Expression pattern analysis of pyridoxal kinase gene from Aeluropus Lagopoides (alaSOS4) under salinity, calcium and abscisic acid treatments.
Soil salinity stresses plants in two ways. High concentration of salts in the soil make it harder for roots to extract water and High concentration of salts within the plant can be toxic .
Plants are classified as glycophytes or halophytes according to their capacity to grow on high salt medium. Most plants are glycophytes and cannot tolerate salt-stress. Halophytes are plants growing on or surviving in saline conditions such as marine estuaries and salt marshes. They respond to salt stress at three different levels: cellular, tissue and the whole plant . In some halophytes, salt excretion represents an avoidance strategy that permits control and regulation of salt content in phytosynthetic ones . In the poaceae family, there are four genera able to excrete salts: Spartina, Aeluropus, Distichlis and Chloris .
Aeluropus lagopoides (poaceae) is a stoloniferous perennial grass with C4 photosynthesis that is distributed in the regions with intermediate salinity and semi-desert climate on Iranian plateau [5-7]. A. lagopoides be able to survive up to 1500 mM NaCl  with a relatively low accumulation of sodium and chloride ions in dry overgrown tissue . A. lagopoides has salt glands consisting of only two cells that predominantly excreted NaCl .
Pyridoxal (PL) kinase encoded in Arabidopsis thaliana (SOS4) , is key enzymes involved in the biosynthetic pathway leading to Pyridoxal 5'-P (PLP). PLP is the active coenzyme product of the vitamin B6 pathway and is essential in many biochemical reactions, including decarboxylation, transamination, deamination, racemization and trans-sulfuration reactions associated primarily with amino acid synthesis . PLP is also involved in enzymes that catalyze some steps in carbohydrate and lipid metabolism and is a cofactor for aminocyclopropane-1-carboxylate synthase. Moreover, vitamine [B.sub.6] has been linked to resistance to oxidative and environmental stresses and is an efficient singlet oxygen quencher and antioxidant [11,13,14]. It is required for postembryonic root development and root hair development in plants [13,14,15,16,17].
SOS4 mutant plants are hypersensitive to [Na.sup.+], [Li.sup.+] and [K.sup.+] ions upon salt stress. SOS4 mutant plants accumulate more [Na.sup.+] and retain less [K.sup.+] than the wild type plants. These results suggest that SOS4 is important for [Na.sup.+] and [K.sup.+] homeostasis in plants . PLP may regulate [Na.sub.+] efflux by SOS', because SOS' contains a putative pyridoxal-5-phosphate binding domain .
In this study expression pattern analysis of alaSOS4 gene was done and then alaSOS4 gene cloned. The purpose of the current study was understanding the role of SOS4 gene in salt stress tolerance mechanisms in such a halophytic plant.
Material and Method
Seeds of A. lagopoides were collected from Ghandi-Abad of Kashan Kavir in the Iran and they were sterilized by commercial sodium hypochlorid 20% and Triton X-100 1% for 10 minutes and were cultured in the hydroponic medium supplemented by 1/2 MS medium. 21 days seedlings in the growthchamber (16/8 h light/dark and 23 [+ or -] 2[degrees]C) treated with NaCl (600 mM), ABA (50 [micro]M), [Ca.sub.2]S[O.sub.4] (5mM), NaCl (600mM) +[Ca.sub.2]S[O.sub.4] (5mM), NaCl (600mM)+ABA (50 [micro]M) and [Ca.sub.2]S[O.sub.4] (5mM)+ABA (50 [micro]M) for 10 days. Then samples were collected and stored at -70 [degrees]C. Each treatment was included 3 biological repetitions and at least 3 technical replicates.
RNA extraction and gene cloning:
Total RNA was extracted from shoots and roots by RNax plus kit (Cinnagene Company, Iran) according to the manufacturer procedure. Then RNAs were treated by DNase 1 RNase free kit consistent with the company method (Fermentas Company, Ukraine) for eliminating of genomic DNA. cDNA was synthesized by cDNA synthesis system (Fermentas Company, Ukraine) and alaSOS4 gene were isolated by specific primers designed based on conserved region of wheat SOS4 gene by Oligo Primer Analusis Software Version 5. alaSOS4 cDNA segments were isolated from A. lagopoides shoots and roots and ligated to pTZ57R/T transmitting plasmid.
Expression patterns analysis of alaSOS4 gene carried out by semi-quantitative RT-PCR in contrast to [beta]-tubuline a house keeping gene in shoots and roots of A. lagopoides. Relative intensity of bands was measured by Total lab software.
Data were analyzed statistically by SPSS vesion 10 software. Variance analysis was carried out to determined for existence of significant difference between means and Duncans tests was performed to compare means at p < 0.05.
Data entry, sequence management, and sequences alignment were performed by DNASTAR software (Madison. WI 53705, USA). Sequence similarity and several structural features were studied by use of online databases including BLASTN and X , pfam  and Blocks .
alaSOS4 gene cloning:
A. lagopoides alaSOS4 EST was cloned in pTZ57RT vector, its size was 520 bp (Fig. 1). After sequencing and making alignment, we found great homology (85%) between its encoded protein and Arabidopsis and Wheat SOS4 proteins (Fig. 1). Clones were sequenced by SeqLab Company, Germany and submitted in GenBank as alaSOS4 with GT734410 accession number.
Expression pattern of alaSOS4 gene:
The expression of alaSOS4 gene was correlated reciprocally to the organ and treatments but interaction effect of the two factors showed no significant difference at p < 0.05 on the alaSOS4 expression levels (Table 1).
The expression of alaSOS4 was increased considerably compared with controls by NaCl, [Ca.sub.2]S[O.sub.4] and ABA+ NaCl tratments in shoots (Fig 2). While its expression was not changed significantly by different treatments in roots but it showed high levels of expression in all of the treatments in relation to the shoots.
Shi and Zhu  and Wang et al  revealed that SOS4 was expressed in hairy roots and other similar structures in Arabidopsis and wheat. The result was evident for an important role of SOS4 in hairy root formation. As shown in Fig 2 the expression level of alaSOS4 was higher in A. lagopoides roots relative to the shoots so it is possible that alaSOS4 is taken part in A. lagopoides hairy roots formation. Also, because of not significant changes in alaSOS4 expression levels in the roots under different treatments, it can conclude that alaSOS4 has an anonymous role in roots.
Pyridoxal (PL) kinase encoded in Arabidopsis thaliana (SOS4) is a key enzyme involved in the biosynthetic pathway leading to Pyridoxal 5'-P (PLP). PLP is the active coenzyme product of the vitamin [B.sub.6] pathway. vitamine B6 has been linked to resistance to oxidative and environmental stresses and is an efficient singlet oxygen quencher and antioxidant and also is a well-known cofactor for numerous enzymes [11,13,14] Graham et al  suggested that the major action of pyridoxine (vitamin [B.sub.6]) in plants may be through its role as a cofactor for numerous enzymes rather than through its direct effect on active oxygen species. Thus, SOS4 gene product plays important and may be various roles in plants.
Although several reports were revealed that SOS pathway is not regulated by ABA in Arabidopsis but Shi et al  and Wang et al  reported its regulation by ABA in Maize. Despite the later reports we found that ABA treatment was not by itself effective on alaSOS4 expression in A. lagopoides shoots. But simultaneously, we found that ABA+NaCl increased its expression significantly in the shoots. According to the similar levels of gene expression in NaCl, [Ca.sub.2]S[O.sub.4] and ABA+NaCl treatments, it seems that ABA is not effective on the gene expression and the increased levels of expression is related to the NaCl effects on gene appearance and more over, alaSOS4 induction by salinity.
Shi et al  reported that SOS4 plays a role in [Na.sup.+], [K.sup.+] and [Ca.sup.2+] homeostasis regulation in Arabidopsis so probably alaSOS4 is effective in Na+ and [Ca.sup.2+] homeostasis and may be has an important role in salt tolerance in A. lagopoides. In the previous report, we demonstrated that sodium content in the shoots of A. lagopoides was significantly higher than roots by 600 mM NaCl treatment. Our results showed that one of the probable salt tolerance mechanisms in A. lagopoides was sodium transferring from roots to shoots or out of plant by salt glands . Some research had been shown that one of the possible halophytic strategies for salt tolerance is loading of excess [Na.sup.+] to xylem and transporting it to shoots .
Also, Shi et al  demonstrated that SOS4 has two possible transcripts in Arabidopsis and their expressions are regulated by NaCl, ABA and cold but drought stress is not effective in shoots and also SOS4 transcripts were down regulated by ABA and NaCl treatments. Accordingly, alaSOS4 was a salt inducible transcript at least in the shoots and showed different functions in the two various studied tissues. In Brassica napus L., external ABA resulted in increasing SOS4 large transcript (PKL) expression and decreasing its small transcripts' (PKL2). Similar to AtSOS4 the PKL expression decreased under different plant hormones treatments except ABA . It is possible by exploiting more sensitive expression analyzing methods like Real-Time RTPCR alaSOS4 changes by exogenous ABA would be detectable.
Additionally, we observed a constitutive expression for alaSOS4 in the roots that did not changed by different conditions. Subsequently, we proposed that it has a special function in the roots that is not responsive to the salinity. As a result, it is possible to reveal it by more physiological experiments and more sensitive molecular methods. Conclusion:
In summary at the first time, we report alaSOS4 expression pattern in A. lagopoides shoots and roots at different conditions, as a halophyte. With regard to the Pyridoxal-5-phosphate (PLP) role in bioshyntetic pathway of vitamine [B.sub.6] as a antistress vitamine is known to be an antagonist of the ATP gated ion channel in animal cells and a well-known cofactor for numerous enzymes . Also, it is concluded that alaSOS4 up-regulation in shoots under [Ca.sub.2]S[O.sub.4] and NaCl treatments may be has important roles in [Na.sup.+] and [Ca.sup.2+] homeostasis and salt tolerance which remain to reveal more physiological and molecular documents.
This study was supported by the National Institute of Genetic Engineering and Biotechnology (NIGEB) grant No. 271.
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(1,2) Masoomeh Jannesa, (1) Khadije Razavi, (2) Azra Saboora
(1) National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
(2) Department of Biology, Faculty of Science, Alzahra University, Tehran, Iran
Khadije Razavi, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
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|Title Annotation:||ORIGINAL ARTICLE|
|Author:||Jannesa, Masoomeh; Razavi, Khadije; Saboora, Azra|
|Publication:||Advances in Environmental Biology|
|Date:||Aug 1, 2012|
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