Expression Analysis of Arabidopsis thaliana Genes ARGAH1ARGAH2 and the Response to Sodium Chloride Stress During Seed Germination  

Xiaoxu Zhang1 , Xinxin Zhang1 , Tetsuo Takano2 , Shenkui Liu1 , Yuanyuan Bu1
1. Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field (SAVER), Ministry of Education, Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry University, Harbin 150040, China
2. Asian Natural Environmental Science Center (ANESC), The University of Tokyo, Tokyo 188-0002, Japan
Author    Correspondence author
Genomics and Applied Biology, 2014, Vol. 5, No. 2   doi: 10.5376/gab.2014.05.0002
Received: 18 Feb., 2014    Accepted: 21 Mar., 2014    Published: 29 Apr., 2014
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This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Zhang et al., 2014, Expression Analysis of Arabidopsis thaliana Genes ARGAH1ARGAH2 and the Response to Sodium Chloride Stress During Seed Germination, Genomics and Applied Biology, Vol.5, No.2, 1-6 (doi: 10.5376/gab.2014.05.0002)


Arabidopsis thaliana possesses two arginase-encoding genes(ARGAH1ARGAH2), which catalyses the catabolism of arginine into L-ornithine and urea. In this research, we focus on the function of the two genes (ARGAH1ARGAH2) encoding Arabidopsis thaliana arginase. The cDNA of cloned ARGAH1 is 1347 bp, and 5' untranslated region (UTR) is 145 bp and the 3' UTR is 166 bp. The open reading frame of ARGAH1 is 1029 bp, encoding 342 amino acids. ARGAH2 is 1284 bp, and 5' untranslated region (UTR) is 34 bp and the 3' UTR is 215 bp. The open reading frame of ARGAH2 is 1035 bp, encoding 344 amino acids. We studied the different expression models of arginase genes under NaCl stress by qRT-PCR and other biotechnology. Interestly, we observed that the expression of ARGAH1 and ARGAH2 is both induced by NaCl stress. At the same time, the enzyme assay of argianse also showed an increase during seed germination. Therefore, we maintain that the argianse genes play a role against to NaCl stress especially during the germination.

Arabidopsis thaliana; ARGAHs; Salt Stress; Seed Germination

The life circle of higher plants begins with the seed germination (Yan, 2014); the seed germination directly concerts the survival rate of seedings and the condition of the follow-up growth (Gutterman, 2002). Therefore, the germination of seed has a significant meaning to the growth and development. As we know, the seed of Arabidopsis thaliana is surrounded by the endosperm, which is made up by only a single layer cell and the episperm (Holdsworth et al., 2008; Weitbrecht et al., 2011). Then the seed germination is defined as two stages. The first stage is completed by the time that the radicle broken through ednosperm. And the second stage is accomplished by the time radicle broken through episperm (Bentsink and Koornneef, 2008). During the seed germination period plants need to carry on a large amount of metabolism for accomplishing the growth and development. However, there are lots of environmental factors that restrict the seed germination and the salt stress is a kind of stresses.

Salt stress is a usual stress type, the half of the world irrigation soil and the twenty percent of cultivated land is suffered to the salt stress at different levels (Zhu, 2001). Salt stress causes ion poison, osmotic stress, and the accumulation of ROS, leading to the peroxidation of lipid and the inactivation of antioxidant enzyme (Mittler, 2002; Tanou et al., 2009). It is reported that salt stress has a serious influence to seed germination. High salt density can decrease the germination rate of seed, delaying the germination time and reducing the rate that seeding grown up (Almansouri et al., 2001). Consequently, researching the influence of salt stress in the seed germination has a significant meaning in order to find out the suitable method to decrease even remove the effect of salt to seed germination.

A lot of nitrogen elements largely in seed is stored in the form of storage proteins, providing an adequate nitrogen source for the subsequent seed germination and seeding growth in the early development period. Studies show that the main form of storage proteins is arginine in the plant seed (Chat Thai and Misra, 1998; King and Gifford, 1997). During the seed germination period, a large number of storage proteins degraded, and we can detect a significant increase in arginine content (Herman and Larkins, 1999). For example, during the seed germination of Loblolly Pine (Pinus taeda L.), the storage proteins existing in megagametophyte degraded, and then the amino acid content of seedings increased largely. Among the amino acid, the part of arginine is the most, almost accounted for half of the nitrogen storage in the storage proteins in megagametophyte (King and Gifford, 1997). Consequently, we can know that arginine play an important role in seed germination progress.

Arginase (E. C. 3. 5. 3. 1) is an enzyme contained manganese metal, and it has three subunits, each of which requires two Mn atoms, and arginase specificity catalyzes L-arginine into L-ornithine and urea (da Silva et al., 2008; Di Costanzo et al., 2005; Reczkowski and Ash, 1992). Currently, the arginase of many plants has been studied. In soybean, there is a sharp increase expression during germination period, which reached the maximum 3 or 5 days later after germination (Goldraij and Polacco, 1999); In rice, the only arginase gene plays a key role in the development of panicles and grain yield, especially when the external source of nitrogen deficiency (Ma et al., 2013); There are two argianse genes in tomato -LeARG1, LeARG2, both of which are abundantly expressed in reproductive tissues such as the bud, not open mature flowers, already open matured flowers and unripe fruits (Chen et al., 2004; Alabadí et al., 1996)); In Arabidopsis thaliana, there are two arginase genes which has been cloned, namely ARGAH1, ARGAH2 (Flores et al., 2008; Krumpelman et al., 1995). ARGAH1 can be transcripted and expressed (Flores et al., 2008). And the expressing of ARGAH2 is induced by Botrytis cinerea and methyl jasmonate (Brauc et al., 2012; Brownfield et al., 2008); during germination and post-germination in loblolly pine, the arginase activity is also increased largely, accompanying with the increase of arginase gene expressing (King and Gifford, 1997; Todd et al., 2001). In addition, argianse activity has a rapid increase during the germination in many plants, such as pumpkin (Splittstoesser, 1969), broad bean (Kollöffel and van Dijke, 1975), soybean (Kang and Cho, 1990), Arabidopsis thaliana (Zonia et al., 1995).

In addition, it is reported that arginase has a response to various biotic stresses and abiotic stress through the regulation of arginine metabolism and polyamine pathway. For example, the mutant of Arabidopsis thaliana arginase accumulated less ROS, which indicated that inhibiting the expression of ARGAHs can improve the tolerance to dehydration, high salt stress and chilling stress (Shi and Chan, 2013). Furthermore, Brown and his team have found out that Botrytis cinere induced the expression of ARGAH2 and the increase expression of ARGAH2 increased the tolerance to Botrytis cinere in Arabidopsis thaliana (Brauc et al., 2012; Brownfield et al., 2008). Similar to this, previous studies have shown that the deletion mutant of ARGAHs genes led to an increase accumulation of nitric oxide, and the overexpression of ARGAH2 enhanced the development of callus tissue when the plant is infected by Clubroot. In tomato, the expression of LeARG2 is induced by wounding. However, LeARG1 is not (Chen et al., 2004). In conclusion, we can summarize that arginase genes are largely expressed during seed germination, and arginase plays a role in the response to many adversity stresses in plants.

In this study, we take Columbia-type Arabidopsis as experimental material. By the means of real-time fluorescence quantification PCR (QRT-PCR) and Semi-quantitative RT-PCR (SqRT-PCR), we analysis the expression characteristics of ARGAHs genes during seed germination, especially under NaCl treatment, and aim to know about the function of arginase genes responding to salt stress during seed germination.

1 Results and Discussion
1.1 Homologous comparison analysis of ARGAH1 and ARGAH2 genes
There are two genes who encode arginase: ARGAH1, ARGAH2. Biological chemistry analysis shows that these two genes both had arginase activity. We successfully cloned these genes: ARGAH1, ARGAH2; meantime, we got matched the sequence data on NCBI. The full length of the cloned cDNA sequence of ARGAH1 is 1347 bp; 5’ non-coding region lasts 145 bp, and the 3’ non-coding region length is 166 bp; the ORF length is 1029 bp, encoding 342 amino acids, and the molecular mass prediction is 37344 Da. The predicted isoelectric point is 6.11. On the other hand, the full length cDNA of ARGAH2 is 1284 bp; its 5’ non-coding region lasts 34 bp, and 3’ non-coding region is 215 bp. The ORF of ARGAH2 is 1035 bp. ARGAH2 encodes 344 amino acids, and the molecular mass prediction is 37980 Da. The predicted isoelectric point is 5.90. The similarity of ARGAH1 and ARGAH2 amino acids is 99%; and 86% with Brassica napus, 81 % with Arginase 1 in tomato.

To further investigate the relationship between these two genes in Arabidopsis thaliana, and the cognate relation with other species, we select the arginase genes from other species to do the comparison. The conclusion will be drawn that the Arabidopsis thaliana arginase1 has the closest relationship with Arabidopsis lyrata and Brassica napus; however, the Arabidopsis thaliana arginase2 has the closest relationship with wheat and Brachypodium distachyon. Two arginases in tomato have a close relationship with each other (Figure 1).



Figure 1 The phylogenetic tree of argianse in selected higher plants

1.2 The expression analysis of ARGAHs genes during different development periods
To clarify the expression characters of arginase gene in different growth periods in Arabidopsis thaliana, this research selects dry seed, normal raised seedling after 36 hours and 2-week seedling as experimental material. Using QRT-PCR and SqRT-PCR methods to research the expression of the arginase genes in different periods of growth. ARGAH1 and ARGAH2 genes expressed a lot in dry seed, and the least in 36-hour seeding. This result shows that the expression of arginase genes is abundant in germination, which is consistent with the findings about these two genes expressed a lot in tomato reproductive tissue (Chen et al., 2004) (Figure 2).



Figure 2 Expression analysis of ARGAH1 gene and ARGAH2 gene during different developmental stages

1.3 Expression Analysis of arginase genes under NaCl treatment during seed germination in Arabidopsis thaliana
The results of the analysis of the arginase genes expression show that the arginase genes expressed abundantly during seed germination. To further investigate the change of the arginase genes expression under the NaCl treatment, this research conducted SqRT-PCR and QRT-PCR methods to do the experiments to study it.

1.4 Expression Analysis of arginase genes under the NaCl treatment in different concentrations
To choose the suitable NaCl density, we read numerous academic papers, finally choose 0, 50, 100, 150, 200 mM as 5 concentration gradients to be treatment conditions.

The result shows that the expression levels of ARGAH1 and ARGAH2 genes had risen with the increasing of NaCl concentration. Under 200 mM NaCl treatment, the expression levels of ARGAH1 and ARGAH2 reached the highest (Figure 3).



Figure 3 Expression analysis of ARGAHs genes under NaCl treatment by SqRT-PCR

1.5 Expression analysis of arginase genes during different germination stages under NaCl treatment
We can see that the expression levels of ARGAH1 and ARGAH2 were increased with increasing NaCl concentration. In order to detect the expression levels of ARGAH1 and ARGAH2 under NaCl treatment, we chose 100 mM as the experimental concentration. The seed sterilized was sown in 1/2 MS medium and 1/2 MS medium adding 100 mM NaCl. The seed was vernalized at 4 ℃ for two days and then transferred to the culture chamber to culture. RNA was extracted from the germinating seed after different culture time (0, 12, 24, 36, 48) h, and then was reverse transcripted to cDNA, the expression levels of ARGAH1 and ARGAH2 was detected by Real-time quantitative PCR. Experimental results are shown in Figure 4, both of ARGAH1 and ARGAH2 were largely expressed in the seed; the expression level of ARGAH1 gene is induced by 100 mM NaCl treatment compared with the control group with no NaCl added. The expression level of ARGAH1 is a little higher after 0 h culture. The speculated reason is that the material was taken out from low temperature (4 ℃), and it is reported that low temperature induce the expression of ARGAHs. In this study, the expression model of ARGAH2 is similar to ARGAH1, except for the time after culture 36 h and 48 h at which time expression of ARGAH2 gene under NaCl treatment was a bit lower than control. It is not clear that the reason causing this situation, so further research is needed to find it out.



Figure 4 Real-time quantitative expression analysis of ARGAHs gene under NaCl treatment during seed germination

1.6 The enzyme activity assays of arginase
In this study, we analyzed the arginase activity under 100 mM NaCl treatment during seed germination. We extracted the whole protein from fresh materials and detected the arginase activity. We can see that the arginase activity in seed was the highest, and the arginase activity is higher under NaCl treatment compared with control.There is an increase in 36 h, while the possible reason needs further analysis to find out (Figure 5).



Figure 5 The analysis of argianse activity during seed germination under 100 mM NaCl treatment in Arabidopsis thaliana

3 Materials and Methods
3.1 Materials and Treatments 
Arabidopsis thaliana ecotype Columbia seed was preserved in the Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry Univeristy, and Harbin, China.

The seed was sterilized with 75% ethanol and sodium hypochlorite, and then was sown in glass containers filled with 1/2 MS medium in the growth chamber after stratification at 4 ℃ for two days in darkness. For experiments about analysis of expression level during different developmental stages, RNA was extracted from seed just after sterilization, 36-hour-old seeding, 3-week-old seeding. For experiments about analysis of expression model under NaCl treatment, seed was sown in glass container filled with 1/2 MS medium, adding different concentrations of NaCl (0,50,100,150,200) mM. For experiments about the analysis of arginase genes under NaCl treatment during seed germination, RNA was extracted from the seed treated by 100 mM NaCl after culture different time (0,12,24,36,48) h.

3.1 Arginase phylogeny
Members of the arginase in plants were obtained by BLAST searches in NCBI. The cDNA of ARGAH1 and ARGAH2 was obtained from TIGR databases. A total of 28 sequences were used for construction of the phylogenetic tree (Figure 1).

3.2 Semi-quantitative reverse-transcription PCR and real-time quantitative PCR
Total RNA was isolated from 0.2 g seed using a modified method as described previously by Martin (Martin et al., 2005), and treated by RNase-free DNase(Takara) to remove genomic DNA. First-strand cDNA was synthesized using Prime Script RT reagent kit (Takara) from 1 μg total RNA, and 1 μl RNA was added to 30 μl PCR mixture for SqRT-PCR. For real-time quantitative PCR, a 20 μl mixture was used with SYBR-green fluorescence (TransGen Biotech) and the comparative △△CT method was used as previously described (Yang et al., 2011). All the primers were referred to Brauc (Brauc et al., 2012).

3.3 Enzyme assays
The method used to measure arginase activity is as described by King (King and Gifford, 1997). Specific operation will not be described.

This work was supported by specific fund for forest scientific research in the public welfare (201404220) and Program for Changjiang Scholars and Innovative Research Team (PCSIRT, IRT13053).

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