The OsAPx2 gene from rice was cloned to produce PBI121::OsAPx2 dual-expression plants, of which expression level would be increasing under stressful conditions. The enzyme ascorbate peroxidase (APX) in the leaves and roots of the plants increased with increasing exposure time to different sodium chloride (NaCl) and hydrogen peroxide (H2O2)concentrations, as indicated by protein gel blot analysis. The increased enzyme yield improved the ability of the plants to resist the stress treatments. The OsAPx2 gene was localized in the cytoplasm of epidermal onion cells as indicated by the instantaneous expression of green fluorescence. An 80% regeneration rate was observed in Medicago sativa L. plants transformed with the OsAPx2 gene using Agrobacterium tumefaciens, as indicated by specific primer PCR. The OsAPx2 gene was expressed at the mRNA level and the individual M. sativa (T#1,T#2,T#5) were obtained through assaying the generation of positive T2 using RNA gel blot analysis. When the seeds of the wild type (WT) and the T2 (T#1,T#5) were incubated in culture containing MS with NaCl for 7 days, the results as shown of following: the root length of transgenic plant was longer than WT plants, the H2O2 content in roots of WT was more than of transgenic plants, the APX activity under stresses increased by 2.89 times compared with the WT, the malondialdehyde (MDA) content of the WT was higher than the transgenic plants, the leaves of the WT turned yellow, but those of the transgenic plants remained green and remained healthy. The chlorophyll content in the WT leaves was less than in the transgenic plants, after soaking in solutions of H2O2, sodium sulfite (Na2SO3), and sodium bicarbonate (NaHCO3). Therefore, the OsAPx2 gene overexpression in transgenic M. sativa improves the removal of H2O2 and the salt-resistance compared with WT plants. A novel strain of M. sativa carrying a salt-resistance gene was obtained.
References
[1]
Ling JS, Zhang JH (1998) Production, Transport and Physiological Fuctions of Stress Signal Abscisic Acid in Roots.plant physiology communications. 34(5): 329–338.
[2]
Gasparsd S, Monzani E, Casella L, Gullotti M, Maritano S, et al. (1997) Inhibition of ascobate oxidase by phenolic compounds. Enzymatic and spectrospic studies.Biochemistry 36(16): 4852–4859.
[3]
Asada K (1992) Ascorbate Peroxidase, A Hydrogen Peroxide Scavenging Enzyme in Plants. Physiologia Plantarum 85: 235–241.
[4]
Shigeoka S, Nakano Y, Kitaoka S (1980a) Metabolism of Hydrogen Peroxide in Euglena gracilis Z by L-ascorbic Acid. Peroxidase. Biochem J 186: 377–380.
[5]
Shigeoka S, Nakano Y, Kitaoka S (1980b) Purification and some properties of L-ascorbic acid peroxidase in Euglena gracilis Z. Arch Biochem Biophys. 201: 121–127.
[6]
Mittler R, Zilinkas BA (1992) Molecular cloning and characterization of a gene encoding pea cytosolic ascorbate peroxidase. J Biol Chem 267: 21802–21807.
[7]
Miyake C, Asada K (1992) Thylakoid-bound ascorbate peroxidase in spinach chloroplasts and photoreduction of its primary oxidation product monodehydroascorbate radical in thylakoids. Plant Cell Physiol 33: 541–553.
[8]
Bunkelmann JR, Trelease RN (1996) Ascorbate peroxidase: a prominent membrane protein in oilseed glyoxysomes. Plant Physiol 110: 589–598.
[9]
Teixeira FK, Menezes-Benavente L, Galv？o VC, Margis R, Margis-Pinheiro M (2006) Rice ascorbate peroxidase gene family encodes functionally diverse isoforms localized in different subcellular comparetments. Planta 224: 333–344.
[10]
Davletova S, Rizhsky L, Liang H, Shengqiang Z, Oliver DJ, et al. (2005) Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of arabidopsis. The Plant Cell 17: 268–281.
[11]
Mittler R (2002) Oxidative stress, antioxidants, and stress tolerance. Ternds Plant Sci 7: 405–410.
[12]
Ghazi HB, Naoyoshi K, Yasuo Y, Emi S, Ryozo, et al (2004) Over-expression of Ascorbate Oxidase in Tobacco Chloroplasts Enhances the Tolerance to Salt Stress and Water Deficit, Physiolgia Plantrum. 121: 231–238.
[13]
Nishikawa F, Kata M, Wang R, Hyodo H, Ikoma Y, et al. (2003) Two APX from Brocoli: Idetification, Expression and Characterization of Their Recombinant Proteins. Postharv Biol Technol 27: 147–156.
[14]
Lu Z, Takano T, Liu S (2005) Purification and characterization of two ascorbate peroxidases of rice (Oryza sativa L.) expressed in Escherichia coli. Biotechnol Lett 27(1): 63–67.
[15]
Yoshimura K, Yabuta Y, Ishikawa T, Shigeoka S (2000) Expression of Spinach Ascorbate Peroxidase Isoenzymes in Response to Oxidative Stresses. Plant Physiol 123: 223–234.
[16]
Ma CL, Wang PP, Cao ZY, Zhao YX, Zhang H (2002) cDNA Cloning and Gene Expression of APX in Suaeda salsa in Response to Salt Stress. J Plant Physio and Molecu Biol 28: 261–266.
[17]
Brawn K, Fridovich I (1981) DNA strand scission by enzymically generated oxygen radicals. Arch. Biochem Biophys 206: 414.
[18]
Chwan YH, Ching HK (2008) NaCl-induced expression of ascorbate peroxidase 8 in roots of rice (Oryza sativa L.) seedlings is not associated with osmotic component, Plant Signaling & Behavior 3: 3, 199–201.
[19]
Agrawal GK, Jwa NS, Iwahashi H, Randeep R (2003) Importance of ascorbate peroxidases OsAPx1 and OsAPx2 in the rice pathogen response pathways and growth and reproduction revealed by their transcriptional profiling Science Gene. 322: 93–103.
[20]
Lu Z, Liu D, Liu S (2007) Two rice cytosolic ascorbate peroxidases differentially improve salt tolerance in transgenic Arabidopsis. Plant Cell Rep 26(10): 1909–1917.
[21]
Xie CP (2003) King of Grass–Alfalfa. Xinjiang Agricultural Science and Technology5: 43.
[22]
Liang Z, Luo A L, Zhao Y, Tang L (1996) Accumulation of Betaine Aldehyde Dehydrogenase Induced by Drought and Salt Stress in Sugar Beet Leaves. Acta Phytophisiologica Sinica 22(2): 161–164.
[23]
Wang J, Zhang H, Allen RD (1999) Over-expression of an Arabidopsis Peroxisomal Ascorbate Peroxidase Gene in Tobacco Increases Protection against Oxidative Stress. Plant Cell Physiology 40(7): 725–732.
[24]
Hightower R, Cathy B, Ranela D (1991) Expression of antifreeze proteins in transgenic plants. Plant Molecular Biology 17 (5): 1013–1021.
[25]
Winicowi Bastoladr (1999) Transgenic Over-expression of the Transcription Factor Alfinl Enhances Expression of the Endogenous MsPRP-gene in Alfalfa and Improves Salinity Tolerance of the Plants. Plant Physiol 120: 473–480.
[26]
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd. New York: Cold Spring Harbor Laboratory Press.pp.1566–1573.
[27]
Bradford M (1976) A rapid and sensitive method for the quantification of g quantities of protein. Anal Chem 72: 248–254.
[28]
Sambrook J, Ruessell DW (2001) Molecular Cloning: A Laboratory Manual, 3rd.Cold Spring Harbor Laboratory New York Press. 1474–1480.
[29]
Jin SJ, Guan QJ, Luo QX, Wang T, Takano T, et al. (2006) Study on High Frequency Regeneration and Transformation System of Alfalfa from Embryo genic Callus.Molecular Plant Breeding. 4(4): 571–578.
[30]
Ralph H, József F (1999) Christine Preis and Karl-Heinz Kogel. Hypersensitive cell death and papilla formation in barley attached by the powdery mildew fungus are associated with hydrogen peroxide but not with salicylic acid accumulation. Plant Physiology 119: 1251–1260.
[31]
Zhao SJ, Dong XC (1998) Laboratory Manual of Plant Physiology, Beijing: China Agriculture Press. 305–306.
[32]
Shigeoka S, Ishikawa T, Tamoi M, Miyagawa Y, Takeda T, et al. (2002) Regulation and Function of Ascorbate Peroxidase Isozymes. J Exp Bot 53: 1305–1319.
[33]
Hodges DM, De Long JM, Forney CF, Prange RK (1999) Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207: 604–611.
[34]
Wu GH, Zhan FJ, Huang ZL, Luo HL, Shao ZF (2001) Study on the Effects of Chilling Stress on the Metabolism of Acacia mangium. Forest Research 14(6): 633–640.
[35]
Cadenas E (1989) Biochemistry of oxygen toxicity. Annu Rev Biochem 58: 79–110.
[36]
Sze K, A braha M, Cse P, Rigo G, Zsigmond L, et al. (2008) Duplicated P5CS Genes of Arabidopsis Play Distinct Roles in Stress Regulation and Developmental Control of Proline Biosynthesis. Plant J 53: 11–28.
[37]
Nounjan N, Nghia PT, Theerakulpisut P (2012) Exogenous proline and trehalose promote recovery of rice seedlings from salt-stress and differentially modulate antioxidant enzymes and expression of related genes. J Plant Physiol 169(6): 596–604.
[38]
Chen JB, Wang SM, Jing RL, Mao XG (2009) Cloning the PvP5CS gene from common bean (Phaseolus vulgaris) and its expression patterns under abiotic stresses. J Plant Physiol 166: 12–19.
[39]
Chou TS, Chao YY, Kao CH (2012) Involvement of hydrogen peroxide in heat shock-and cadmium induced expression of ascorbate peroxidase and glutathione reductase in leaves of rice seedlings. J Plant Physiol 169(5): 478–486.
[40]
Trinh TH, Ratet P, Kondorosi E, Durand P, Kamaté K, et al. (1998) Rapid and efficient transformation of diploid Medicago truncatula and Medicago sativa ssa.falcate lines improved in somatic embryogenesis. Plant Cell Reports 17: 345–355.
[41]
Deak M, Kiss G, Koncz C, Dudits D (1986) Transformation of Medicago by Agrobacterium mediated gene transfer. Plant Cell Reports 5(2): 97–100.
[42]
Ganesh KA, Nam SJ, Hitoshi I, Randeep R (2003) Importance of ascorbate peroxidases OsAPx1 and OsAPx2 in the rice pathogen response pathways and growth and reproduction revealed by their transcriptional profiling. Science Gene 322: 93–103.
[43]
Li Bl, Mei HS (1989) Relationship between Oat Leaf Senescence and Activated Oxygen Metabolism. Acta Photophysiologica Sinica 15(1): 62–72.
[44]
Zhao J, Wang C, Bedair M, Welti R, Sumner LW, et al. (2011) Suppression of Phospholipase Dys Confers Increased Aluminum Resistance in Arabidopsis thaliana. PLoS ONE 6(12): e28086.
[45]
Qian H, Lu T, Peng X, Han X, Fu Z, et al. (2011) Enantioselective Phytotoxicity of the Herbicide Imazethapyr on the Response of the Antioxidant System and Starch Metabolism in Arabidopsis thaliana. PLoS ONE 6(5): e19451.
[46]
Yang YX (2002) Main Characteristics, Progress and Prospect of International Wetlands Science Research.Progress in Geography. 21(2): 111–120.
Xu ZZ, Yu ZW, Dong QY, Qi XH, Yu SL (1997) Effects of Water on Cell Membrane and the Ultrastructure of Flag Leaf Cell Winter Wheat. Acta Agronomica Sinica 23(3): 370–375.
[49]
Du XM, Yin WX, Zhang H, Zhao YX (2003) The Researching Progress of superoxide Dismutase.Journal of Chinese Biotechnology. 23(1): 49–51.
