Background

High salinity is a universal stress state that affects plant growth and development. The saline soils of Northeast China are formed by the accumulation of Na₂CO₃ and NaHCO₃ (Wang et al., 2009; Wang et al., 2012). Plants are equipped various biochemical and physiological responses in order to grow (Seki et al., 2003).

Reactive oxygen species (ROS) produced by salinity stress are mainly composed of superoxide radicals (O₂^-) and hydrogen peroxide (H₂O₂) (Hasegawa et al., 2000). Oxidative stress induced by ROS on different cell components mainly includes membrane lipids, proteins and nucleic acids (Halliwell and Gutteridge, 1986). The alternative pathway is known to be involved in salt stress, the AOX pathway can prevent harmful ROS production. It controls the mitochondrial ROS production by steadily reducing the rate of electron transport (Mhadhbi et al., 2013). In metabolic imbalances, this pathway will be activated as an early response (Clifton et al., 2005), Alternative oxidase (AOX) is a pivotal enzyme in the respiratory chain of plants. It can regulate the yanide-resistant pathway that branches from the main respiratory chain at the level of ubiquinone (Arnholdt-Schmitt et al., 2006; Florez-Sarasa et al., 2009; Mhadhbi et al., 2013). AOX was first discovered in thermogenic plants (aroids) during a thesis, AOX is responsible for thermogenesis (attract insects for pollination) and stress tolerance (Meeuse, 1975).

The expression of the first AOX gene in plants was studied in 1991 (Rhoads and McIntosh, 1991). In higher plants, typical AOX genes consist of two subfamilies: AOX1 and AOX2 (Whelan et al., 1996). The AOX1-type genes exist in all plants, while the AOX2-type genes exist only in dicotyledon plants (Vanlerberghe et al., 2009). The expression of AOX genes in response to environmental, developmental, and other cell signals are different (Finnegan et al., 1997; Considine et al., 2001; Saish et al., 2001). The 1-type AOX proteins are linked to stress, the functioning of 2-type AOX proteins are relate to developmental processes (Considine et al., 2002).

In this study, we cloned an LpAOX1 gene from L. pumilum, and study the relation between the LpAOX1 gene and salt stress in the yeast, understanding the importance of LpAOX1 gene expression in resisting saline-alkali stress.

1 Materials and Methods

1.1 Cloning of the open reading frame (ORF) region of LpAOX1

We extracted the total RNA from the L. pumilum leaves using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Reverse transcription of total RNA into cDNA with Prime-Script Reverse Transcriptase (Takara, Tokyo, Japan). We amplified a transcript fragment from the cDNA by PCR with the forward primer (5'-ATGTTGAGCTCTCGCGCCGC-3') and reverse primer (5'-TCAGTGGTACCCGAGCGGCG-3') based on the transcriptome contract sequencing results of L. pumilum under 20 mM NaHCO₃ and untreated. We purified the PCR product from agarose gel by the DNA Gel Extraction Kit (Generay, Shanghai, China), attached the product to the pMD18-T vector (Takara, Tokyo, Japan) and sequenced, then nucleotide sequences and deduced protein sequences were input into NCBI for Blast analysis. Then, nucleotide sequences were input into NCBI for Blast analysis.

DNAMAN8 software was used for multiple sequence alignment. Phylogenetic analyses were performed in MEGA 3.0 using default values.

1.2 Construction of expression vectors and yeast transformation

We amplified the LpAOX1 gene using pMD18T-LpAOX1 plasmid DNA as a template with BamHI sense primer 5'-GGATCCATGTTGAGCTCTCGC-3' (restriction site underlined for all restriction enzymes) and XhoI antisense primer 5-'CTCGAGTCAGTGGTACCCGAG-3', The PCR amplified products were digested with two enzymes BamHI and XhoI and then the recycled fragment is then connected to the same site of the pYES2 expression vector (Clontech, Tokyo, Japan), the attachment product is called pYES2-LpAOX1.

The fragment of Green fluorescent protein (GFP) was digested from the plasmid pEGFP DNA (Clontech) with two enzymes BamHI and NotI, and then connected the recovered product to the same site of pYES2 vector to construct the control vector (pYES2-GFP). To construct the pYES2-LpAOX1-GFP expression vector, we amplified the LpAOX1 gene using pMD18T-LpAOX1 plasmid DNA as a template with HindIII sense primer 5'-AAGCTTATGTTGAGCTCTCGC-3' and BamHI antisense primer 5'-GGATCCGTGGTACCCGAG-3' and the fragments were digested with two enzymes HindIII and BamHI and then connected the recovered product to the same site of pYES2-GFP.

The pYES2-LpAOX1 and pYES2-LpAOX1-GFP plasmid DNAs were transformed into the yeast strain INVSC1 (Saccharomyces cerevisiae) (Clontech) using the electroshock transformation method.

1.3 LpAOX1 protein intracellular localization in transgenic yeast

The transformed yeast cells containing pYES2-LpAOX1-GFP were cultured in liquid YPD medium (1% yeast extract + 2% peptone + 2% D-glucose) at 30°C for 12 h. The overnight cultured cells were collected through centrifuge and washed twice with liquid YPG medium (1% yeast extract + 2% peptone + 2% galactose). Then these cells were cultured in a liquid YPG medium to induce the LpAOX1 gene and the GFP gene expression as well at 30°C for 8 h. Mitochondria were stained with MitoTracker Red. GFP fluorescence and MitoTracker Red fluorescence from the transgenic yeast with LpAOX1/GFP fusion protein was detected by the use of FluoView FV500 confocal laser scanning microscope (Olympus, Tokyo, Japan).

1.4 The LpAOX1 gene expression in response to different stresses

pYES2-LpAOX1 and pYES2 (control) transgenic yeast cells were incubated in the YPD medium at 30°C one night. The overnight culture yeast concentration was adjusted to OD₆₀₀ = 0.6. Serial diluted culture solutions (10, 10⁻¹, 10⁻², 10⁻³ and 10⁻⁴) were cultured onto YPD agar plates supplemented with different stresses (0.6 M NaCl, 0.8 M NaCl, 1 M NaCl, 10 mM Na₂CO₃, 20 mM Na₂CO₃, 30 mM Na₂CO₃, 20 mM NaHCO₃, 35 mM NaHCO₃, 50 mM NaHCO₃, pH 9, pH 10, pH 11, 3 mM H₂O₂, 5 mM H₂O₂, or 7 mM H₂O₂) respectively.

2 Results

2.1 Molecular cloning and sequence analysis an open reading frame of LpAOX1

The open reading frame (ORF) of gene was cloned from the L. pumilum cDNA. The ORF fragment contains 1,023 base pair (Figure 1) and encodes a 340 amino acid polypeptide.

The protein possessed the AOX1 domains throught the BLAST searching in the National Center for Biotechnology Information (NCBI) database. The amino acid sequence had the highest similarity with the AOX1 protein (Figure 2).

The construction of a neighbor-joining phylogenetic tree had been done based on LpAOX1 amino acid sequence. As shown in Figure 3, the most closely related protein identified from phylogenetic tree was Phoenix dactylifera AOX1 protein and Ananas comosus AOX1 protein. So this gene was designated as LpAOX1.

2.2 Intracellular localization of LpAOX1 in transgenic yeast

Vector including pYES2-LpAOX1-GFP fusion protein was successfully transformed into the yeast strain INVISC1 cells. The green fluorescence channel (GFP) and red fluorescence protein channel (Mitoracker) of pYES2-LpAOX1-GFP fusion protein had the same subcellular location, indicating that the LpAOX1 protein was localized at the mitochondrion in yeast (Figure 4).

2.3 Expression of LpAOX1 gene in transgenic yeast in response to stresses

Two yeast lines were constructed: one yeast line was transformed with LpAOX1 gene and another yeast line contained empty pYES2 (control). There is no yeast expression difference between control and LpAOX1-transgenic line under no stress applied and different pH value. However, the growth rate of transgenic yeast cells had significant differences in the presence of various abiotic stresses, the LpAOX1-transgenic lines grew better than the control especially in the presence of 1 M NaCl, 30 mM Na₂CO₃, 50 mM NaHCO₃, 5 mM H₂O₂ (Figure 5).

3 Discussion

A type 1 alternative oxidase gene (LpAOX1) was isolated from L.pumilum, and its expression in yeast was analyzed to understand their roles in the stress response. LpAOX1 deduced protein has high sequence similarity with the other plant species AOX1 protein, this results showed that the LpAOX1 belong to the 1-type AOX protein. The AOX is an integral protein of the inner mitochondrial membrane, is localized on the matrix side of the mitochondrial inner membrane (Rogov et al., 2014). GFP fluorescence was detected in the mitochondria of yeast transformed with pYES2-LpAOX1-GFP, the result demonstrate that LpAOX1 was localizated in mitochondria in vivo.

The AOX protein level different between a sensitive and a tolerant Vigna unguiculata root after hydroponic with high salt concentration (Costa et al., 2007), High expression of AOX1 induces enhanced salt tolerance capability in Medicago truncatula through regulation of ROS and protection of photosystem (Zhang et al., 2012; Mhadhbi et al., 2013). When Arabidopsis thaliana grew under salinity stress conditions, showed increaser AtAOX1a transcription levels and lower ROS formation (Smith et al., 2009).

AOX transcription increased in pea under salt stress (Marti et al., 2011), these results showed that AOX make plants more resistant to environmental pressures and play an important role in salt stress.

We speculated that the LpAOX1 also has relation to the stress, so we analyzed its expression in yeast under different stress, there was no difference between LpAOX1-transgenic line and control under different pH value, this maybe pH value have no effect on LpAOX1 expression. The LpAOX1 gene transgenic yeast grow better than control yeast under NaCl, Na₂CO₃, NaHCO₃, and H₂O₂ stresses, these indicated the LpAOX1 gene expression can improve the tolerance to the stresses, maybe LpAOX1 expression in the yeast prohibited excessive ROS production and control harmful ROS contents generation, the alleviation of ROS generation could increase yeast resistance to different stress.

Authors’ contributions

JSM conceived and designed the experiments; XC, CSY and HH performed the experiments; XC analyzed the data; JSM in contributed reagents/materials/analysis tools; JSM and XC wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This work was supported by Fundamental Research Funds for the Central Universities (2572016EAJ6).

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