Cloning and Characterization of PutSTE24 Gene from Puccinellia tenuifolra Which Expressed in Response to Abiotic Stresses

The Puccinellia tenuifolra cDNA library was expressed in yeast (Saccharomyces cerevisiae) and screened on agar plates containing toxic concentrations of aluminum. Ninteen cDNAs were isolated that enhanced the aluminum tolerance of yeast. One cDNA, named PutSTE24, has a ORF of 1 275 bp, encoding a predicted protein containing 424 amino acid, and has a high similarity of 77% with STE24 in Arabidopsis thilinana. PutSTE24 and AtSTE24 were transformed into yeast cells separately and were treated with AlCl3, salt, drought, low pH and oxidation and metal ions stresses. Results revealed that these two recombinant yeast cells showed similarly and grew better in AlCl3, salt and oxidation stresses than control cells, but no obvious difference in low pH and drought stresses. Additionally, on the responsive to the metal ions, these two genes have obvious resistance to the stresses of K, Mg and Cu, are somewhat resistant to Fe, Cd, and have no obvious responsive relationship with Ca, Mn and Ba, but to the metal ions of Co, Ni and Zn, these two recombinant yeast cells are sensitive, growing worse than the control cells, especially the Zn. It is basically confirmed the gene STE24 is related to metal stresses, which has no report in the previous studies.


Background
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), Al 3+ 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 Ca 2+ homeostasis, hormone transport and signal transduction (Matsumoto, 2000). Previous studies showed that aluminumactivated root malate or citrate exudation from plasma membrane or vacuolar membrane played an important role in plant Al 3+ 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 Al 3+ tolerance . 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 Al 3+ 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 Al 3+ related genes with AlCl 3 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' posttranslational 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 Ⅰ 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) . Finally, the exposed isoprenyl-cysteine is methylated by and prenyl-dependent carbo-xylmethyltransferase (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 overexpression 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 PGGTⅠ, 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 Al 3+ tolerance and other stresses in yeast.

Cloning and sequence analisys of PutSTE24
In the previous studies, full length cDNAs overexpressing library of Puccinellia tenuifolra was constructed in yeast (Saccharomyces cerevisiae). A clone was screened out from this yeast library with medium containing AlCl 3 . 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.

Over-expressing of PutSTE24 and AtSTE24 respectively in yeast and Al 3+ tolerance analysis
In this study, PutSTE24 was screened out with AlCl 3 stress, therefore, to further analyze the responsive relationship of it and its homologue AtSTE24 with Al 3+ 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; Figure 4 and Figure 5). In the presence of different concentrations of AlCl 3 , the growth of these transformants showed differently (Figure 3). The growth of these two transformants showed similarly. At 6 mmol/L of AlCl 3 , they grew much better than the control yeast; but at 6.5 or 7 mmol/L of AlCl 3 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 Al 3+ stress at a degree. Al 3+ 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 H 2 O 2 (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 H 2 O 2 . The results indicate that STE24 protease plays a role in response to salt and oxidation stresses and its role in Al 3+ tolerance may be not specific.

Responsion of PutSTE24 and AtSTE24 overexpressing cells to various of metal cations
To further discuss the responsive relationship of STE24 with metal ions except Al 3+ , 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 + , Mg 2+ and Cu 2+ ; some better than the control with the Fe 3+ and Cd 2+ ; and was almost the same as that of the control in the presence of Ca 2+ , Mn 2+ and Ba 2+ . Interestingly, the AtSTE24 in the stress of AlCl 3 Note: Yeast cells containing pAUR123, pAUR123-PutSTE24 and pAUR123-AtSTE24 were, respectively, incubated as described in materials and methods; Serial dilutions were spotted onto solid yeast YPD medium supplemented without or with additional AlCl 3 (6 mmol/L, 6.5 mmol/L and 7 mmol/L), growth were monitored for 3~6 d at 30℃ Figure 4 Growth assay of yeast expressing PutSTE24 and AtSTE24 in the different stresses Note: Yeast cells containing pAUR123, pAUR123-PutSTE24 and pAUR123-AtSTE24 were, respectively, incubated as described in materials and methods; Serial dilutions were spotted onto solid yeast YPD medium supplemented without or with additional stresses, such as low pH (pH values 4.2), sorbitol 1.6 mol/L, NaCl 0.9 mol/L and H 2 O 2 4.8 mmol/L, growth were monitored for 3~6 d at 30℃ Figure 5 Growth assay of yeast expressing PutSTE24 and AtSTE24 in the stresses of various of metal ions Note: Yeast cells were incubated as described in materials and methods; Serial dilutions were spotted onto solid yeast YPD medium supplemented with or without metal cations, including K + 1 mol/L, Mg 2+ 0.8 mol/L, Cu 2+ 8 mmol/L, Fe 3+ 10 mmol/L, Cd 2+ 180 µmol/L, Ca 2+ 100 mmol/L, Mn 2+ 1.2 mmol/L, Ba 2+ 6 mmol/L, Co 2+ 0.5 mmol/L, Ni 2+ 1.5 mmol/L and Zn 2+ 4 mmol/L, growth were monitored for 3~7 d at 30℃ growth of the two STE24 transformants seemed hyper-sensitive in the presence of Co 2+ , Ni 2+ and Zn 2+ . These results indicate that STE24 is a gene related to some metal ions stresses besides Al 3+ , 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.

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.

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.
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.