Aluminum is a non-toxic element in the earth’s crust at normal pH values, but in the acid soils, at the low pH values (pH<5.5), Al³⁺ is solublilized from aluminosilicate clay minerals and is toxic to crop plants (Kochian et al., 2004). Toxic aluminum can disrupt a series of cellular processes, such as nutrient acquisition, cell wall loosening, nuclear division, cytoskeleton stability, cytoplasmic Ca²⁺ homeostasis, hormone transport and signal transduction (Matsumoto, 2000). Previous studies showed that aluminum-activated root malate or citrate exudation from plasma membrane or vacuolar membrane played an important role in plant Al³⁺ tolerance (Hoekenga et al., 2006). For instance, genes AtALMT1 and TaALMT1 discovered in Arabidopsis thilinana and wheat (Triticum aestivum), that encode aluminum-dependant malate tranporters, are the most important way to Al³⁺ tolerance (Kobayashi et al., 2007). Besides these, there are some genes or enzymes else existing in plants, including ZmMATE, OsSTARA1/2, AtSTOP1, AtBCB (Arabidopsis blue copper-binding protein), parB (tobacco glutathione S-transferase) and catalase et al (Satoshi et al., 2007). 

The modern studies focus on the Al³⁺ toxicity in acid soils, but aluminum can be also toxic in alkali circumstance, existing in complicated ionic ways. In this study, Puccinellia tenuifolra, a typical plant in alkali soils, was use to construct its full length cDNA library expressed in yeast, screened the Al³⁺ related genes with AlCl₃ in the medium. PutSTE24, showed a high similarity to AtSTE24 (77%), was screened out.

The CAAX protease STE24, first identified in a genetic screen in yeast for mutants defective in the production of a biologically active a-mating pheromone, is a prenylation-dependent protease catalising a kind of eukaryotic proteins’ post-translational modifications essential to their targeting (Apolloni et al., 2000). These proteins end by the residues recombination CAAX, named as CAAX proteins, and their post-translational modifications usually include the following three sequential, enzymatic steps. First, the proteins are prenylated by one of two prenyltransferases named geranylgeranyltransferase â… or farnesyltransferase (Galichet and Gruissem, 2003), which happens in cytoplasm. In yeast and animal cells, prenylation is followed by proteolytic removal of the last three amino acids of the protein (AAX) by either of the two endoproteases, RCE1 and STE24 (AFC1) (Boyartchuk et al., 1997; Young et al., 2001), which is thought to take place on the cytoplasmic surface of the endoplasmic reticulum (ER) (Schmidt et al., 1998). Finally, the exposed isoprenyl-cysteine is methylated by and prenyl-dependent carboxylmethyltransferase (PCM) (Clarke, 1992; Romano et al., 1998).

In the recent ten years, the protein prenylation in plant has been clarified specifically, and genes encoding the above enzymes have cloned in Arabidopsis thilinana. There has been some reports showed that over-expression of some genes is related to stress tolerance of plant. In Arabidopsis, loss-of-function mutations in the ERA1 gene, encoding the β-subunit of PFT, ggb1 gene, encoding the β-subunit of PGGTâ… , or plp gene, which encode α-subunit of these two enzymes, cause an enhanced response to abscisic acid (ABA) in seed germination and stomatal closure assays (Cutler et al., 1996; Pei et al., 1998; Running et al., 2004; Johnson et al., 2005). The above two enzymes involved in negative regulation of signaling in guard cells. AtSTE24, an Arabidopsis homologue of the CAAX protease STE24, was cloned and expressed in rce1∆ ste24∆ mutant yeast to demonstrate functional complementation (Bracha et al., 2002). To date, there are few studies were reported on AtSTE24, and fewer reports introducing its relationship with stresses tolerance and responsion reaction with metal ions.

This paper reports on the cloning and characterization of PutSTE24 and AtSTE24, indicating that STE24 is a protease related to Al³⁺ tolerance and other stresses in yeast.

1 Results and Analysis
1.1 Cloning and sequence analisys of PutSTE24
In the previous studies, full length cDNAs over-expressing library of Puccinellia tenuifolra was constructed in yeast (Saccharomyces cerevisiae). A clone was screened out from this yeast library with medium containing AlCl₃. By PCR using the specific primers described in materials and methods and sequencing, results showed that PutSTE24 cDNA contained full length of 1 700 nucleotides and had a open reading frame (ORF) of 1 275 bp nucleotides encoding a predicted 424 amino acids (Figure 1). The predicted protein was calculated to have a molecular mass of 48.3 kD and pI of 6.84.

The Blast algorithm identified three proteins with higher similarity to PutSTE24 (Figure 2). They are AtSTE24 from Arabidopsis thaliana (At4g01320, 77% amino acid identity), CAAX prenyl protease 1 from Zea mays (100286144, 79% amino acid identity), and putative STE24 from Ricinus communis (8286673, 77% amino acid identity). Like AtSTE24, PutSTE24 possesses two conservative sequence motifs: HEXXH that is a signature of zinc metalloproteases and a C-terminal KKXX, the ER membrane retention signal (Figure 2).

Figure 1 Nucleotide sequences and the encoded amino acid sequences of PutSTE24

Figure 2 Alignment of deduced amino acid sequences of PutSTE24 with its homology in Arabidopsis thaliana, Zea mays and Ricinus communis

1.2 Over-expressing of PutSTE24 and AtSTE24 respectively in yeast and Al³⁺ tolerance analysis
In this study, PutSTE24 was screened out with AlCl₃ stress, therefore, to further analyze the responsive relationship of it and its homologue AtSTE24 with Al³⁺ stress, yeast transformed lines were constructed. One was transformed with empty vector pAUR123 as a control. The two else transformants were over-expressed PutSTE24 and AtSTE24 respectively (Figure 3; Figrue 4 and Figure 5). In the presence of different concentrations of AlCl₃, the growth of these transformants showed differently (Figure 3). The growth of these two transformants showed similarly. At 6 mmol/L of AlCl₃, they grew much better than the control yeast; but at 6.5 or 7 mmol/L of AlCl₃ stress, this growth advantage disappeared, and they seemed similar to the control, even worse. The results indicated over-expressing of PutSTE24 and AtSTE24 can alleviate Al³⁺ stress at a degree. 

Figure 3 Growth assay of yeast expressing PutSTE24 and AtSTE24 in the stress of AlCl3

Figure 4 Growth assay of yeast expressing PutSTE24 and AtSTE24 in the different stresses

Figure 5 Growth assay of yeast expressing PutSTE24 and AtSTE24 in the stresses of various of metal ion

Al³⁺ stress can also cause some other stresses at the same time, such as low pH and oxidation stresses, therefore, in this study, growth of these transformed yeast lines was observed in the conditions of pH 4.2, sorbitol, NaCl and H₂O₂ (Figure 4). The growth of the PutSTE24 and AtSTE24 transformants was the same as that of the control in the presence of low pH and sorbitol, but was better than that of the control on the media containing NaCl and H₂O₂. The results indicate that STE24 protease plays a role in response to salt and oxidation stresses and its role in Al³⁺ tolerance may be not specific.

1.3 Responsion of PutSTE24 and AtSTE24 over-expressing cells to various of metal cations
    To further discuss the responsive relationship of STE24 with metal ions except Al³⁺, serial dilutions were spotted onto solid yeast YPD medium supplemented without or with various of metal cations and the growth was monitored (Figure 5). As shown in Figure 5, the growth of the two STE24 transformants was much better than that of the empty vector transformant on the media containing K⁺, Mg²⁺ and Cu²⁺; some better than the control with the Fe³⁺ and Cd²⁺; and was almost the same as that of the control in the presence of Ca²⁺, Mn²⁺ and Ba²⁺. Interestingly, the growth of the two STE24 transformants seemed hyper-sensitive in the presence of  Co²⁺, Ni²⁺and Zn²⁺. These results indicate that STE24 is a gene related to some metal ions stresses besides Al³⁺, which have not been reported previously. This responsive relationship is deduced to caused by the post-translation modification of some cations transporters under the action of STE24 protease.

2 Discussions
In this study, the growth of yeast transformed with PutSTE24 and AtSTE24 was assayed in the presence of various of abiotic stresses. We have got the conclusions that STE24 is a gene related to some metal ion stresses, but the molecular mechanism involved in have not been clear.

3 Materials and methods
3.1 Materials
Yeast full-length cDNA library of Puccinellia tenuiflora (1 865 000 clones), cDNAs of Arabidopsis thaliana, Escherichia coli strain JM109, Yeast strain (Saccharomyces cerevisiae) InVSCI.

3.2 Cloning PutSTE24 and AtSTE24 from plant and sequence analysis
The ORF portion of Put STE24 was amplified from the yeast expression library of P. tenuiflora with the primers F-F: 5’-GCAGCTGTAATACGACTCAC-3’ and F-R: 5’-TTACATGATGCGGCCCTCTA-3’. The ORF portion of AtSTE24 was amplified from the yeast expression library of Arabidopsis thaliana with the primers F-F: 5’-GGTCACTCTTTTCTCAGCCATG-3’ and F-R: 5’-ACAAGAGACGAGTTAAGCGGAC-3’. Homologous comparison was obtained with other plants according to the amino acid sequence of the two genes.

3.3 Plasmids construction of pAUR123-PutSTE24 and pAUR123-AtSTE24 and yeast transformation
The modified form of PutSTE24 was constructed: SgsI-PutSTE24-SfaAI. The forward primer (F-P: 5’-GCGATCGCGCACTGTAATACGACTCAC-3’) was designed to add SgsI site and the reverse primer (R-P: 5’-CTCGAGTTACACAAAAAAGCTTG-3’) was designed to add SfaAI. 

The modified form of AtSTE24 was constructed: SgsI-AtSTE24-SfaAI. The forward primer (F-P: 5’-GGTACCTTTTCTCAGCCATG-3’) was designed to add SgsI site and the reverse primer (R-P: 5’- GGCGCGCCTCTAGATGCATGCTCGAG-3’) was designed to add SfaAI. 

All amplified fragments were cloned into the pAUR123 vector (Invitrogen) and the constructed vectors were introduced into yeast mutant InVSCI using the LiAc/PEG method. The yeast transformants were selected on medium supplied with Aureobasidin A. 

3.4 Tolerance of PutSTE24/AtSTE24-overexpressing cells to various stress
For growth response assay, the yeast transformants of pAUR123, pAUR123-PutSTE24 and pAUR123-AtSTE24, were cultured in liquid YPD medium until OD₆₀₀≈0.6 respectively, and diluted 10^-1, 10^-2, 10^-3, 10^-4 and 10^-5 fold with ddH₂O. Then, aliquots of each dilution were spotted onto solid yeast YPD medium supplemented with different concentrations of AlCl₃, NaCl, H₂O₂, pH, sorbitol, KCl, MgCl₂, FeCl₃, MnCl₂, ZnCl₂, CaCl₂, CuCl₂, CdCl₂, NiSO₄, _BaCl2 and CoCl₂ as indicated. The yeast transformant of pAUR123 empty vector was used as a control, growth were monitored for 3~7 d at 30℃.

Authors’ contributions
MHZ, XXZ and LYW designed and conducted this experiments; LHD, BS and TT participated the experiment design and data analysis; SKL is the person who takes charge of this project, including experiment design, data analysis, writing and modifying of the manuscript. All authors have read and approved the final manuscript.

Acknowledgements
This work was supported by the Heilongjiang Provincial Program for Distinguished Young Scholars (JC200609) and State Forestry Administration 948 Program of PR China (No. 2008429) to Shenkui Liu. Authors appreciate two anonymous reviewers for their useful critical comments and revising advice to this paper. And also we mentioned some reagent suppliers and sequencing service providers in this work, that doesn’t mean we would like to recommend or endorse their products and services.

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