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THE BETTER GROWTH PHENOTYPE OF DvGS1-tranSGenic
arabiDopSiS thaliana IS ATTRIBUTED TO THE IMPROVED
EFFICIENCY OF NITROGEN ASSIMILATION
Chenguang Zhu*, Guimin Zhang, Shilin Chen, Wei Wang, Yuanping Tang and Rentao Song
Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, Shanghai, China
*Corresponding author: cgzhu@shu.edu.cn
Abstract: The overexpression of the algal glutamine synthetase (GS) gene DvGS1 in Arabidopsis thaliana resulted in higher plant biomass and better growth phenotype. The purpose of this study was to recognize the biological mechanism for the growth improvement of DvGS1-transgenic Arabidopsis. A series of molecular and biochemical investigations related to nitrogen and carbon metabolism in the DvGS1-transgenic line was conducted. Analysis of nitrogen use efficiency (NUE)-related gene transcription and enzymatic activity revealed that the transcriptional level and enzymatic activity of the genes encoding GS, glutamate synthase, glutamate dehydrogenase, alanine aminotransferase and aspartate aminotransferase, were significantly upregulated, especially from leaf tissues of the DvGS1-transgenic line under two nitrate conditions. The
DvGS1-transgenic line showed increased total nitrogen content and decreased carbon:nitrogen ratio compared to wild-type plants. Significant reduced concentrations of free nitrate, ammonium, sucrose, glucose and starch, together with higher concentrations of total amino acids, individual amino acids (glutamate, aspartate, asparagine, methionine), soluble proteins and fructose in leaf tissues confirmed that the DvGS1-transgenic line demonstrated a higher efficiency of nitrogen assimila-tion, which subsequently affected carbon metabolism. These improved metabolisms of nitrogen and carbon conferred the
DvGS1-transgenic Arabidopsis higher NUE, more biomass and better growth phenotype compared with the wild-type plants.
Key words: glutamine synthetase; Arabidopsis thaliana; nitrogen use efficiency; transgenic; biological mechanism Received: April 14, 2015; Revised: May 5, 2015; Accepted: May 5, 2015
INTRODUCTION
Nitrogen is the limiting nutrient during the growth of plants. It is absorbed from soil mainly in the form of nitrate (NO3−), ammonia/ammonium (NH
3/NH4 +) or urea (CO(NH2)2). Insufficient nitrogen severely af-fects the yield of crops, whereas oversupply has no ef-fect on yield and contributes significantly to nitrogen pollution (Amiour et al., 2012; Liao et al., 2012).
Nitrogen use by plants involves two steps: up-take and utilization. The utilization can be fur-ther compartmentalized into assimilation and translocation⁄remobilization (Masclaux-Daubresse et al., 2010). The nitrate and ammonium are usu-ally major forms of nitrogen resources taken up by plants (Good et al., 2004; Miller et al., 2007). Nitrate
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aspartate aminotransferase (AspAT), respectively (Hodges, 2002). Another aminotransferase, the ala-nine aminotransferase (AlaAT) can serve as a marker of nitrogen use efficiency (NUE) (Cañas et al., 2010). AlaAT-overexpressed rice plants grown in nitrogen-limiting conditions show increased biomass and yield, as well as increases in total nitrogen and key metabo-lites (Gln, Glu and Asn) (Shrawat et al., 2008).
Current attempts at improving plant NUE via transgenic approaches focus on altering nitrogen uptake, assimilation, remobilization and regulation. They include the transgenic expression of genes en-coding AlaAT (Shrawat et al., 2008), GS (Brauer et al., 2011), NADH-GOGAT (Chickova et al., 2001), NADP(H)-GDH (Abiko et al., 2010), AspAT (Zhou et al., 2009), ASN (Lam et al., 2003), etc. Expression of additional factors, including those involved in ni-trogen assimilation, carbon-nini-trogen balance and signaling, may also have roles in improving NUE (McAllister et al., 2012). However, transgene tar-geted metabolic engineering may also be limited by the complexity and correlation of many metabolic processes. Currently, the regulation of genes that are involved in nitrogen metabolism and starvation re-sponse is not well-understood (Fischer et al., 2013). It is thought that changes in the expression of GS (either GS1 or GS2) as well as changes in the activ-ity of GS have an effect on nitrogen metabolism in plants, potentially affecting NUE (Eckes et al., 1989; Fei et al., 2003). Our previous work also reports that overexpression of the algal GS gene DvGS1 in Arabi-dopsis thaliana results in higher biomass and better growth phenotype. The fresh and dry weights, leaf GS activity and soluble protein concentration of the DvGS1-transgenic Arabidopsis showed various de-grees of increase compared with the wild-type plants (Zhu et al., 2014). Therefore, a series of molecular and biochemical investigations related to nitrogen and carbon metabolism in the DvGS1-transgenic Arabidopsis were respectively conducted in this study, in order to understand the molecular mechanism for the improvement of growth phenotype, which would be useful in engineering transgenic crops with a high efficiency of nitrogen utilization.
MATERIALS AND METHODS
Plant growth conditions
The seeds of DvGS1-transgenic Arabidopsis T3 ho-mozygotes were germinated on 1/2 MS agar plates after vernalization, and grew vertically until the root length was about 1-1.5 cm. Then the seedlings were transferred to plant nutrient solution (PNS) as previ-ously described (Zhu et al., 2014) for hydroponic cul-ture. During the growth, two concentrations of nitrate (9 mM and 2 mM KNO3) were used. A regular day/ night cycle was set to a day length (illumination of 4000 lux at 22°C) of 16 h and a dark period (at 20°C) of 8 h. The air humidity was maintained at the level of 60-70%. After 30 days of PNS culture, the plants were harvested, including leaves, roots and stems. The plant materials were frozen in liquid nitrogen and stored at -80°C until use.
Determination of total nitrogen and carbon contents
After drying and weighing, the plant materials were ground to a homogenous powder. A sample of 10-20 mg was carefully weighed in tin capsules to deter-mine total nitrogen and carbon contents using a Vario MICRO cube organic elemental analyzer (Elementar Analysensysteme GmbH, Hanau, Germany).
Amino acid and saccharide analysis
Total amino acid content was determined after ex-traction from fresh leaf tissues with 2% (w/v) solu-tion of 5-sulfosalicylic acid by the Rosen colorimetric method (Rosen, 1957) using glycine as a reference. The concentrations of free amino acids were moni-tored by ion exchange chromatography on an L-8900 amino acid analyzer according to instructions of the manufacturer (Hitachi Ltd, Tokyo, Japan). The con-tent of hexoses, sucrose and starch in the plant tissues was analyzed using kits for the detection of saccharide content according to the protocol (colorimetric as-say) provided by the manufacturer (Suzhou Comin Biotechnology Co., Ltd, Suzhou, China).
Real-time quantitative PCR
The total RNA from DvGS1-transgenic Arabidopsis plants was extracted using Trizol reagent (Invitrogen), followed by treatment with DNase I to remove the contaminating genomic DNA. Reverse transcription was performed using the reverse transcriptase Rever-Tra Ace (TOYOBO) to obtain a cDNA template. The quantitative PCR (qPCR) was performed using SYBR Green Real-time PCR Master Mix (TOYOBO) accord-ing to the manufacturer’s recommendations. Primers for genes in A. thaliana were designed according to the corresponding sequences in the genome database of Arabidopsis. All the primers are listed in Supplemen-tary Table 1. Real-time qPCR was carried out with a Realplex system (Eppendorf, Hamburg, Germany) us-ing SYBR Green to monitor the dsDNA synthesis. The UBQ2 (At2G36170) and EF1α (At5G60390) genes were used as the internal standards for normalization of all the treatments. The relative expression levels of the target genes are presented after normalization to the internal standards from the technical repeats of three independent biological repeats.
Enzymatic assays
Enzymes were extracted from frozen plant materials that were previously stored at -80°C. All the extrac-tions were performed at 4°C. The AspAT and AlaAT activities were measured in NADH-coupled colori-metric assays according to the protocol described in Cazetta et al. (1999). The GS activity was assayed by the previously described approach (Zhu et al., 2014). The GDH activity was assayed for the amination reaction according to the method of Turano et al. (1996). The NR, NiR and Fd-GOGAT activities were determined according to the method described in De-bouba et al. (2007). The activity of NADH-GOGAT was measured in NADH-coupled colorimetric assay according to the protocol reported by Ertan (1992). Nitrate and ammonium determination
Free ammonium was extracted from fresh leaves with 60% (v:v) methanol and was determined by the
salic-ylate dichloroisocyanurate assay (Berthelot reaction) (Husted et al., 2000). Free nitratewas extracted from fresh leaves with hot deionized water (80°C) and the supernatant was determined by salicylic acid-H2SO4 method (Cataldo et al., 1975) using KNO3 as the stan-dard.
Statistical analyses
Statistical analysis for all the biochemical detection was carried out using two-way analysis of variance (ANOVA). Data followed a normal distribution.
Table 1. The concentrations of free individual amino acids from leaf tissues of DvGS1-1 line and wild type Arabidopsis under two concentrations of nitrate.
Amino acid
(nmol/mg DW)
Low nitrate (2 mM) Normal nitrate (9 mM)
WT DvGS1-1 WT DvGS1-1
Asp 14.22±0.62 19.12±0.91 * 13.16±2.73 19.81±2.88 * Thr 9.13±0.85 8.94±1.35 9.10±1.57 9.88±1.34 Ser 8.76±1.42 8.99±1.03 10.10±1.71 10.26±1.47 Asn 18.59±1.73 36.16±2.11 * 17.66±3.29 34.71±4.88 * Glu 12.50±0.26 43.12±0.58 * 14.39±2.67 27.57±1.97 * Gln 9.01±0.55 12.27±0.70 8.50±0.47 12.05±1.82 Gly 2.50±0.33 2.68±0.13 2.80±0.55 3.38±0.75 Ala 16.09±0.28 16.78±1.62 16.99±2.39 17.33±2.57 Val 9.38±0.62 10.62±0.74 10.20±1.59 12.60±1.31 Cys 0.42±0.08 0.31±0.04 0.41±0.09 0.59±0.06 Met 0.70±0.11 1.77±0.54 * 0.72±0.13 1.15±0.11 * Ile 4.97±0.31 4.64±0.87 5.50±0.61 7.24±0.59 Leu 7.49±1.26 7.11±0.12 8.17±0.72 10.49±1.30 Tyr 2.46±0.52 2.52±0.25 2.50±0.22 3.32±0.81 Phe 5.28±0.23 5.71±0.85 5.85±0.84 8.83±1.11 Trp 4.70±0.72 4.62±0.37 4.32±0.92 4.27±0.66 Lys 3.69±0.33 5.63±0.45 6.50±0.94 8.84±0.71 His 1.38±0.40 1.48±0.09 1.74±0.78 2.40 ±0.69 Arg 4.37±0.34 3.92±0.78 4.76±1.50 6.92±0.49 Pro 4.00±0.81 4.10±0.71 3.94±0.67 4.64±0.95 aData represent the means±standard deviation of three
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Means and standard errors were calculated by Excel software. Significances were determined using the two-tailed t-test on the mean of the technical repeats of biological repeats.
RESULTS
DvGS1-transgenic arabidopsis displays higher nitrogen content and lower C:N ratio
In this study, with the purpose of understanding the biological mechanism for the improvement of growth
Fig. 1. The differences in biomass, concentrations of carbon, nitrogen and nitrogen-containing compounds between DvGS1-1 line and wild-type Arabidopsis. The aboveground parts of plants were harvested for measuring their fresh weights (A) and dry weights (B). The soluble protein (C), total amino acid (D), nitrate (E) and ammonium (F) concentrations in leaf tissues, together with the total nitrogen content (G), carbon content (H) and C:N ratio (I) in 30-day-old transgenic DvGS1-1 line (shaded columns) and wild-type (white columns) Arabidopsis
grown under normal (9 mM) and low (2 mM) nitrate supplies. Data represent the means±standard deviation from five biological repeats using ten plants for each repeat. Significant differences are calculated by two-tailed t-test at the levels of *P<0.05, **P<0.01, ***P<0.001.
In order to determine the nature of C:N changes in the transgenic line DvGS1-1, the concentrations of soluble proteins, total amino acids, ammonium and nitrate in the leaves of the transgenic line DvGS1-1 were detected. The soluble protein concentrations and total amino acid concentrations from leaf tis-sue of the DvGS1-1 line were increased by 16-18% and 36-53% compared to those of the wild type un-der both nitrate conditions (Fig. 1C, D). The nitrate and ammonium contents from the leaf tissue of the DvGS1-1 line were 15-19% and 8-15%, respectively, lower than those in the wild type under both nitrate supplies (Fig. 1E, F). The concentrations of soluble and insoluble saccharides from different tissues of the transgenic line DvGS1-1 were also investigated. Results showed that relative to the wild type, higher fructose concentrations were found in the transgenic line DvGS1-1 under both normal (9 mM) and low ni-trate (2 mM) supplies (Fig. 2B, D). However, reduced concentrations of sucrose and glucose were observed in DvGS1-1 under both nitrate conditions (Fig. 2A, C, E, G). The starch concentrations of the transgenic
line DvGS1-1 were reduced in stem and root tissues, and unchanged in leaf tissue under normal nitrate supply (Fig. 2F). Under low nitrate supply, the starch concentration of transgenic line DvGS1-1 was re-duced in the stem, increased in leaf and unchanged in the root tissue (Fig. 2H).
Steady-state transcriptional levels of NUE-related genes
The mRNA accumulation of a number of genes that are involved in nitrogen assimilation and amino acid (Asn, Asp, Ala, Glu, Gln) biosynthesis was quantified in root, stem and leaf tissues of Arabidopsis DvGS1-1 line and wild type under both nitrate conditions. Under normal nitrate supply, the most significant differences in transcript abundance were observed in the leaf tissue. The transcriptional levels of GDH2, AlaAT1, AspAAT and ASN1 in the leaf of the DvGS1-1 line were increased from 3- to 6-fold as compared to the transcriptional levels in the wild type; the lev-els of GDH1, GDH3, Gln1-2, Gln1-3, AspAT2 and
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GOGAT1 in the DvGS1-1 line were also increased by 100%, the levels of ASN2, ASN3, NR1 and NiR were significantly downregulated by 1- to 4-fold (Fig. 3E). In the stem, the transcriptional levels of Gln1-4, As-pAAT, AlaAT1, AlaAT2, Fd-GOGAT1 and NADH-GOGAT were increased by 41-69% in the DvGS1-1 line, but the levels of NR1, Gln2, GDH3 and Gln1-1 were downregulated by 44-68% (Fig. 3C). In the root tissue, the most significant increase in transcriptional level (39-66%) appeared in the ASN1, NADH-GO-GAT, Gln1-1, Gln1-3, AspAT2 and AspAT4, the levels of GDH3 and NR1 were downregulated by more than 80%, and the levels of other genes including Gln1-2, AspAT1, NR2 and NiR were also reduced compared with the wild type (Fig. 3A). Under low nitrate supply,
the most significant differences in transcript abun-dance were observed in leaf and stem tissues. The transcriptional levels of AlaAT1, ASN1 and GDH2 in leaf tissue of the DvGS1-1 line were 3- to 5-fold higher than in the wild type (Fig. 3F). The levels of AspAT1, AspAT3, AspAT4, Fd-GOGAT1, Gln1-1 and Gln1-3 in the DvGS1-1 line were also increased by 45-128%. The levels of NR1, NR2, NiR, ASN2, ASN3, GDH3 and AspAT5 were significantly downregulat-ed by 61-86% (Fig. 3F). In the stem tissue, the tran-scriptional levels of ASN1, AspAT3, AlaAT2, AspAT4, GDH2 and Gln1-4 in the DvGS1-1 line were 2- to 7-fold higher as compared to those in the wild type, but the levels of NR1, NR2, NiR, Fd-GOGAT1, As-pAT1 and Gln2 were downregulated by 37-78% (Fig.
Fig. 3. Quantitative PCR detection of NUE-related gene expressions in leaves, stems and roots of transgenic DvGS1-1 line (black columns) and wild-type (white columns) Arabidopsis 30-day-old plants grown under normal (9 mM) and low nitrate (2 mM) supplies. Quantitative PCR result of NUE-related gene expressions in root (A), stem (C) and leaf (E) grown under normal nitrate supply. Quantitative PCR result of NUE-related gene expressions in root (B), stem (D) and leaf (F) grown under low nitrate supply. Data represent the means ± standard deviation from the technical repeats of biological repeats (n=3). Significant differences are calculated by two-tailed t-test at the levels of *P<0.05, **P<0.01, ***P<0.001.
3D). In the root tissue, the most significant increase in transcriptional levels (62-170%) appeared in the GDH1, AspAT1, ASN1 and Gln1-3 (Fig. 3B). The lev-els of NR1, NiR, GDH3, NADH-GDH, Gln1-1, Gln2 and AspAT2 were downregulated by more than 50%, the levels of other genes including Gln1-4, AspAT5, ASN2, NR2, Fd-GOGAT2 and NADH-GOGAT were also reduced compared with the wild type (Fig. 3B). The enzymatic activities of NUE-related genes
According to the mRNA levels, seven types of genes (GS, GDH, AlaAT, AspAT, ASN, NADH-GOGAT, Fd-GOGAT) in the DvGS1-1 line showed significantly in-creased transcriptional levels compared with the wild type under either normal or low nitrate conditions. Consequently, the activities of these enzymes (except ASN, as there is no reference describing an approach for ASN assay) together with the NR and NiR, were assayed to test the concordance between mRNA level and enzymatic activity. In leaf tissue, it was observed that the activities of GS, GDH, Fd-GOGAT, AspAT, and AlaAT in the DvGS1-1 line were generally in-creased by 13-21% under both nitrate supplies (Fig. 4A-E, G, I-L). The NR and NiR activities were re-duced by 10-18% (Fig. 4M-P), and the NADH-GO-GAT activity had no significant change compared with the wild type under both nitrate supplies (Fig. 4F, H). In stem tissue, the activities of GS and GDH were increased by 12-16% under low nitrate supply but showed no significant changes under normal ni-trate condition (Fig. 4A-D). The AspAT activity was increased by 14-20%, the activities of Fd-GOGAT and NADH-GOGAT had no significant changes compared with wild type under both nitrate supplies (Fig. 4E-H, J, L). The AlaAT activity was increased by 13% under normal nitrate conditions (Fig. 4I), the NR activity was reduced by 12% under low nitrate supply (Fig. 4O) and the NiR activity was reduced by 18% under normal nitrate supply (Fig. 4N). In root tissue, the activities of GS and NADH-GOGAT were enhanced by 29% and 57%, respectively, under nor-mal nitrate supply (Fig. 4A, F), and the GDH activity was increased by 13% under low nitrate supply (Fig. 4D). The AlaAT activity was reduced by 13% under
normal nitrate conditions but increased by 27% under low nitrate conditions (Fig. 4I, K). The NR and NiR activities were reduced by 7-10% under both nitrate supplies (Fig. 4M-P). The activities of AspAT and Fd-GOGAT were not significantly changed under either nitrate supply (Fig. 4E, G, J, L).
Free amino acid profiles
The free amino acid profiles in fresh leaves of the Arabidopsis DvGS1-1 line and wild type under both nitrate conditions are presented in Table 1. Under normal nitrate conditions, the concentrations of Asp, Asn, Glu and Met showed a significant increment in the DvGS1-1 line. The Asn and Glu showed the most increased levels; their concentrations in the DvGS1-1 line were increased by 97% and 92%, respectively, compared with those in wild type (Table 1). The con-centrations of Asp and Met were increased by 51-60%, and the other amino acids showed no significantly different concentrations compared with those in the wild type (Table 1). Under low nitrate conditions, the concentrations of Asp, Asn, Glu and Met in DvGS1-1 line were significantly increased. The highest con-centration of Glu, more than 3-fold higher than in the wild type value, was detected in the DvGS1-1 line (Table 1). The concentration of Asp was increased by 34%, while the concentrations of Asn and Met were increased by 95-153%. The other amino acids showed similar concentrations and no significant differences compared to the wild type.
Discussion
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that more inorganic nitrogen was assimilated into amino acids, which resulted in the synthesis of more amounts of soluble proteins. The total nitrogen con-tent of the DvGS1-1 line was higher than in the wild-type plants. This indicated that expression of DvGS1 actually affected the host’s nitrogen metabolism. An increased nitrogen content and at the same time de-creased or almost identical concentration of carbon resulted in the decreased C:N ratio of the DvGS1-1
line compared with the wild type under both nitrate supplies. The reduction in the C:N ratio probably ef-fected the better growth phenotype of the DvGS1-1 transgenic line. It is known that a high C:N ratio in the cell usually results in reduced proliferation of lat-eral roots and smaller shoots of plants (Malamy and Ryan, 2001; Kant et al., 2008). In trying to improve the NUE, it has been widely recognized that the link between carbon and nitrogen is crucial (Makino et
Fig. 4. Differences of enzymatic activities in various tissues of transgenic DvGS1-1 line (black columns) and wild-type (white columns)
Arabidopsis plants. The activities of GS (A, C), GDH (B, D), Fd-GOGAT (E, G), NADH-GOGAT (F, H), AlaAT (I, K), AspAT (J, L), NR (M, O) and NiR (N, P) in leaves, stems and roots of 30-day-old plants grown under normal (9 mM) and low nitrate (2 mM) conditions for 30 days after sowing. Data represent the means±standard deviation of three biological samples, and three technical repeats for each sample. Significant differences are calculated by two-tailed t-test at the levels of *P<0.05, **P<0.01, ***P<0.001.
al., 1997; Reich et al., 2006). The lower concentra-tions of glucose, sucrose and starch, together with the higher accumulation of fructose in different tissues of the DvGS1-1 line confirmed that the increased total nitrogen content caused by the overexpression of DvGS1 also affected the metabolism of total sac-charides directly or indirectly. Sucrose occupied the maximum ratio of concentration of the four saccha-rides and the reduction in sucrose concentration in leaf and stem tissues under both nitrate conditions was in correlation with the reduction of total carbon content in the DvGS1-1 line. Generally, we showed that the lower C:N ratio of the whole plant and syn-thesis of increased amounts of soluble proteins in leaf tissue resulted in the production of a higher plant biomass (higher fresh weight and dry weight in DvGS1-1 line) in the DvGS1-1 line as compared to the wild-type plaTo further elucidate the molecular mechanism for the NUE improvement of DvGS1 -transgenic Arabidopsis, the transcriptional levels and enzymatic activities of NUE-related genes in various tissues of the DvGS1-1 line together with the concen-trations of individual free amino acids from leaf tissue of the DvGS1-1 line under both nitrate conditions, were subsequently investigated. It was concluded that overexpression of DvGS1 directly resulted in up- or downregulated transcriptional levels of several NUE-related genes that play important roles in nitrogen assimilation and amino acid biosynthesis. As for the leaf tissues, the higher enzymatic activities of Fd-GO-GAT, GDH, AlaAT, AspAT and GS in the DvGS1-1 line under both nitrate supplies revealed that these increased activities were attributable to the higher levels of gene expression, because the transcriptional levels of AlaAT1, GDH2, Fd-GOGAT1, AspAT1/As-pAAT and Gln1-3 were significantly upregulated un-der both nitrate supplies (Fig. 3E, F). Previous inves-tigations showed that GS/GOGAT systems play key roles in ammonium assimilation in plants (Masclaux-Daubresse et al., 2010; Foyer et al., 2011). It was also shown that GDH is involved in ammonium assimila-tion to form glutamate, and is confirmed to fuel the tricarboxylic acid (TCA) cycle (Miyashita and Good, 2008). In plants, the primary anabolism of alanine, the nontoxic storage form of nitrogen, appears to
en-1116
zymatic activities of NR and NiR to maintain more ef-ficient and economical metabolism of nitrogen inside the leaf and root tissues of the DvGS1-1 line.
The higher concentrations of total amino acids and individual amino acids in the leaves of the Ara-bidopsis DvGS1-1 line were also closely related to the improved NUE. The concentrations of Asp, Glu and Asn in the DvGS1-1 line were significantly increased under both nitrate conditions. These amino acids rep-resent nontoxic storage forms of nitrogen in plants and also play important roles in nitrogen transport and remobilization (Miyashita et al., 2007; McAllis-ter et al., 2012). The Met also showed significantly increased levels (153% and 60%, respectively, higher than wild-type leaves under low and normal nitrate conditions). This sulfur-containing amino acid is an essential amino acid for protein biosynthesis and plays an important role in seed germination, plant growth and the quality of crops (Müntz, 1998). As the total amino acid concentration of the DvGS1-1 line was increased by 36-53% compared to the wild type under both nitrate conditions, the four types of amino acids (Asp, Asn, Glu, Met) should account for the major proportion of the total amino acids in the leaves. Therefore, it was concluded that the sig-nificantly increased concentrations of Glu, Asp, Asn and Met resulted in a higher concentration of total amino acids, and the higher concentrations of amino acids were associated with the rise of soluble protein concentration. The increased efficiency of nitrogen assimilation also affected the biosynthesis of carbo-hydrates, which resulted in the reduction of the C:N ratio in the whole plant.
CONCLUSIONS
Our study confirmed that the overexpression of DvGS1 in A. thaliana directly enhanced the expres-sion of GS, GOGAT, GDH, ASN,AlaAT and AspAT genes, which were important to nitrogen assimilation and remobilization. The enhanced enzymatic activi-ties of the proteins encoded by these genes resulted in higher concentrations of total amino acids and soluble proteins. The higher activities of AlaAT,
As-pAT and GDH simultaneously affected carbon me-tabolism in the Arabidopsis DvGS1-1 transgenic line, which resulted in increased total nitrogen content and decreased C:N ratio in the whole plant. Finally, these improved metabolisms of nitrogen and carbon con-ferred the DvGS1-transgenic Arabidopsis higher NUE, more global biomass and better growth phenotype compared to wild-type plants.
Acknowledgments: This work was supported by grants from Shanghai Municipal Natural Science Foundation (14ZR1414500) and Ministry of Agriculture of China (2014ZX08003-005).
Authors’ contributions: CZ is the main author who con-tributed to the design of the research and conception of the project, organization and analysis of the data and writing of the manuscript. GZ, SC, WW and YT contributed to the design of the research and data analyses. CZ and RS were supervisors of the research project and reviewed several drafts of the manuscript. All authors read and approved the final manuscript.
Conflict of interest disclosure: The authors declare that there are no conflicts of interest.
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