Introduction
Plants may be subjected to kinds of abiotic stresses in the process of growth. Aluminum toxicity and low pH is a major environmental factor limiting plant growth and production on acid soils and 50% of the world’s arable land is under the threat of soil acidification (Shinozaki et al., 2003). Aluminum toxicity may be triggered in both acid and alkaline conditions due to the complex chemistry of Al. The trivalent Al species (Al³⁺) dominates in acid conditions (pH < 5.5), whereas Al(OH)4- in alkaline conditions and an extremely toxic polynuclear Al species, Al₁₃ can also form when Al solutions are partially neutralized with a strong base. All those Al species can inhibit root growth and then limit the growth and production, so it’s of great value to clarify the molecular mechanism of aluminum toxicity in the saline-alkalized land (Foy, 1988). Aluminum of trace amount can promote growth of plants to some degree (Peng et al., 2003), but it turns to be toxic when the concentration increases to the range of micromole (Doncheva et al., 2005). Studies on maize have indicated that the high concentration of aluminum Al³⁺ can inhibit cell divisions in short time (Matsumoto, 2000). The most obvious symptoms of the plants being cultivated with the micromolar concentration of Al³⁺ is the inhibition of the elongation of root (Zhang and Rengel, 1999), and the root become short and thick (Horst, 1995). The root apex accumulates more Al and suffers greater physical damage than the other parts of the root (Foy et al., 1967). The aluminum toxicity can not only hinder minerals absorption and restrain cellular metabolism (Kollmeier et al., 2000), but also break the balance of hormone and exert influence on activities of enzymes, and inhibit mitosis and the synthesis (Matsumoto, 2000). As far as the cells and tissues are concerned, aluminum toxicity can destroy the structure and function of the membrane (Zhang et al., 1997) and the stability of the cell wall (Matsumoto, 2000) and cytoskeleton (Sasaki et al., 1996), and it will be an inhibition of cell-elongation (Willats et al., 2001). From the level of organs ,it performed to inhibit the growth of plant root, stem, leaf (Ryan et al., 1993; Samuels et al., 1997; Sivaguru and Horst, 1998), and prompt tissue necrosis (Shen and Yan, 2001). In recent years, the researchers cloned a series of genes associated with aluminum toxicity. At the molecular level there has a certain explanation how plant response the aluminum toxicity (Yang et al., 2004), however the main stress mechanism of aluminum toxicity in plant is still unknown. Puccinellia tenuifolra, puccinellia of gramineous, also known as the star grass, it is a perennial herbs that mainly distributed in saline-alkali land in the northeast China plain. Because of its strong adaptability, puccinellia can grow in the extremely severe salinization of soil, and even in pH up to 11 of the soil it can also be normal growth. Puccinellia is important to restore the saline soil vegetation (Wang and Guo, 2001), but at present the research on mechanism of aluminum toxicity in puccinellia was not reported. This study screened PutSTE24 gene of puccinellia with aluminum resistance. Using biology software to predict the subcellular localization of this gene, using yeast InvsI to express PutSTE24 gene to clarify the positioning results. By real-time fluorescent quantitative PCR technique to detect PutSTE24 mRNA expression features. Construction of plant expression vector to transform Arabidopsis thaliana, take advantage of resistance experiment in seed germination to confirm the relationship between Puccinellia and aluminum toxicity. This study laid the foundation for further researching the interactions between PutSTE24 and the related gene of Puccinellia and regulatory mechanism.

1 Results and Discussion
1.1 Homologous comparison analysis of PutSTE24 gene
cDNAs library of Puccinellia has been done by our lab, we obtained the PutSTE24 gene KJ598490 length is about 1 800 bp under the aluminum resistance screening, coding frame is 1 275 bp, coding protein size is 424 amino acid, predict the molecular weight of 48.3 kD. The isoelectric point is 6.84. According to the NCBI, using the BLAST search tool, we identified an expressed sequence of PutSTE24. We can find higher homology 12 species such as Brachypodium distachyon. Using MEGA software to analyze the evolutionary tree (Figure 1), the evolutionary tree is in order to resolve the relationship between genes and other species. Results show that the Puccinellia PutSTE24 shares 90% identity with Brachypodium distachyon and barley, 80% identity with rice, sorghum, and maize, 70% identity with this CAAX protease of STE24 in Arabidopsis thaliana and canola.

Figure 1 Phylogenetic tree analysis of PutSTE24 gene

 
1.2 Subcellular localization of converting PutSTE24 gene of yeast cells
Through bioinformatics software to predict the positioning of Puccinellia PutSTE24 genes encode proteins within the cells. The results show that the PutSTE24 genes encode proteins is utmost in the endoplasmic reticulum (Figure 2A). At the same time, this study further used the Polyethylene glycol (PEG)/lithium acetate (PEG/LiAc) to transform thepYES2: GFP, pYES2: PutSTE24: GFP into wine yeast in INVScI. After galactose induced by laser confocal microscopy to detect the fluorescence. We can observe the control pYES2::GFP abundantly expressed in the cytoplasm in yeast (Figure 2, pYES2), and PutSTE24:: GFP fusion protein showed a GFP fluorescence can be observed (Figure 2, pYES2-PutSTE24), and we can also see the cell structure that present similar circular or oval, it is a typical structure of the endoplasmic reticulum, at the same time we combined with the predictions of a biological software, presumably the genes that are located inendo plasmic reticulum. According to the AtSTE24 of Arabidopsis that are located in the endoplasmic reticulum and it is the homologous gene with PutSTE24 genes of Puccinellia. And in other yeast and animal cells, the CAAX protein located in endoplasmic reticulum (Dai et al., 1998; Hildebrandt et al., 2013; Manolaridis et al., 2013), so you can preliminary confirmed that the PutSTE24 gene located in the endoplasmic reticulum.

Figure 2 The subcellular predication of the deduced PutSTE24 and PutSTE24 located in yeast cells

 
1.3 PutSTE24 mRNA expression in stress analysis
To investigate correlation of PutSTE24 and abiotic stress response, the real-time fluorescent quantitative PCR was used to analyze gene expression characteristics under aluminum poisoning and salinity stress. The result showed that the expression of PutSTE24 significantly was induced by AlCl₃, NaCl and NaHCO₃ stress. Firstly, the expression of PutSTE24 in different tissues and organs (root, stem, leaf, flower and sheath) was analyzed using qPCR. The results displayed the expression level of PutSTE24 was the highest in root, then the second is in leaf and sheath, and the expression level was least in stems and flowers. The expression of root was about 3 times of stems and flowers, and the expression of leaves and sheath was about 2 times of stems and flowers (Figure 3). The homologous gene of Arabidopsis AtSTE24 in various tissues expression characteristic analysis showed the stem and leaf expression of AtSTE24 was similar with PutSTE24, and leaf expression was also higher than stem (Bracha et al., 2002).

Figure 3 Real-time quantitative expression analysis of PutSTE24 tissue specificity in puccinellia (root, stem, leaf, flower and sheath)

 
In 100 μM AlCl₃ treatment, the expression of PutSTE24 in root increased in the processing time, and the expression reached the highest in 48 h. It was about 8 times with the control (Figure 4A). The expression had a large amount of growth in the leaf from 6 h, which was about 4-fold higher than the contrast. The expression slightly decreased in 12 h, but still higher than control. Thus the expression was the least within 24 h, which was not different with the control. But the expression level increased in 48 h (Figure 4B). The expression of PutSTE24 reached its highest value at 6 h in the 100 mM NaCl treatment and was 5 fold higher than that of the control. Subsequently, the expression declined gradually at 12 h, 24 h, and 48 h, and remained similar with the control (Figure 4C). In NaCl treatment, the expression increased in 6 h, then decreased in 12 h and the expression rose in 24 h, finally was slowly decreased in 48 h, but the expression is about 1.5 times of the control (Figure 4D). The results of analyses showed that the expression level of PutSTE24 changed after 80 mM NaHCO₃ treatment and that the degree of the change was in direct proportion to the treated time (Figure 4E). Compared with the control (0 h), the expression rose at 6 h and 12 h treatments. However, the expression clearly increased at 24h treatment. The highest expression occurred at 48 h, which was approximately 7-fold higher than that of the control. The expression was detected at different times, and the results are shown in (Figure 4F). The expression level did not change compared with that of the control group. The highest value appeared at 6 h, and the change was significant. The results showed that PutSTE24 can be induced by AlCl₃, NaCl and NaHCO₃ stress. Especially, the gene expression of root greatly increases with the increase of time under AlCl₃ and NaHCO₃ stress, which was about 7.5 times greater than that of the control. In AlCl₃, NaCl and NaHCO₃ stress, the gene expression of leaf began to increase in 6 h, but had a slight decline in 12 h, while the expression rose again in 24 h and 48 h. It was suggested that PutSTE24 is the related gene with AlCl₃, NaCl and NaHCO₃ treatments, may participate in adversity stress response and improve the resistance.

Figure 4 Real-time quantitative expression analysis of PutSTE24 gene under stresses of aluminum (AlCl3), salt (NaCl), alkali (NaHCO3)

 
1.4 Overexpression of PutSTE24 gene tolerance analysis in Arabidopsis
PutSTE24 overexpressing transgenic lines (3#, 4#, 5#, 6#, 8#) expressing the full-length PutSTE24 cDNA from the cauliflower mosaic virus (CaMV) 35S promoter were generated, tested, and confirmed to accumulate the PutSTE24 by northern-blot analysis. The results showed that transformation of T2 generation of Arabidopsis (T2-4#, 5#, 6#) strain can be detected (Figure 5). The growth of wild-type (WT) was similar with the overexpressors. But the phenotype of WT and overexpressors were suppressed after AlCl₃ treatments. The root became short and the growth of leaf is restrained. The overexpressing transgenic lines growth of root and leaf was significantly better than that of WT under 200 μm, 300 μm and 400 μm AlCl₃ treatment (Figure 5). Under 400 μm AlCl₃ (Figure 5D), WT cannot grow, while overexpressors still can grow. Meanwhile, fresh weight of overexpressors distinctly was higher than WT in 300 μm and 400 μm AlCl₃. The results suggested that PutSTE24 overexpressing transgenic had better tolerance of AlCl₃ than WT. So PutSTE24 may be induced to alleviate the Al toxicity.

Figure 5 Resistant analysis of transgenic Arabidopsis over-expressing PutSTE24 in Al-stress

 
2 Material and Methods
2.1 Material
The cDNAs library was constructed and preserved in the Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry University, and Harbin, China. Alkaline grass seeds were cultured with hydroponics. The leaf and root were collected and cryopreservation in -80℃ for RNA extraction.

2.2 Sequence analysis
The analysis of complete nucleotide sequence of PutSTE24 was performed using Blast X in the nonredundant aminoacid sequence database of the NCBI website (http://www.ncbi.nlm.nih.gov). The open reading frame (ORF) and the deduced amino acid sequence were analyzed using DNA star v7.1 software (http://dnastar.com/t-products-lasergene.aspx), to examine the alignments of multiple sequences (Larkin et al., 2007). The maximum likelihood (ML) phylogenetic tree was constructed in the PHYML website (http://atgc.lirmm.fr/phyml/).

2.3 Construction of expression vector and transformation
To understand the subcellular localization of PutSTE24, we predicted its encoded proteins by using bioinformatics analysis software (Prediction of Protein Localization Sites version 6.4) this gene was amplified from constructed cDNA library. The correctly modified gene was inserted into yeast expression vector pYES2 to construct pYES2-PutSTE24-GFP, and then the expression vector was transformed into saccharomyces cerevisiae strains INVScI. After induced by galactose, yeast was detected fluorescence expression by laser scanning confocal microscope for further clarify the subcellular localization of PutSTE24.

2.4 Expression analysis of PutSTE24 under aluminum stress condition
Alkaline grass seeds were pre-cultured with hydroponics for two weeks, then they were cultured in 100 mmol/L NaCl, 80 mmol/L NaHCO₃ and 100 μM AlCl₃. Leaf and root were collected at regular intervals (6 h, 12 h, 24 h, 48 h), and then extracted the total RNA based on the practice of the literature. CDNA was synthesized by reverse transcription according to the manufacturer’s instructions of First-Strand cDNA Synthesis Kit (TakaRa, China). RT-PCR was performed using MxPro-Mx3000P to detect the mRNA expression levels of PutSTE24 gene under different stress conditions and different tissues. A pair of gene PutSTE24 specific primers, F: 5'-ACACCCTTGCGTTCTTAGCAGG-3', R: 5'-AGCCACAATTGGCGGTGCGA G-3' was used to amplify a DNA fragment of 1 275 bp. Another pair of specific primers for the actin of Puccinellia tenuiflora, 5'-TTGAACAAGAAATGGCAACTGCTG-3' (actin-F) and 5'-CAAGGAAAGATGGTTGGAAAAGTG-3' (actin-R), was used to amplify a DNA fragment of 1 275 bp as the internal control. The PCR reaction was performed in a 20 μL centrifuge tube containing 2×BrilliantⅢ SYBR Green QPCR Master Mix (Agilent), 1 μL of cDNA, 1 μL each of the forward and reverse primers (10 μM). The reaction procedure was performed as follows: 40 cycles of 30 s at 95℃, 1 min at 60℃and 30 s at 72℃. Two replicates were performed. Statistical analysis was performed using software SPSS 13.0, and the graph displayed expression levels of gene was drawn according to these data.

2.5 Analysis of putative transformants
The PutSTE24 gene was amplified again from constructed cDNA library using primers associated BamHI. The correctly modified gene was inserted into plant expression vector pBI121 to construct pBI121-PutSTE24, and then the expression vector was introduced into Agrobacterium strain EHA105 by electroporation method. Arabidopsis were transferred with this plant expression vector via Agrobacterium tumefaciens–mediated methods. The putative transgenic seeds (T1) were inoculated on 1/2 MS containing 50 mg/L Kana for resistance screening and detected with northern blot. The positive progeny were cultivated to T3 plants for stress experiment.

2.6 Over-expression of PutSTE24 in transgenic Arabidopsis under aluminum stress
For testing the tolerance for Al stress, positive T2 plants (T2-4#, 5#, 6#) and untransformed (control, WT) plants were aseptically cultured in 1/2 MS included varies of AlCl3 (0 μM, 200 μM, 300 μM, 400 μM) for 14 days. Three independent biological replicates were performed. The effects of phenotypic change and root length under Al stress were observed and recorded. These data were presented as the mean ± standard error from three independent experiments.

Acknowledgements
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 in University of China (PCSIRT) (IRT13053).

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