Background

Nitrogen is one of the most important nutrient resource and limiting factors for plant growth, it is required for the synthesis of many cellular components, including amino acids, proteins, nucleic acids, lipids, chlorophyll and other metabolites compounds (Numes et al., 2010) Currently, about half of all nitrogen used for crop production is applied as urea in agriculture, however, urea fertilizer has adverse effects on seed germination, seedling growth, and early plant growth (Sumner, 1926; Uheda and Maejima, 2009; Yu and Zhang, 2012). Previous study has been reported that urea can be originates from Arginine breakdown, which involved the degradation of storage proteins during seed germination under salt stress condition (Herman and Larkins, 1999). Seed germination is the most important stage for survival during the life of a plant in saline environments. Thus, better characterization of the Arginase gene responses to salt stress during seed germination may therefore facilitate the development of crops in saline stress conditions.

Arginase is a kind of dinuclear manganese protein and has two subtypes (Ⅰ and Ⅱ) (Mori, 2007), it is widely distributed among animals, plants and microorganisms. Currently, it is found and cloned in soybean, tomato, rice, loblolly pine, etc in plants, but not all species have two subtypes. A copy of the arginase gene was found in rice (Ma, 2013), soybean and loblolly pine. In addition, this gene has a great relationship with seed germination and seedling development in soybean (Goldraij and Polacco, 1999) and loblolly pine (Todd et al., 2001). In tomato, LeARG1 or LeARG2 genes are highest in reproductive tissue and high specificity for L-arginine. But, only the LeARG2 gene was strongly expressed in leaves after injury and mediated by jasmonic acid (Chen et al., 2004). In Arabidopsis, AtArgAH1 (At4g08900) and AtArgAH2 (At4g08870) arginase genes have been cloned and identified, and they can complement the car1 (Saccharomyces cerevisiae) mutation in yeast. AtArgAH1 (Krumpelman et al., 1995) and AtArgAH2 (Flores et al., 2008) were significantly different in amino acid sequences in the N-terminal, but also homologous up to 85%, this may be due to the recent gene duplication. GFP-fusion proteins showed that two genes are both mitochondrial proteins (Palmieri et al., 2006).

In this study, we analysed the expression pattern of ArgAHA1 and ArgAHA2 genes in response to salt stress and different nitrogen nutrient during seed germination in Arabidopsis thaliana. Our results show that ArgAH2 was highly expressed in siliques, ArgAH1 was highly expressed in root under normal conditions, respectively. Under salt stress condition, the expression levels of ArgAH1 and ArgAH2 was up-regulated in both roots and cotyledons. In addition, under different nitrogen sources, the expression levels of ArgAH1 and ArgAH2 were shown different pattern in both the roots and cotyledons.

1 Materials and Methods

1.1 Plant materials and growth conditions

Arabidopsis thaliana (wild-type, Col-0) was used in this study. Seeds were sown on half Murashige and Skoog (1/2MS) medium containing 0.8% sucrose (w/v) with or without different stresses. After 4°C for 2 days in darkness treatment, the seedlings were grown at 22°C growth chamber under a 12 h-light/12 h-dark cycle.

1.2 Gene expression analysis

Different organs (roots, stems, leaves, panicle and siliques) of 3 weeks plants were sampled for qRT-PCR. A second batch of seedlings were treated with different concentrations of various stresses (NaCl, NH₄Cl, Urea, Arginine). The roots and cotyledons were sampled after 0, 24, 48, or 72 h treatment. Total RNA was isolated using Trizol (Thermo, USA), cDNA was synthesized using reverse transcriptase (TAKARA). Using Arabidopsis ACTIN gene as an internal standard(Table 1), relative quantification were performed with SYBR green using the real-time qPCR (Agilent, MX3000p).

Table 1 sequence of the primers used for quantitative real-time PCR

2 Results and Discussion

2.1 Tissue specific analysis of AtArgAH1 and AtArgAH2 in Arabidopsis

According to Figure 1-A, AtArgAH1 was mainly expressed in root, leaf, siliques while stem and panicle include small amount of AtArgAH1. The expression of AtArgAH2 was highest in siliques, while it was very low in root and stem (Figure 1-B). It can be concluded that the expression of AtArgAH2 is dominant and high at the mature stage, indicating some differences between the two genes. The pollen contained only ArgAH1 in a tissue-specific manner (Brownfield et al., 2008). In contrast, only ArgAH2 expression was induced after MeJA treatment.

2.2 Expression analysis of AtArgAH1 and AtArgAH2 in response to NaCl stress

Under the different concentrations of NaCl treatment, both genes were significantly up-regulated, except for AtArgAH1 in cotyledons under 75 mmol/L (Figure 2-A-a). But, the increasing level in AtArgAH2 was higher than AtArgAH1, and the expression level of two genes in cotyledons higher than in young roots in the same way.

In Figure 2-B, two genes in the control group (0 mM NaCl) were gradually down-regulated. In the treatment group (125 mM NaCl), the expression of two genes showed a similar trend, that is, up-regulated in cotyledons (Figure 2-B-a, c) and down-regulated in young roots (Figure 2-B-b, c). In addition, the expression of AtArgAH2 in cotyledons increased more significantly than AtArgAH1.

2.3 Expression analysis of AtArgAH1 and AtArgAH2 in response to NH₄Cl treatment

Under the different concentrations of NH₄Cl stress, AtArgAH1 was up-regulated in cotyledons (Figure 3-A-a), while AtArgAH2 in cotyledons showed a downward trend, particularly, at 100, 125 mmol/L (Figure 3-A-c). The AtArgAH1 in the young roots showed no obvious change, except for a significant increase at 100 mM (Figure 3-A-b). In contrast, AtArgAH2 followed a increasing trend at first reaching a highest point at 100 mM, and then followed a dramatic decrease (Figure 3-A-d).

In Figure 3-B, in the control group, the expression of AtArgAH1 in cotyledons was slightly down-regulated in most times, but the considerable reduction was only recorded at 48 h (Figure 3-B-a). The rest of control group showed substantial increases. In the treatment group, both genes were significantly up-regulated in most cases, except for AtArgAH1 at 72 h in cotyledons (Figure 3-B-a). Overall, there was no significant difference between two genes under the treatment.

2.4 Expression analysis of AtArgAH1 and AtArgAH2 in response to Urea treatment

Under the urea treatment, the expression of AtArgAH1 was moderately down-regulated in cotyledons at 100 mmol/L, and then increased dramatically at higher concentrations (Figure 4-A-a). On contrast, AtArgAH1 in young roots showed a significant downward tendency (Figure 4-1-B), which was the same with AtArgAH2 in cotyledons (Figure 4-A-c). AtArgAH2 in young roots was gradually up-regulated (Figure 4-A-d) although the expression decreased dramatically at highest concentration (150 mmol/L), but it was almost indistinguishable from the control (0 mmol/L).

In Figure 4-B, in the control group, both genes were up-regulated in cotyledons, but significantly down-regulated at 72 h (Figure 4-B-a, c). The expression of AtArgAH1 was substantially down-regulated in young roots at first, but it was slightly up-regulated again as time passed (Figure 4-B-b). The AtArgAH2 gene in roots showed a dramatic upward trend, and the expression decreased at 72 h (Figure 4-B-d). In the treatment group, AtArgAH1 was down-regulated in cotyledons, up-regulated in young roots, and the highest point was at 24 h. The expression of AtArgAH2 increased significantly in both cotyledons and young roots.

2.5 Expression analysis of AtArgAH1 and AtArgAH2 in response to arginine treatment

Under the arginine treatment, the expression of two genes in cotyledons showed a similar trend in which they were considerably up-regulated (Figure 5-A-a, c). In root, on contrast, there was only one significant increase at 1.25 mM in both genes, and the rest of concentrations had no noticeable change (Figure 5-A-b, d). However, the expression level of AtArgAH1 gene relative to control (0 mM) in cotyledons was higher than in AtArgAH2.

In Figure 5-B, in the control group, AtArgAH1 was up-regulated in both cotyledons (Figure 5-B-a) and young roots (Figure 5-B-b), while AtArgAH2 showed a significant downward tendency as time went by in cotyledons (Figure 5-B-c). On the other hand, AtArgAH2 was dramatically up-regulated in young roots (Figure 5-B-d), except for a slight decrease at 24 h. In the treatment group, both genes were all up-regulated in most times. However, there were some decreases in both genes in cotyledons at 72 h and the AtArgAH1 in roots at 0 h.

According to the data, there were some differences between two genes under different abiotic stresses. In addition, the expression of genes in different tissues was also different. Under the salt stress, gene expression was significantly different, which is consistent with (Shi et al., 2013) conclusion. They believe that the mutant salinity is due to the excessive synthesis of NO and polyamine. This is probably because that arginase competes with other enzymes that degrade arginine, and strength of enzyme activity determines the direction of arginine decomposition (Yang and Gao, 2007).

3 Conclusion

This study mainly focused on the expression analysis of arginase genes in respons to different abiotic stress during the early-seed germination and seedling growth stage using real-time quantitative PCR. The expression of AtArgAH1 and AtArgAH2 was highly expressed in root and siliques under normal conditions, however, AtArgAH2 was higher expression than AtArgAH1 in mature stage. In salt stress, the expression levels of ArgAH1 and ArgAH2 was up-regulated in both roots and cotyledons. But the expression of AtArgAH2 relative to AtArgAH1 is more significant. Under the NH₄Cl stress, AtArgAH1 gene was up-regulated at higher concentrations. Under the urea stress, differences in the expression of two genes were large and irregular. Under the arginine stress, difference was not obvious.

Authors’ contributions

Y. Bu and S. Liu designed the study. X. Shen performed the experiments and analyzed the data. Y. Bu, S. Liu and T. Takano supervised the study and critically revised the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This work was supported by the Special Fund for Forest Scientific Research in the Public Welfare (201404220) , National Natural Science Foundation (NSFC) of China (No. 31500501) and Fundamental Research Funds for the Central Universities (2572016CA14) awarded to Yuanyuan Bu. Further supported by the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13053) awarded to Shenkui Liu.

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