of seedlings as described previously (Abiko et al. 2005), and their NADP(H)-GDH activities were determined according to the method of Abiko et al. (2010). The soluble protein of shoots of transgenic and wild-type plants was examined by western blotting using an anti-Flag monoclonal antibody (Abmart, Shanghai, China). For each enzyme activity, the data points are the average of three replicates. Three experiments were performed, with consistent results. The results from one set of experiments are presented here. Subcellular localization assays For detection of the subcellular localization of MgGDH, the coding region of MgGDH was cloned into the PA7YFP vector, in which the YFP-coding sequence was fused in frame to the 30 -end of MgGDH gene sequence. The construct was transformed into Arabidopsis (Arabidopsis thaliana) protoplasts by the PEG-mediated transformation method as previously described (Yoo et al. 2007). Localization of the YFP-tagged MgGDH protein was visualized in protoplasts by confocal laser scanning microscopy (Olympus FV1000, Olympus, Tokyo, Japan). Water-loss rate assay The water-loss rate from detached leaves was measured according to the method of Zhang et al. (2012). The

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second fully expanded leaves were excised and exposed to air at room temperature (approximately 25 °C), and the weight was determined every 1 h. The rate of water loss was calculated as the weight of water lost divided by the initial leaf weight. Each data point represents the average of three replicates and the experiments were repeated twice. The results from the repeated experiments were consistent, and those from one set of experiments were shown. Scanning electron microscopy Fragments of leaves from wild-type to transgenic plants were fixed in 2.5 % (v/v) glutaraldehyde in a phosphate buffer at 4 °C for 4 h, rinsed and incubated overnight in 1 % (w/v) OsO4 at 4 °C. Then, the samples were dehydrated serially in 30, 50, 70, 85, 95 and 100 % (v/v) ethanol solutions, and subjected to critical-point drying (Cao et al. 2007). Finally, the materials were coated with gold for the observation of guard cells by scanning electron microscopy (SEM). Two observation experiments were performed and in each experiment, the numbers of different types of stomata were calculated from the same field of one leaf segment, and three corresponding leaf segments from three different leaves were observed. The result from one set of experiments is presented here. Real-time PCR analysis For real-time PCR analysis, total RNA was isolated from rice leaves using TRIzol reagent (Invitrogen, Carlsbad, USA). First-stand cDNAs were synthesized from RNasefree DNase I-treated (TaKaRa, Shiga, Japan) total RNA using SuperScriptTM III reverse transcriptase (Invitrogen, Carlsbad, USA) following the manufacturer’s instruction. Real-time PCR was performed with Mx3000 Real-time PCR System (Stratagene, Amsterdam, The Netherlands) using SYBR green (TaKaRa, Shiga, Japan) to monitor dsDNA synthesis, according to the manufacturer’ s instruction. The primers used for real-time PCR analysis were as follows (in parentheses): J033099M14 (50 -AGATT GGCTTTGGGCAGGGTT-30 and 50 -GTTAAACAACAT ATCGTAAAGAGCC-30 ), J033031H21 (50 -GGTGCTGTT GGTGTTGGGAGG-30 and 50 -CGTCAAGCTGGCTAAA TAACGTGTC-30 ), 03g44230 (50 -TTTAGGCTGGATTGG TGGGACA-30 and 50 -GGTAGCTTTAGAACTCCATCGT CCC-30 ), 07g01090 (50 -CTCATCTCCGAGGATACAGC G-30 and 50 -CGTGAAGGCGAGCAAGGAG-30 ), OsLEA3 (50 -GCAGCGTCCTCCAACAGGC-30 and 50 -TGGCAGA GGTGTCCTTGTTGG-30 ), OsRab16A (50 -GCTCGTCTG AGGATGATGGAATGG-30 and 50 -CTGCTGCTCGCCC TTGTTGC-30 ). Rice Actin gene was used as internal

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control with primers (50 -ACCCAAGAATGCTAAGCCAA GAG-30 and 50 -ACTTTGTCCACGCTAATGAAGAAAC30 ). The relative expression levels were measured as described previously (Livak and Schmittgen 2001). Each data point represents the average of three replicates. Three experiments were performed with consistent results. The result from one set of experiments is presented here.

Measurement of ammonium, glutamate, proline and sugar contents For ammonium content assay, the shoots and roots of transgenic and wild-type plants were ground in liquid nitrogen, respectively, and extracted in 10 volumes of 10 mM ice-cold formic acid. The ammonium concentration was measured with the use of Nessler’s reagent according to Krug et al. (1979). The concentration of glutamate in the shoots and roots of transgenic and wild-type plants was determined using commercial kits (NJJCBIO, Nanjing, China) as described previously (Zhou et al. 2014b). Briefly, leaves (approximately 0.1 g FW) were homogenized in 0.9 mL plant protein extraction reagent (CWBIO, Beijing, China) for 20 min on ice. Then, the homogenates were centrifuged at 13,500 g for 20 min. The supernatants were collected and determined the concentration of glutamate according to instructions of the manufacturer. Free proline content in shoots was measured using the protocol described previously (Ye et al. 2009). Briefly, about 50 mg shoots of transgenic and wild-type plants were, respectively, homogenized in 1 mL 3 % (w/v) aqueous sulfosalicylic acid and the homogeneous mixture was centrifuged at 15,800g for 15 min at 4 °C. About 200 lL supernatant was mixed with 200 lL acid ninhydrin (0.1 g ninhydrin dissolved in 2.4 mL glacial acetic acid and 1.6 mL 6-ortho-phosphoric acid) and 200 lL acetic acid in a new microcentrifuge tube. The reaction mixture was placed in a boiling water bath for 30 min and cooled to 4 °C for 30 min. Then, the solutions were extracted with 400 lL toluene by vigorous shaking. The toluene phase was transferred to a colorimetric tube for absorbance measurement at 520 nm with a spectrophotometer (SHIMADZU, Kyoto, Japan). Total soluble sugars in shoots were determined according to Xiang et al. (2007) with minor modification. About 0.08 g leaves were homogenized in 6.4 mL double-distilled water, then the mixture was boiled twice in a water bath at 100 °C for 30 min. The extract (approximately 400 lL) was removed to a new microcentrifuge tube which added with 1.2 mL double-distilled water, 0.8 mL 9 % (v/ v) phenol and 4 mL sulfuric acid. The reaction mixture was kept at room temperature for 30 min, the absorbance was

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then determined at 485 nm with a spectrophotometer (SHIMADZU, Kyoto, Japan). For the above parameters, each data point represents the average of three replicates. Two experiments were performed, and the results are consistent. The result from one set of experiments is presented here.

Results Analysis of the recombinant His-TF-MgGDH and HisTF-OsGDH protein for kinetic properties To investigate the kinetic properties of MgGDH and OsGDH, the proteins were expressed as His-TF-MgGDH and His-TF-OsGDH fusions in E. coli BL21 (DE3) and purified, respectively. The recombinant proteins His-TFMgGDH and His-TF-OsGDH, purified to near homogeneity, were identified as a 106.5 and 126.1 kDa band, respectively; while the His-tagged Trigger Factor chaperone protein (His-TF) as a control gave a 55 kDa band (Fig. 1a, b). In vitro NADP(H)-GDH activities were determined using purified His-TF-MgGDH and His-TFOsGDH. The NADP(H)-GDH activities of His-TFMgGDH were significantly higher than those of His-TFOsGDH (Fig. 1c). Furthermore, the amination activities of both His-TF-MgGDH and His-TF-OsGDH were markedly higher (t test, P \ 0.01) than their deamination activities (Fig. 1c). The purified His-TF-MgGDH was assayed for its ability to use ammonium, 2-oxoglutarate (2-OG), NADPH, glutamate and NADP? as substrates (Table 1). It is noteworthy that the affinity of His-TF-MgGDH for ammonium (Km = 0.67 ± 0.05 mM) was substantially higher than that for glutamate (Km = 22.3 ± 0.81 mM). The Vmax value of His-TF-MgGDH for ammonium was 0.038 ± 0.002 mM min-1 lg soluble protein-1, whereas that for glutamate was 0.012 ± 0.0006 mM min-1 lg soluble protein-1. The results indicate that His-TFMgGDH tends to utilize ammonium to produce L-glutamate. Subsequently, the Km value of His-TF-OsGDH for ammonium was also determined. The Km value of HisTF-MgGDH for ammonium was dramatically lower (t test, P \ 0.01) than that of His-TF-OsGDH for ammonium (Fig. 1d), indicating that the capacity for ammonium assimilation of His-TF-MgGDH was higher than that of His-TF-OsGDH. Furthermore, the Km value of MgGDH (0.67 ± 0.05 mM) is lower than those of previous reported fungal gdhA (1.16 ± 0.13 mM; Abiko et al. 2010) and PcGDH (3.73 ± 0.23 mM; Zhou et al. 2014b), previously shown to improve ammonium assimilation in transgenic rice, indicating that MgGDH has a higher capacity to assimilate ammonium than gdhA and PcGDH.

Characteristics of transgenic rice plants over-expressing MgGDH To test the biological functions of MgGDH in plants, an expression vector which carried the MgGDH full-length gene under the control of ubiquitin promoter was constructed and transformed into rice (Oryza sativa L. cv. ‘kitaake’; Fig. 2a). The MgGDH transcript level was measured in shoots of wild-type and transgenic plants by RT-PCR. As expected, the exogenous MgGDH was overexpressed in shoots of transgenic plants but not in the wildtype plants (Fig. 2b). Meanwhile, the relative abundance of MgGDH protein was determined by western blot with an anti-Flag antibody. The results showed that MgGDH-Flag fusion protein had accumulated in transgenic plants but not in the wild-type plants (Fig. 2c), indicating that MgGDH was heterologously expressed in transgenic rice successfully. To confirm the subcellular localization of MgGDH, the YFP-tagged MgGDH construct under the control of the CaMV 35S promoter was made and expressed transiently in Arabidopsis protoplasts. Confocal laser scanning microscopy showed that YFP-MgGDH was mostly distributed in the cytoplasm, while in the control, the YFP signal was located primarily in the nucleus, plasma membrane, and cytoplasm (Fig. 2d), which indicated that MgGDH is a cytoplasm-localized protein. In agreement with this observation, it was recently reported that fungal GDH protein gdhA (Abiko et al. 2010), CeGDH (Zhou et al. 2014a) and PcGDH (Zhou et al. 2014b) were also cytoplasm-localized. Over-expression of MgGDH gene in rice enhanced the tolerance to dehydration stress Under stress conditions, increased proteolytic activity gives rise to increased intracellular hyperammonia, which is toxic if not eliminated efficiently (Lutts et al. 1999). Because MgGDH has a high affinity for ammonium (Table 1), it may ameliorate the toxicity of ammonia in transgenic rice plants under such stress. To test this hypothesis, wild-type and transgenic plants were subjected to salt stress (200 mM NaCl) and dehydration stress [20 % (v/v) PEG4000]. Although no significant effect on improved tolerance to salt stress was detected (data not shown), obvious improvements of tolerance to dehydration stress were detected in transgenic plants (Fig. 3a). Under normal growth conditions, all plants grew normally and no significant differences were observed (Fig. 3a). Whereas, when these plants were treated with 20 % (v/v) PEG for 15 days, the transgenic plants exhibited less severe wilting than wild-type plants (Fig. 3a). Subsequently, the plants treated with 20 % (v/v) PEG were transplanted in normal 1/2 MS nutrient solution (Murashige and Skoog 2006) for

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Fig. 1 Protein purification and kinetic analysis in vitro of MgGDH. a Expression and purification of His-TF-OsGDH from E. coli by affinity resin. b Expression and purification of His-TF-MgGDH from E. coli by affinity resin. c NADP(H)-GDH activities of purified His-

TF-OsGDH and His-TF-MgGDH. d The Km value for ammonium of purified His-TF-OsGDH and His-TF-MgGDH. Data are presented as mean values ± SD (n = 3). *P B 0.05; **P B 0.01, Student’s t test

Table 1 Kinetic property of recombinant His-TF-MgGDH protein

transgenic plants compared with wild-type plants (Fig. 3c). After recovery for 3 days, the survival rates of transgenic plants reached 81–86 %, whereas the survival rate of the wild-type plants was only 5 % (Fig. 3d). The results demonstrate that over-expression of MgGDH in rice plants improved the tolerance to dehydration stress at the seedling stage. To further investigate the effects of exogenous MgGDH on the tolerance to dehydration stress in transgenic rice plants, the water status in leaves from wild-type and transgenic plants were determined. Under dehydration stress conditions, the leaves of transgenic plants had significantly lower water-loss rates (t test, P \ 0.05) compared with wild-type plants (Fig. 3e). The result suggests that the enhanced dehydration stress tolerance in transgenic plants might result from an increased ability to retain water. Stomata are known to play an important role in responses to abiotic stresses (Hetherington and Woodward 2003), so the stomatal apertures of transgenic and wildtype plants were also examined. The 3-week-old transgenic and wild-type plants were subjected to drought stress for

Substrate NH4?

Km (mM) 0.67 ± 0.05

2-oxoglutarate

1.08 ± 0.11

NADPH

0.18 ± 0.02

Glutamate NADP?

22.3 ± 0.81

-1

Vmax (mM min protein-1)

lg soluble

0.038 ± 0.002

0.012 ± 0.0006

0.023 ± 0.002

The calculation was accomplished by using the Lineweaver–Burk double-reciprocal protocol. Values represent the means of three independent experiments. Data are presented as average values ± SD (n = 3)

recovery. After recovery for 5 days, the survival rates of transgenic plants reached 61–65 %, whereas the survival rate of the wild-type plants was only 13 % (Fig. 3b). To further accurately evaluate the effects of transgenic plants on dehydration stress tolerance, a soil drought experiment was performed in deep polyvinylchloride (PVC) pipes. The results showed that leaf rolling was substantially delayed in

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Fig. 2 Characteristics of MgGDH transgenic rice plants. a Schematic representation of the over-expression vector pCAMBIA1301GWMgGDH. RB Right border, Tnos terminator of nopaline synthase gene (nos), HPT hygromycin resistance gene, 35S CaMV 35S promoter, FLAG FLAG-tag sequence, MgGDH NADP(H)-GDH gene derived from Magnaporthe grisea, Ubi ubiquitin promoter derived from Zea mays, AsRed red fluorescence protein gene, LB left border. b Expression of MgGDH in transgenic and wild-type plants, as determined by reverse transcription-PCR. The ACTIN gene was amplified as a control. c Western blot analysis MgGDH of transgenic and wild-type plants using Flag-tag antibody. d Subcellular localization of MgGDH protein

4 days before they were used for stomatal analysis. The results showed that 41.9 and 43.6 % of stomata were completely closed in Ubi::MgGDH-13 and Ubi::MgGDH14 transgenic lines, respectively, but only 20.1 % were completely closed in wild-type plants. On the other hand, only 19.3 and 16.8 % of stomata were completely opened in Ubi::MgGDH-13 and Ubi::MgGDH-14, respectively, but 43.2 % were completely opened in wild-type plants. However, the percentages of partially open stomata were not obviously different between Ubi::MgGDH-13, Ubi::MgGDH-14 and wild-type plants (36.7–39.6 %; Fig. 3f, g). The results suggest that heterologous expression of MgGDH in rice could decrease water loss from plants by an increase in stomatal closure. Increased GDH amination activity and glutamate content, and reduced ammonium content in transgenic plants under dehydration stress conditions Abiotic stress gives rise to increased GDH in vitro amination activity (Lutts et al. 1999; Kumar et al. 2000; Lasa

et al. 2002; Hoai et al. 2003; Skopelitis et al. 2006). To clarify the physiological mechanism of the tolerance to dehydration stress in transgenic plants, the NADP(H)-GDH activities of transgenic and wild-type plants were investigated. No significant difference in NADP(H)-GDH activities was detected between shoots and roots of wild-type plants under normal growth conditions (Fig. 4a, b). Under dehydration stress conditions, wild-type plans displayed increased NADP(H)-GDH activities in shoots and roots (Fig. 4a, b). Moreover, the amination activity was significantly higher than the deamination activity in both shoots and roots of wild-type plants under dehydration stress at 6 and 8 days (Fig. 4a, b). After dehydration stress for 8 days, amination activities in shoots and roots of wild-type plants increased approximately 4.2-fold and 9.9-fold, respectively, compared to those before dehydration stress, while deamination activities of wild-type plants increased only 2.7-fold and 4.8-fold in shoots and roots, respectively (Fig. 4a, b). The results indicate that endogenous NADP(H)-GDH of rice was involved in response to dehydration stress. However, it was noteworthy that the NADP(H)-GDH activities of transgenic plants in shoots and roots were significantly higher than those in the wildtype plants (Fig. 4c, d). Particularly, in shoots of transgenic plants, the amination activities were dramatically (t test, P \ 0.01 in both Ubi::MgGDH-13 and Ubi::MgGDH-14) higher than the deamination activities (Fig. 4c, d). The results suggest that enhanced dehydration stress tolerance of transgenic plants might be due to the increased NADP(H)-GDH amination activity. The amination activity of NADP(H)-GDH results in assimilation of ammonium to produce L-glutamate. Therefore, the contents of NH4? and glutamate were measured in shoots and roots in transgenic and wild-type plants under normal growth and dehydration stress conditions. Under normal growth conditions, although no obvious difference of NH4? content in roots was detected between transgenic and wild-type plants, the NH4? content in shoots of transgenic plants was significantly lower (t test, P \ 0.05; Fig. 4e, f). Under normal growth conditions, on the other hand, there was no significant difference in the glutamate content in shoots and roots between transgenic plants and wild-type plants (Fig. 4g, h). Compared with wild-type plants, dehydration stress resulted in a significantly less (t test, P \ 0.05) accumulation of NH4? but dramatically higher (t test, P \ 0.05) accumulation of glutamate in the shoots and roots of transgenic plants (Fig. 4e, f, g, h). For example, after dehydration stress for 9 days, the contents of NH4? in the shoots and roots of wild-type plants were 1.6-fold and 1.8-fold higher than those in the shoots and roots of transgenic plants, respectively. After dehydration stress for 5 days, the contents of glutamate in the shoots and roots of transgenic plants were

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Fig. 3 MgGDH improves dehydration stress tolerance of transgenic rice plants. a PEG4000 [20 % (v/v)] treatment of 15-day-old seedlings of transgenic and wild-type plants for 15 days and then recovered for 8 days. b Survival rates of transgenic and wild-type plants after 15 days of 20 % (v/v) PEG4000 treatment and 8 days of recovery. Values are mean ± SD (n = 3, *P B 0.05; **P B 0.01, Student’s t test). c Drought treatment of 20-d-old seedlings of transgenic and wild-type plants for 9 days and then recovered for 3 days. d Survival rates of transgenic and wild-type plants after

9 days of drought stress and 3 days of recovery. Values are mean ± SD (n = 3, *P B 0.05; **P B 0.01, Student’s t test). e Water-loss rates in detached leaves of transgenic and wild-type plants. f Scanning electron microscopy images of three levels of stomatal opening. Bar 5 lm. g The percentage of three levels of stomatal opening in transgenic and wild-type plants (n = 95 stomata for wild-type; n = 93 stomata for MgGDH-13; n = 96 stomata for MgGDH-14)

1.6-fold and 1.3-fold higher than those in the shoots and roots of wild-type plants, respectively. The results demonstrate that increased amination activity may result in the reduction of NH4? toxicity and increase of glutamate content in transgenic plants under dehydration stress conditions.

elucidate the physiological changes in response to dehydration stress, proline and soluble sugar contents were determined in the transgenic and wild-type plants under normal growth and dehydration stress conditions. Under normal growth conditions, no significant difference of proline and soluble sugar contents were detected between transgenic and wild-type plants (Fig. 5a, b). However, under dehydration stress conditions, the transgenic plants accumulated dramatically higher (t test, P \ 0.05) contents of proline and soluble sugars compared with the wild-type plants (Fig. 5a, b). After dehydration stress for 6 days, transgenic plants accumulated approximately fivefold more proline (Fig. 5a) and twofold more soluble sugar (Fig. 5b)

Increased proline and soluble sugar contents in transgenic plants under dehydration stress conditions In response to abiotic stresses, plants often adjust their metabolism, e.g., by accumulation of proline and soluble sugars (Liu and Zhu 1997; Abraham et al. 2003). To

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Planta Fig. 4 NADP(H)-GDH activities, ammonium ion and glutamate contents in transgenic and wild-type plants under normal growth and dehydration stress conditions. a, b NADP(H)-GDH activities in shoots and roots of wild-type plants. c and d NADP(H)-GDH activities in shoots and roots of transgenic and wild-type plants. e and f Ammonium ion contents in shoots and roots of transgenic and wild-type plants. g and h Glutamate contents in shoots and roots of transgenic and wild-type plants. Data are presented as mean values ± SD (n = 3). *P B 0.05; **P B 0.01, Student’s t test

than those before dehydration stress. Such increases were dramatically higher than those in wild-type plants, in which proline and soluble sugars were increased less than 3.6-fold and 1.5-fold, respectively, after dehydration stress. The results suggest that the accumulation of proline and soluble sugars in transgenic plants under the dehydration stress conditions may contribute to their improved tolerance to dehydration stress. To further elucidate the molecular mechanism of dehydration stress response via exogenous MgGDH in rice,

transcript levels of several stress-inducible genes were assayed. The genes included two putative D1-pyrroline-5carboxylate synthetase genes (J033099M14 and J033031H21; Xiang et al. 2007), two putative proline transporter genes (03g44230 and 07g01090; Xiang et al. 2007) and two dehydrate-inducible genes of LEA proteins (OsLEA3 and OsRAB16A; Fukao et al. 2011). Under normal growth conditions, OsLEA3 had markedly higher expression level in transgenic plants than that in wild-type plants, while other genes showed no obvious difference in

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Fig. 5 Contents of free proline and soluble sugars in transgenic and wild-type plants under normal growth and dehydration stress conditions. a Free proline contents in transgenic and wild-type plants. b Soluble sugars contents in transgenic and wild-type plants. c Expression levels of putative proline synthetase genes (J033099M14 and J033031H21), proline transporter genes

(03g44230 and 07g01090) and dehydrate-inducible genes (OsLEA3 and OsRAB16A) in transgenic and wild-type plants under normal growth and dehydration stress conditions by real-time PCR. Data are presented as mean values ± SD (n = 3). *P B 0.05; **P B 0.01, Student’s t test

expression levels between transgenic and wild-type plants (Fig. 5c). However, under dehydration stress conditions, the expression levels of these stress-responsive genes were markedly higher in transgenic plants than those in the wildtype plants (Fig. 5c). The results demonstrate that MgGDH over-expression activated other stress-related genes under dehydration stress conditions, which may have contributed to their improved tolerance to this abiotic stress.

contributes to the degradation of oxidatively damaged proteins and results in hyperammonia in the mitochondria (Sweetlove et al. 2001; Skopelitis et al. 2006). If not scavenged efficiently, hyperammonia can have a detrimental effect on metabolism in plant cells (Vines and Wedding 1960). Assimilation of ammonium into glutamine and glutamate involves coupled reactions catalyzed by GS and GOGAT pathway (Lea and Miflin 1974). Ammonium ions can also be incorporated into glutamate via GDH in the presence of NADP(H) as a cofactor (Wootton 1983). Although the GS/GOGAT pathways in plants were discovered in the 1970s, there has been debate on the role of GDH in assimilation of ammonium. However, NADP(H)GDH and GS both play important roles in ammonium

Discussion Abiotic stress can result in reactive oxygen species (ROS) generation, which induces protease activity and in turn

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assimilation in microorganisms (Kinghorn and Pateman 1973; Kanamori et al. 1987). Although the GS/GOGAT couple is the main pathway mediating ammonium assimilation under normal conditions in plant cells (Lea and Miflin 1974; Skopelitis et al. 2006), it is the amination activity of GDH that responds to salt stress (Skopelitis et al. 2006). Previous study showed that the improved drought tolerance of wheat plants could be related to the increased assimilation of ammonium resulted from increased GDH activities under low and high osmotic stress (Zhang et al. 2009). Since the hyperammonia are toxic to plant cells (Britto and Kronzucker 2002), the capacity to assimilate ammonium is an important element in moderate the effect of stress (Hoai et al. 2003). The results suggested that GDH acts as antistress enzyme play an important role in ammonia detoxification in plants under stress conditions (Skopelitis et al. 2006). In present study, NADP(H)-GDH activities, particularly the amination activities, were enhanced in shoots and roots of wild-type rice plants under dehydration stress condition (Fig. 4a, b). The result is consistent with the increased GDH activities observed by previous workers under stressful conditions of darkness (Laurie`re and Daussant 1983; Yamaya and Oaks 1987), senescence (Calle et al. 1986), salinity (Gulati and Jaiwal 1996) and heavy metal toxicity (Mattioni et al. 1997). GDH has quite a high Km (5.2–70 mM) for ammonium in higher plants (Miflin and Lea 1980; Srivastava and Singh 1987). In contrast, the affinity of NADP(H)-GDH from microorganism for ammonium is much higher (Wang and Tian 2001; Noor et al. 2005; Abiko et al. 2010; Du et al. 2014; Zhou et al. 2014b). For example, the Km value of NiGDH for ammonium has been reported to range from 0.3 to 0.45 mM (Wang and Tian 2001), while that of gdhA ranges from 1.05 mM (Noor et al. 2005) to 1.25 mM (Abiko et al. 2010). The Km value of PcGDH for ammonium is 3.73 mM (Zhou et al. 2014b). The results suggested that NADP(H)-GDH of microorganism can assimilate ammonium ions more efficiently than that of higher plants. In this study, the Km value of MgGDH for ammonium was shown to be 0.67 ± 0.05 mM, which is dramatically lower than that of OsGDH (Km = 12.43 ± 1.84 mM; Fig. 1d). In addition, the Km value of MgGDH for glutamate is 22.3 ± 0.81 mM (Table 1). The results suggested that MgGDH has a higher affinity for ammonium than OsGDH and tends to assimilate ammonium to produce L-glutamate. Tobacco transformed with NADP(H)-GDH (gdhA) from bacterial showed increased tolerance to herbicides, toxic concentration of ammonia (Ameziane et al. 2000; Nolte et al. 2004), and water deficit (Mungur 2002; Mungur et al. 2006). Transgenic Zea mays plants carried a gene for NADP(H)-GDH (gdhA) from E. coli showed improved drought tolerance (Lightfoot et al. 2007). More recently,

over-expression of fungal SsGDH in rice improved the tolerance to herbicide compared to the control plants (Du et al. 2014). The results of present study indicate that overexpression of fungal MgGDH in rice could improve the tolerance to dehydration stress (Fig. 3a, c). Although GDH catalyzes both the amination of 2-OG and the deamination of glutamate, it is the amination role that is enhanced under adverse environmental conditions (Robinson et al. 1991; Gulati and Jaiwal 1996). Intracellular hyperammonia derived from senescence-induced high proteolytic activities (Masclaux et al. 2000; Loulakakis et al. 2002; Terce´-Laforgue et al. 2004b) or abiotic stress (Lutts et al. 1999; Hoai et al. 2003) results in increased GDH amination activity in vivo. In seedlings of salt-tolerant cultivars, GDH amination activity increases with increasing salt stress, whereas it decreases in sensitive cultivars (Kumar et al. 2000). Similarly, the GDH amination activity is high in roots of pea (Pisum sativum) which is an ammonium-tolerant plant (Lasa et al. 2002). Thus, plant cells cope with stress conditions by increasing GDH amination activity, which detoxifies the high intracellular ammonia generated by the proteolytic and deamination activity (Skopelitis et al. 2006). In present study, the amination and deamination activities of NADP(H)-GDH in transgenic plants were found to be significantly higher than those in wild-type plants (Fig. 4c, d). Moreover, the amination activity in shoots of transgenic rice plants was dramatically higher (t test, P \ 0.01 in both Ubi::MgGDH13 and Ubi::MgGDH-14) than the deamination activity (Fig. 4c, d). The results indicated that the enhanced dehydration stress tolerance of transgenic plants might be due to the increased NADP(H)-GDH activities, especially the amination activity. Additionally, under normal growth conditions, the ammonium ion contents were significantly lower in the shoots of transgenic plants than that in the wild-type plants (Fig. 4e, f). It was noteworthy that the ammonium ion contents were significantly lower in both shoots and roots of transgenic plants than those of the wildtype plants under dehydration stress conditions (Fig. 4e, f). The results suggested that heterologous MgGDH played an important role in assimilation and re-assimilation of ammonia to remove the toxicity of ammonia during dehydration stress. In response to abiotic stresses, plants often accumulate compatible osmolytes, such as proline (Liu and Zhu 1997; Armengaud et al. 2004; Ito et al. 2006) and soluble sugars (Gilmour et al. 2000; Garg et al. 2002; Tabuchi et al. 2004; Gupta and Kaur 2005; Chyzhykova and Palladina 2006), to protect subcellular structures from damage by adjusting the intracellular osmotic potentials. Over-production of proline could give rise to increased tolerance to osmotic stress in P5CS-overexpressing tobacco (Kishor et al. 1995). Accumulation of proline in stressed plants has been associated

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with increased tolerance to conditions of abiotic stress (Nanjo et al. 1999a, b). On the contrary, the suppression of proline synthesis in P5CR-suppressed transgenic soybean plants increased the sensitivity to water deficit (De Ronde et al. 2000). As described above, the MgGDH transgenic rice plants had significantly higher contents of proline and soluble sugars than wild-type plants under dehydration stress conditions (Fig. 5c, d). The transcripts of proline synthetase and transport genes were also significantly more abundant in transgenic plants than those in wild-type plants under dehydration stress conditions (Fig. 5e). Clearly, heterologous expression of MgGDH increased proline and soluble sugar contents in transgenic plants under dehydration stress conditions, which presumably also contributed to the improvement of tolerance to dehydration stress. It is noteworthy that the glutamate synthesized under stress conditions by GDH is directed towards proline synthesis (Skopelitis et al. 2006). Because the expression of salt stress ornithine omega-aminotransferase is repressed, proline biosynthesis from glutamate seems to be the critical pathway under stress conditions (Delauney and Verma 1993; Delauney et al. 1993). In present study, it was found that the amount of glutamate in MgGDH transgenic plants were significantly higher than that in wild-type plants under dehydration stress conditions (Fig. 5a, b), indicating that the higher amount of proline were a result of higher amount of glutamate in transgenic plants. Rice LEAs, LEA3 and RAB16A (RAB21) were reported to be strongly induced by drought treatment (Fukao et al. 2011), and over-expression of OsLEA3 in rice showed increased drought tolerance under field conditions (Xiao et al. 2007). Indeed, the transcript levels of OsLEA3 and OsRAB16A are often considered to be molecular indicators of drought stress in plants (Fukao et al. 2011). In present study, it was found that dehydration stress treatment highly induced the accumulation of OsLEA3 and OsRAB16A mRNA in transgenic plants (Fig. 5e), which suggested that heterologous MgGDH activated the expression of OsLEA3 and OsRAB16A. Previous study reported that the transcription of OsLEA3 and OsRAB16A is also strongly enhanced by ABA (Fukao et al. 2011), implying that heterologous MgGDH might be involved in ABA signaling pathway in rice. In conclusion, over-expression of MgGDH can help prevent toxic accumulation of ammonium and thereby enhance tolerance to dehydration stress in rice. Author contribution Conceived and designed the experiments: JZL and XML. Performed the experiments: YBZ and CSZ. Partially participated in the experiments: YZY, YCP, DYT, XYZ and YHZ. Analyzed the data: YBZ, CSZ and JZL. Wrote the manuscript: YBZ and JZL. All authors read and approved the manuscript.

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Acknowledgments The authors thank Dr. Hong Liu for providing the fungi Magnaporthe grisea and assistance of cloning MgGDH. This research was supported by Important National Science and Technology Specific Projects (No. 2009ZX08001-030B), National Science Foundation of China (No. 31170172), Hunan Provincial Natural Science Foundation of China (No.12JJ3024), Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (No. 20134486), Planned Science and Technology Project of Hunan Province (No. 2014WK2005 and No. 2014NK3001).

References Abiko T, Obara M, Ushioda A, Hayakawa T, Hodges M, Yamaya T (2005) Localization of NAD-isocitrate dehydrogenase and glutamate dehydrogenase in rice roots: candidates for providing carbon skeletons to NADH-glutamate synthase. Plant Cell Physiol 46:1724–1734 Abiko T, Wakayama M, Kawakami A, Obara M, Kisaka H, Miwa T, Aoki N, Ohsugi R (2010) Changes in nitrogen assimilation, metabolism, and growth in transgenic rice plants expressing a fungal NADP(H)-dependent glutamate dehydrogenase (gdhA). Planta 232:299–311 Abraham E, Rigo G, Szekely G, Nagy R, Koncz C, Szabados L (2003) Light-dependent induction of proline biosynthesis by abscisic acid and salt stress is inhibited by brassinosteroid in Arabidopsis. Plant Mol Biol 51:363–372 Ameziane R, Bernhard K, Lightfoot D (2000) Expression of the bacterial gdhA gene encoding a NADPH glutamate dehydrogenase in tobacco affects plant growth and development. Plant Soil 221:47–57 Armengaud P, Thiery L, Buhot N, Grenier-De March G, Savoure A (2004) Transcriptional regulation of proline biosynthesis in Medicago truncatula reveals developmental and environmental specific features. Physiol Plant 120:442–450 Boyer JS (1982) Plant productivity and environment. Science 218:443–448 Britto DT, Kronzucker HJ (2002) NH4? toxicity in higher plants: a critical review. J Plant Physiol 159:567–584 Calle F, Martin M, Sabater B (1986) Cytoplasmic and mitochondrial localization of the glutamate dehydrogenase induced by senescence in barley (Hordeum vulgare). Physiol Plant 66:451–456 Cao WH, Liu J, He XJ, Mu RL, Zhou HL, Chen SY, Zhang JS (2007) Modulation of ethylene responses affects plant salt-stress responses. Plant Physiol 143:707–719 Chen QJ, Zhou HM, Chen J, Wang XC (2006) Using a modified TA cloning method to create entry clones. Anal Biochem 358:120–125 Chyzhykova OA, Palladina TO (2006) The role of amino acids and sugars in supporting of osmotic homeostasis in maize seedlings under salinization conditions and treatment with synthetic growth regulators. Ukr Biokhim Zh 78:124–129 De Ronde JA, Spreeth MH, Cress WA (2000) Effect of antisense L41-pyrroline-5-carboxylate reductase transgenic soybean plants subjected to osmotic and drought stress. Plant Growth Regul 32:13–26 Delauney AJ, Verma DPS (1993) Proline biosynthesis and osmoregulation in plants. Plant J 4:215–223 Delauney AJ, Hu C-AA, Kavi Kishor PB, Verma DPS (1993) Cloning of ornithined d-aminotransferase cDNA from Vigna aconitifolia by trans-complementation in E. coli and regulation of proline biosynthesis. J Biol Chem 268:18673–18678

Planta Du C, Lin J, Yang Y, Liu H, Li C, Zhou Y, Li Y, Tang D, Zhao X, Zhu Y, Liu X (2014) Molecular cloning, characterization and function analysis of a GDH gene from Sclerotinia sclerotiorum in rice. Mol Biol Rep 41:3683–3693 Fukao T, Yeung E, Bailey-Serres J (2011) The submergence tolerance regulator SUB1A mediates crosstalk between submergence and drought tolerance in rice. Plant Cell 23:412–427 Garg AK, Kim JK, Owens TG, Ranwala AP, Choi YD, Kochian LV, Wu RJ (2002) Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. Proc Natl Acad Sci USA 99:15898–15903 Gilmour SJ, Sebolt AM, Salazar MP, Everard JD, Thomashow MF (2000) Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiol 124:1854–1865 Gulati A, Jaiwal PK (1996) Effect of NaCl on nitrate reductase, glutamate dehydrogenase and glutamate in Vigna radiata calli. Bio. Plant 38:177–183 Gupta AK, Kaur N (2005) Sugar signalling and gene expression in relation to carbohydrate metabolism under abiotic stresses in plants. J Biosci 30:761–776 Hetherington AM, Woodward FI (2003) The role of stomata in sensing and driving environmental change. Nature 424:901–908 Hoai NTT, Shim IS, Kobayashi K, Usui K (2003) Accumulation of some nitrogen compounds in response to salt stress and their relationships with salt tolerance in rice (Oryza sativa) seedlings. Plant Growth Regul 41:159–164 Ito Y, Katsura K, Maruyama K, Taji T, Kobayashi M, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2006) Functional analysis of rice DREB1/CBF-type transcription factors involved in cold-responsive gene expression in transgenic rice. Plant Cell Physiol 47:141–153 Kanamori K, Weiss RL, Roberts JD (1987) Role of glutamate dehydrogenase in ammonia assimilation in nitrogen-fixing Bacillus macerans. J Bacteriol 169:4692–4695 Kinghorn JR, Pateman JA (1973) NAD and NADP L-glutamate dehydrogenase activity and ammonium regulation in Aspergillus nidulans. J Gen Microbiol 78:39–46 Kishor P, Hong Z, Miao GH, Hu C, Verma D (1995) Overexpression of 41-pyrroline-5-carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants. Plant Physiol 108:1387–1394 Krug FJ, Ru˚zˇicˇka J, Hansen EH (1979) Determination of ammonia in low concentrations with Nessler’s reagent by flow injection analysis. Analyst 104:47–54 Kumar RG, Shah K, Dubey RS (2000) Salinity induced behavioral changes in malate dehydrogenase and glutamate dehydrogenase activities in rice seedlings of differing salt tolerance. Plant Sci 156:23–34 Lasa B, Frechilla S, Aparicio-Tejo PM, Lamsfus C (2002) Role of glutamate dehydrogenase and phosphoenolpyruvate carboxylase activity in ammonium nutrition tolerance in roots. Plant Physiol Biochem 40:969–976 Lauriere C, Daussant J (1983) Identification of the ammoniadependent-isoenzyme of glutamate dehydrogenase as the form induced by senescence or darkness stress in the first leaf of wheat. Physiol Plant 48:89–92 Lea PJ, Miflin BJ (1974) Alternative route for nitrogen assimilation in higher plants. Nature 18:614–616 Li C, Yu L, Liu Z, Zhu L, Hu Y, Zhu M, Zhu X, Shi Y, Meng S (2006) Schistosoma japonicum: the design and experimental evaluation of a multivalent DNA vaccine. Cell Mol Biol Lett 11:449–460 Lightfoot DA, Mungur R, Ameziane R, Nolte S, Long L, Bernhard K, Colter A, Jones K, Iqbal MJ, Varsa E, Yong B (2007) Improved drought tolerance of transgenic Zea mays plants that express the

glutamate dehydrogenase gene (gdhA) of E. coli. Euphytica 156:103–116 Lin JZ, Zhou B, Yang YZ, Mei J, Guo XH, Huang XQ, Tang DY, Liu XM (2009) Piercing and vacuum infiltration of the mature embryo: a simplified Method for Agrobacterium mediated transformation of Indica rice. Plant Cell Rep 28:1065–1074 Liu J, Zhu JK (1997) Proline accumulation and salt-stress-induced gene expression in a salt-hypersensitive mutant of Arabidopsis. Plant Physiol 114:591–596 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2[-4 4C(T)] method. Methods 25:402–408 Loulakakis KA, Primikirios NI, Nikolantonakis MA, RoubelakisAngelakis KA (2002) Immunocharacterization of Vitis vinifera L. ferredoxin-dependent glutamate synthase and its spatial and temporal changes during leaf development. Planta 215:630–638 Lutts S, Majerus V, Kinet JM (1999) NaCl effects on proline metabolism in rice (Oryza sativa) seedlings. Physiol Plant 105:450–458 Masclaux C, Valadier MH, Brugie‘re N, Morot-Gaudry JF, Hirel B (2000) Characterization of the sink/source transition in tobacco (Nicotiana tabacum L.) shoots in relation to nitrogen management and leaf senescence. Planta 211:510–518 Mattioni C, Gabbrielli R, Vongronsveld J, Clijsters H (1997) Nickel and cadmium toxicity and enzymatic activity in Ni tolerant and non-tolerant populations of Silene italica pers. J Plant Physiol 150:173–177 Miflin BJ, Lea PJ (1980) Ammonia assimilation. In: Miflin BJ (ed) The biochemistry of plants, vol 5. Academic Press, New York, pp 169–202 Mungur R (2002) Metabolic profiles of GDH transgenic crops. MS thesis, SIUC Carbondale Mungur R, Wood AJ, Lightfoot DA (2006) Water potential is maintained during water deficit in Nicotiana tabacum expressing the E. coli glutamate dehydrogenase gene. Plant Growth Regul 50:231–238 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497 Nanjo T, Kobayashi M, Yoshiba Y, Kakubari Y, YamaguchiShinozaki K, Shinozaki K (1999a) Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana. FEBS Lett 461:205–210 Nanjo T, Kobayashi M, Yoshiba Y, Sanada Y, Wada K, Tsukaya H, Kakubari Y, Yamaguchi-Shinozaki K, Shinozaki K (1999b) Biological functions of proline in morphogenesis and osmotolerance revealed in antisense transgenic Arabidopsis thaliana. Plant J 18:185–193 Nolte SA, Young BG, Mungur R, Lightfoot DA (2004) The glutamate dehydrogenase gene gdhA increased the resistance of tobacco to glufosinate. Weed Res 44:335–339 Noor S, Punekar NS (2005) Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon–nitrogen interface. Microbiology 151:1409–1419 Ohta S, Mita S, Hattori T, Nakamura K (1990) Construction and expression in tobacco of a b-glucuronidase (GUS) reporter gene containing an intron within coding sequence. Plant Cell Physiol 31:805–813 Robinson SA, Slade AP, Fore GG, Phillips R, Radcliffe RG, Stewart GR (1991) The role of GDH in plant nitrogen metabolism. Plant Physiol 95:509–516 Skopelitis DS, Paranychianakis NV, Paschalidis KA, Pliakonis ED, Delis ID, Yakoumakis DI, Kouvarakis A, Papadakis AK, Stephanou EG, Roubelakis-Angelakis KA (2006) Abiotic stress generates ROS that signal expression of anionic glutamate dehydrogenases to form glutamate for proline synthesis in tobacco and grapevine. Plant Cell 18:2767–2781

123

Planta Srivastava HS, Singh RP (1987) Role and regulation of L-glutamate dehydrogenase activity in higher plants. Phytochemistry 26:597–610 Sweetlove LJ, Mowday B, Hebestreit HF, Leaver CJ, Millar AH (2001) Nucleoside diphosphate kinase III is localized to the intermembrane space in plant mitochondria. FEBS Lett 508:272–276 Tabuchi A, Kikui S, Matsumoto H (2004) Differential effects of aluminium on osmotic potential and sugar accumulation in the root cells of Al-resistant and Al-sensitive wheat. Physiol Plant 120:106–112 Terce´-Laforgue T, Dubois F, Ferrario-Mery S, Crecenzo MAP, Sangwan R, Hirel B (2004a) Glutamate dehydrogenase of tobacco 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 Terce´-Laforgue T, Mack G, Hirel B (2004b) New insights towards the function of glutamate dehydrogenase revealed during sourcesink transition of tobacco (Nicotiana tabacum) plants grown under different nitrogen regimes. Physiol Plant 120:220–228 Toki S, Hara N, Ono K, Onodera H, Tagiri A, Oka S, Tanaka H (2006) Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. Plant J 47:969–976 Valliyodan B, Nguyen HT (2006) Understanding regulatory networks and engineering for enhanced drought tolerance in plants. Curr Opin Plant Biol 9:189–195 Vines HM, Wedding RT (1960) Some effects of ammonia on plant metabolism and a possible mechanism for ammonia toxicity. Plant Physiol 35:820–825 Wang F, Tian B (2001) Neurospora NADP glutamate dehydrogenases and its expression in E. coli and transgenic plant. Chin Sci Bull 46:137–140 Wootton JC (1983) Re-assessment of ammonium-ion affinities of NADP-specific glutamate dehydrogenases. Biochem J 209:527– 531 Xiang Y, Huang YM, Xiong LZ (2007) Characterization of stressresponsive CIPK genes in rice for stress tolerance improvement. Plant Physiol 144:1416–1428

123

Xiao B, Huang Y, Tang N, Xiong L (2007) Over-expression of a LEA gene in rice improves drought resistance under the field conditions. Theor Appl Genet 115:35–46 Yamaya T, Oaks A (1987) Synthesis of glutamate by mitochondria— an anaplerotic function for glutamate dehydrogenase. Physiol Plant 70:749–756 Ye H, Du H, Tang N, Li X, Xiong L (2009) Identification and expression profiling analysis of TIFY family genes involved in stress and phytohormone responses in rice. Plant Mol Biol 71:291–305 Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572 Yoshiba Y, Kiyosue T, Nakashima K, Yamaguchi-Shinozaki K, Shinozaki K (1997) Regulation of levels of proline as an osmolyte in plants under water stress. Plant Cell Physiol 38:1095–1102 Zhang HN, Wang ZQ, Cui GJ, Lin TB (2009) Difference in seedlings ammonium assimilation of wheat cultivars with different drought resistance under osmotic stress. Ying Yong Sheng Tai Xue Bao 20:2406–2410 Zhang SX, Haider I, Kohlen W, Jiang L, Bouwmeester H, Meijer AH, Schluepmann H, Liu CM, Ouwerkerk PBF (2012) Function of the HD-Zip I gene Oshox22 in ABA-mediated drought and salt tolerances in rice. Plant Mol Biol 80:571–585 Zhou XC, Lin JZ, Zhou YB, Yang YZ, Liu H, Zhang CS, Tang DY, Zhao XY, Zhu YH, Liu XM (2014a) Changes in nitrogen utilization and growth in transgenic rice over-expressing a fungal NADP(H)-dependent glutamate dehydrogenase (CeGDH). Crop Sci, pp 1–22 Zhou YB, Liu H, Zhou XC, Yan YZ, Du CQ, Li YX, Liu DR, Zhang CS, Deng XL, Tang DY, Zhao XY, Zhu YH, Lin JZ, Liu XM (2014b) Over-expression of a fungal NADP(H)-dependent glutamate dehydrogenase PcGDH improves nitrogen assimilation and growth quality in rice. Mol Breed 34:335–349