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Identification of Glutathione Peroxidase (GPX) Gene Family in Rhodiola crenulata and Gene Expression Analysis Under Stress Conditions

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Identification of Glutathione Peroxidase (GPX) Gene Family in Rhodiola crenulata and Gene Expression Analysis Under Stress Conditions

Lipeng Zhang et al. Int J Mol Sci.

Abstract

Glutathione peroxidases (GPXs) are important enzymes in the glutathione-ascorbate cycle for catalyzing the reduction of H₂O₂ or organic hydroperoxides to water. GPXs play an essential role in plant growth and development by participating in photosynthesis, respiration, and stress tolerance. Rhodiola crenulata is a popular traditional Chinese medicinal plant which displays an extreme energy of tolerance to harsh alpine climate. The GPXs gene family might provide R. crenulata for extensively tolerance to environment stimulus. In this study, five GPX genes were isolated from R. crenulata. The protein amino acid sequences were analyzed by bioinformation softwares with the results that RcGPXs gene sequences contained three conserve cysteine residues, and the subcellular location predication were in the chloroplast, endoplasmic reticulum, or cytoplasm. Five RcGPXs members presented spatial and temporal specific expression with higher levels in young and green organs. And the expression patterns of RcGPXs in response to stresses or plant hormones were investigated by quantitative real-time PCR. In addition, the putative interaction proteins of RcGPXs were obtained by yeast two-hybrid with the results that RcGPXs could physically interact with specific proteins of multiple pathways like transcription factor, calmodulin, thioredoxin, and abscisic acid signal pathway. These results showed the regulation mechanism of RcGPXs were complicated and they were necessary for R. crenulata to adapt to the treacherous weather in highland.

Keywords: Rhodiola crenulata; expression pattern; gene family; glutathione peroxidase; interaction proteins.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Amino acid sequences alignment of plant glutathione peroxidase by DNAMAN. The highly conserved domains were presented by line and digital alphabet I, II, and III The conserved three Cys residues of plant GPX proteins are indicated by triangles. Abbreviations of plant species: Rc, Rhodiola crenualta; At, Arabidopsis thaliana. The GPX protein sequences of Arabidopsis thaliana were as followed: AtGPX1 (AT2G25080.1); AtGPX2 (AT2G31570.1); AtGPX3 (AT2G43350.1); AtGPX4 (AT2G48150.1); AtGPX5 (AT3G63080.1); AtGPX6 (AT4G11600.1); AtGPX7 (AT4G31870.1); AtGPX8 (AT1G63460.1).
Figure 2
Figure 2
Architecture and phylogenetic tree of glutathione peroxidases of Rhodiola crenulata. The structures of RcGPXs were predicted by MEME software. Distribution of conserved domains was presented by different colors (A). Hidden Markov model logos obtained using MEME (B). The phylogenetic tree of RcGPXs was constructed using the neighbor-joining method of CLUSTALW by Mega 5.0, with 1000 bootstraps, and the bar indicates 0.05 substitutions per site. Each ellipse shows a clade (C).
Figure 3
Figure 3
Phylogenetic analyses of thirty three plant glutathione peroxidase (GPX) proteins of R. crenulata and other species. The tree was constructed using the neighbor-joining method of CLUSTALW by Mega 5.0, with 1000 bootstraps, and the bar indicates 0.05 substitutions per site. Each ellipse shows a clade. The protein sequences of GPXs were followed: AtGPX1 (AT2G25080.1); AtGPX2 (AT2G31570.1); AtGPX3 (AT2G43350.1); AtGPX4 (AT2G48150.1); AtGPX5 (AT3G63080.1); AtGPX6 (AT4G11600.1); AtGPX7 (AT4G31870.1); AtGPX8 (AT1G63460.1); VvGPX1 (XM002285528); VvGPX2 (XM002263291); VvGPX5 (XM002276220); VvGPX8.1 (XM002272900); VvGPX8.2 (XM010662496); VvGPX (XM010662503); OsGPX1 (Os04g0556300); OsGPX2 (Os06g0185900); OsGPX3 (Os02g0664000); OsGPX4 (Os03g0358100); OsGPX5 (Os11g18170); PtGPX1 (POPTR_0006s28120); PtGPX2 (POPTR_0007s02160); PtGPX5 (POPTR_0014s13490); PtGPX8 (POPTR_0001s09280); MdGPX1 (XP008379282.1); MdGPX5 (XP008355452.1); MdGPX6 (NP001280872.1); MdGPX7 (XP008347490.1); MdGPX8 (XP008384017.1). Plant species included: Rc (Rhodiola crenulata); At (Arabidopsis thaliana); Os (Oryza sativa); Pt (Populus trichocarpa); Vv (Vitis vinifera); and Md (Malus domestica).
Figure 4
Figure 4
Tissue-specific expression analysis of RcGPXs. Six organs and tissues were used for real-time PCR (A). The expression levels of RcGPXs under different development stages or tissues were presented by colors (B). mRNA levels were normalized with respect to RcGAPDH and are expressed relative to the values of callus tissue, which were given an arbitrary value of 1. Data represent the means ± SE of at least three replicates.
Figure 5
Figure 5
Expression patterns of Rhodiola crenulata glutathione peroxidase (RcGPXs) genes for leaves under abiotic stresses. (A) Drought stress was used by PEG-4000 (20%); (B) UA stress was used the 234 nm ultraviolet ray wavelength; (C) cold stress was under ice-water environment for 0 °C; (D) flooding stress was used to build hypoxia circumstance. The total RNA was extracted from young leaves. mRNA levels were normalized with respect to RcGAPDH and were expressed relative to the values at 0 day (control), which were given an arbitrary value of 1. Data represent the means ± SE of at least three replicates.
Figure 6
Figure 6
Time-course expression levels of RcGPXs genes for leaves under abscisic acid treatment. Plant hormone abscisic acid (ABA) was used to treat whole seedlings of R. crenulata at 50 μM. Five time periods of conditions were selected at 0 h, 6 h, 12 h, 24 h, and 36 h. The total RNA was extracted from young leaves. mRNA levels were normalized with respect to RcGAPDH and were expressed relative to the values at 0 h (control), which were given an arbitrary value of 1. Data represent the means ± SE of at least three replicates.
Figure 7
Figure 7
Expression levels of RcGPXs genes for leaves under heavy metal conditions. Heavy metal compounds were used to treat whole seedlings of R. crenulata for 6 h. The seedlings were exposed to MS (Murashige and Skoog) media contained different heave mental ions and the concentrations were 25 μM Hg+ (HgCl), 50 μM Cu2+ (CuSO4), 50 μM Co2+ (CoCl2), and 50 μM Ag+ (AgNO4). CK indicted the negative control which seedlings were exposed for MS media for 6 h. The total RNA was extracted from young leaves. mRNA levels were normalized with respect to RcGAPDH and were expressed relative to the CK values, which were given an arbitrary value of 1. Data represent the means ± SE of at least three replicates.
Figure 8
Figure 8
The putative interaction proteins of RcGPXs were investigated by yeast two—hybrid assays. Y2H gold yeast strains containing RcGPXs as bait and Arabidopsis genes as prey were grown on SD solid media lacking Trp and Leu for 5 days (left panel) and were assayed for reporter gene His expression by growing on SD solid media lacking Trp, Leu, and His (right panel). Prey and bait empty vectors were used as negative control. The Arabidopsis genes: AtABI2 (AT5G57050.1); AtCML38 (AT1G76650); AtNAC102 (AT5G63790); AtTrxm2 (AT4G03520.1); AtTrxm3 (AT2G15570.2); AtTrxh7 (AT1G59730.1); AtTrxf2 (AT5G16400.1); AtTrx1 (AT1G76760); AtTrx4 (AT1G19730.1); AtTrx5 (AT1G45145.1); and AtTrxz (AT3G06730.1).

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