1 Introduction

Salt stress is one of the important environment factors that affects seed germination and seedling growth (Connor et al., 2012). In general, salt stress cause cell membrane damaged, and the activity of the enzyme to reduce, the chloroplast degradation, photosynthesis is restrained in cells, results in both a delay and a reduction of seed germination (Lin et al., 2011; Lin et al., 2014; Lin et al., 2016). In addition, high salinity accumulation in soil have toxic effect on plants and severly inhibited seed germination, lead to metabolic changes (Krasensky et al., 2012). In agriculture, seed germination is a key period that affect plants growth and yieldin saline environment (Weitbrecht et al., 2011). Better characterization of the molecure responses to salt stress during seed germination will provide a sound basis foundation to improve agriculture productivity with increased tolerance to NaCl stress.

Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme in ammoniumassimilation, which is widely diatributed in microorganisms, higher plants and animals. A variety of GS isoenzyme distributed in higher plants, mainly divided into cytoplasmic glutamine synthetase (GS1) and plasmid glutamine synthetase (GS2) (Oliveira, 2002). In higher plants, the cytosolic glutamine synthetase isoform (GS1) encoded by a multigene family, and the chloroplastic glutamine synthetase isoform (GS2) encoded by one gene (Gln2), which have specific, apparently non-redundant, physiological roles in ammonium assimilation (Leegood et al., 1995; Cren et al., 1999; Ishiyama et al., 2004; Canovas et al., 2007). In Arabidopsis, five pupative genes for GS1, AtGln1;1, AtGln1;2, AtGln1;3, AtGln1;4, and AtGln1;5, are encoded in the genome. The exact functional roles and physiological diversities of the individual GS1 isoenzymes in Arabidopsis have not been well characterized.

In this study, we analysed the effect of salt stress and on the growth of AtGS1.5 transgenicand knock out mutant (atgs1.5) plants, the activity of GS and NH₄⁺content of AtGS1.5 transgenic Arabidopsis, atgs1.5 and WT plants. Our results show that overexpression of AtGS1.5 geneimproves salt stress tolerance in Arabidopsis. Furthermore, overexpression of AtGS1.5 gene increased the activity of glutamine synthase and decreased NH₄⁺ content in transgenic plants.

2 Materials and Methods

2.1 Plant materials

A. thaliana (wild-type Col-0), a T-DNA-disrupted AtGS1.5 mutant (atgs1.5: SALK_086579c) and threetransgenic plants were used in this study. The mutant was obtained from the Arabidopsis Biological Research Center (ABRC), were identified by PCR using atgs1.5 primer (Table 1). Transgenic plants was performed by the Agrobacterium-mediated floral-dip method (Clough et al., 1998), and T3generation plants were used for analyses.

2.2 Growth conditions

To reduce variation in germination, we sorted the seeds with an 80-mesh sieve to remove smaller seeds, and used seeds between 250 and 300 um in size. After 2d of cold (4°C) treatment, the seedlings were grown at 22°C under a 12-h-light/12-h-dark cycle in a growth chamber. Seeds were surface-sterilized and plated on different types of solid one-half-strength Murashige and Skoog (1/2MS) medium with or without salt stress: 1/2MS+ NaCl (0, 125, 135, 150) mM. Root length and fresh weight were measured after 2-3 weeks cultivation.

2.3 Total DNA and RNA extraction and gelblot analysis

Total DNA isolated using CTAB were used for PCR and DNA gel boltting. Gene-specific primers were used, respectively: for atgs1.5 primer as Table 1. The homozygous lines of the mutant were screened with a primer binding at the left border of the T-DNA insert (LBP for the Salk Institute collection) in combination with a gene-specific primer. For southern blot analysis, the DNA were digestion by SacI, a probe labeled with digoxigenin (DIG, Roche, USA) with DNA gel blot analysis. Total RNA extraction was performed using RNA extraction kit (Thermo,USA). RNA gel blot analysis was performed as described previously (Guo et al., 2013).

2.4 Measurementof ammonium content

The NH₄⁺ was extracted and concentration was determined by the Ammonia Assay Kit (Cominbio, China) according to manufacturer’s instructions. About 0.1 g sample was used and added 1ml extracting solution with sufficient mixing, centrifugal 10 min with 16000 rpm and get the supernatant finally, then add 300µl buffer1, 10 µl buffer2, and 200 µl supernatant was boiling in water bath for 5 min, room temperature cooling 10 min, add 500 µl buffer3, measuredat OD=580nm.

2.5 Glutamine synthetase activity assay

The GS synthetic activity was determined by the GS Activity Assay Kit (Cominbio, China) according to manufacturer’s instructions. About 0.1 g sample was used and added 1ml extracting solution with sufficient mixing, centrifugal 10 min with 8 000 g and get the supernatant, 320 µl buffer1 and buffer2 were separately added in testing tube and contrast tube, then 140 µl buffer3 and 140 µl sample were added both in testing tube and contrast tube, mixing and water bath with 25°C 30min，200 µl buffer4 were added, mixing and standing 10 min, centrifugal 10 min with 5 000 g, get the supernatant and measure at OD= 540 nm.

3 Results and Discussion

3.1 Characterization of AtGS1.5 transgenic and mutant Plants

The T-DNA insertion is located in the third exon of AtGS1.5 (Figure 1A), as confirmed by PCR-based genotype analyses (Figure 1B). RT-PCR and Northern blotting analyses of the homozygous T-DNA insertion atgs1.5 mutants show that they completely lack AtGS1.5 transcripts (Figure 1A; Figure 1B). These results indicate that the homozygous T-DNA insertion in the AtGS1.5 locus resulted in a null mutant. Three Arabidopsis transgenic plants that overexpressed AtGS1.5 under the control of the CaMV35S promoter (#1–3) were identified by northern (Figure 1C) and southern blotting (Figure 1D). Control samples showed weak AtGS1.5 signals, whereas the transgenic plants had higher expression confirming that the plants had been successfully transformed with AtGS1.5. Southern blot analysis show thatthreetransgenic plants with multiple copies than that of WT plants (Figure 1D).

3.2 Overexpressionof AtGS1.5 gene enhances tolerance to salt stress in Arabidopsis

The seeds of WT, atgs1.5, and AtGS1.5 were germinated on 1/2 MS medium supplemented with different concentrations of NaCl (Figure 2A), the growth of WT, atgs1.5, and AtGS1.5 were not obviously different in medium without salt stress (0 mM NaCl). Moreover, in the presence of different concentrations of NaCl (125, 135 and 150 mM), the root lengths (Figure 2B) and fresh weights (Figure 2C) of AtGS1.5 were markedly higher than those of atgs1.5 mutants and WT plants. These results demonstrate that overexpression of AtGS1.5 enhances salt tolerance during seed germination.

3.3 Effect of GS activity on seed germination under salt stress

Total glutamine synthetase (GS) activity was determinded from the WT, AtGS1.5 transgenic and atgs1.5mutant plants under NaCl stress during seeds stratification or after seeds stratification. On the control medium, the GS activity were slightly higher in AtGS1.5 transformants than those of WT and atgs1.5 mutant seeds within 12 or 48 h of seeds stratification, whereas the GS activity levels began to significantly increase in the AtGS1.5 transformants seeds at 48 h of seeds stratification after expose to 125 mMNaCl, comparable to that of WT and atgs1.5 mutant seeds (Figure 3A). After seeds stratification, the levels of GS activity clearly higher in AtGS1.5 transgenic plants (Figure 3B), while the atgs1.5 mutant exhibited lower levels under 0 mM NaCl condition. In the presence of 125 mM NaCl, the GS activity levels were significantly increase in AtGS1.5 transgenic plants, however the atgs1.5 mutant exhibited lower levels (Figure 3B). These results suggest that increase GS activity improved salt tolerance during seed germination.

3.4 Effect of ammonium on seed germination under salt stress

Glutamine synthetase is a key enzyme in plant nitrogen metabolism responsible of the first step of ammonium assimilation. NH₄⁺ as the final component in nitrogen metabolism (Wu et al., 2001), we also detedcted the NH₄⁺ concentration in WT, AtGS1.5 transgenic and atgs1.5 mutant plants under NaCl stress during seeds stratification or after seeds stratification. On the control medium, NH₄⁺ concentration were slightly higher in atgs1.5 mutant seeds than those of WT and AtGS1.5 transformants within 24 or 48 h of seeds stratification, whereas the NH₄⁺ levels began to significantly increase in WT and atgs1.5 mutant seeds at 48 h of seeds stratification after expose to 125 mM NaCl, comparable to that of the AtGS1.5 transformants seeds (Figure 4A). After seeds stratification, the NH₄⁺ levels were clearly higher in atgs1.5 mutant, while the AtGS1.5 transgenic exhibited lower levels under 0 mM NaCl condition. In the presence of 125 mM NaCl, the NH₄⁺ levels were significantly increase in atgs1.5 mutant plants, however the AtGS1.5 transgenic plants exhibited lower levels (Figure 4B). These results suggest that decrease NH₄⁺ levels improved salt tolerance during seed germination.

In Arabidopsis, >90% of seed N is incorporated into storage proteins (Baud et al., 2002; Li et al., 2006b). When these storage proteins are degraded during germination (Hong et al., 2012), ammonium is produced and needs to be reassimilated into Gln for subsequent remobilization to support seedling growth (Rentsch et al., 2007). However, excessive nitrogen accumulation produce NH₄⁺ can cause toxic effects. In our present study, we found that overexpression of AtGS1.5 gene in Arabidopsis thaliana can obviously increase the tolerance of plants to salt stress during seed germination stage, suggesting that enhancing GS activity improves salt tolerance during seed germination by lowing the concentration of NH₄⁺.

Acknowledgements

This work was supported by the Fundamental Research Funds for the Central Universities (2572016CA14) and Heilongjiang Province foundation for Returnees (LC201405) awarded to Yuanyuan Bu. Further supported by the Program for Changjiang Scholars and Innovative Research Team in University (IRT13053) awarded to Shenkui Liu.

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