Transgenic barley with overexpressed PTrx increases aluminum resistance in roots during germination

Transgenic barley with overexpressed PTrx increases aluminum resistance in roots during germination 1 first-author 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:xmzxyj@126.comshaohongbochu@126.com †E-mail:xmzxyj@126.comshaohongbochu@126.com xmzxyj@126.com 2 3 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:xmzxyj@126.comshaohongbochu@126.com †E-mail:xmzxyj@126.comshaohongbochu@126.com xmzxyj@126.com 1 1 1 1 National Engineering Research Center for Wheat, Henan Agricultural University, Zhengzhou 450002, China National Engineering Research Center for Wheat, Henan Agricultural University, Zhengzhou 450002, China Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China Institute of Life Sciences, Qingdao University of Science and Technology (QUST), Qingdao 266042, China Institute of Life Sciences, Qingdao University of Science and Technology (QUST), Qingdao 266042, China A transgenic barley line (LSY-11-1-1) with overexpressed Phalaris coerulescens thioredoxin gene ( PTrx ) was employed to measure the growth, protein oxidation, cell viability, and antioxidase activity in barley roots during germination on the presence of 2 mmol/L AlCl 3 on filter paper. The results show that (1) compared with the non-transgenic barley, LSY-11-1-1 had enhanced root growth, although both were seriously inhibited after AlCl 3 treatment; (2) the degree of protein oxidation and loss of cell viability in roots of LSY-11-1-1 were much less than those in roots of non-transgenic barley, as reflected by lower contents of protein carbonyl and Evans blue uptakes in LSY-11-1-1; (3) activities of catalase (CAT), glutathione peroxidase (GPX), ascorbate peroxidase (APX), and glutathione reductase (GR) in LSY-11-1-1 root tips were generally higher than those in non-transgenic barley root tips, although these antioxidase activities gave a rise to different degrees in both LSY-11-1-1 and non-transgenic barley under aluminum stress. These results indicate that overexpressing PTrx could efficiently protect barley roots from oxidative injury by increasing antioxidase activity, thereby quenching ROS caused by AlCl 3 during germination. These properties raise the possibility that transgenic barley with overexpressed PTrx may be used to reduce the aluminum toxicity in acid soils. Phalaris coerulescens thioredoxin gene ( PTrx ) Aluminum Transgenic barley Oxidative stress Introduction 1 Numerous environmental stresses induce the formation of reactive oxygen species (ROS) in plant cells (Boscolo et al., 2003 2003 ). Excessive metallic ions are a well-known example of environmental stress, and aluminum is a particularly important ion because it is involved in a number of physiological processes, such as increasing antioxidase activity (Cakmak and Horst, 1991 1991 ; Yamamoto et al., 2001 2001 ), and inducing the expression of various genes related to oxidative stress (Richards et al., 1998 1998 ; Maron et al., 2008 2008 ; Goodwin and Sutter, 2009 2009 ). Corresponding with this, there is a complex scavenging system to cope with excessive production of ROS in plant cells, including low molecular weight compounds such as ascorbic acid and glutathione, and various antioxidases (Vieira Dos Santos and Rey, 2006 2006 ) such as guaiacol peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX), and glutathione reductase (GR) (Mittler, 2002 2002 ). The ROS scavenging system efficiently reduces ROS under normal conditions, but complete reduction might not occur in stress environments, and the result may be a state of oxidative stress causing oxidative damage of biomolecules, such as lipids, proteins, and DNA, or even causing cell death (Delisle et al., 2001 2001 ; Pan et al., 2001 2001 ; Boscolo et al., 2003 2003 ; Shao et al., 2007a 2007a ; 2007b 2007b ; 2008 2008 ; 2009 2009 ). Thioredoxin (Trx) is a family of small, 12 kDa proteins, and can be found in all plants. The active site (Trp-Cys-Gly(Pro)-Pro-Cys) of these proteins contains a redox-active disulfide group (Lemaire et al., 2003 2003 ; Wong et al., 2004 2004 ). The exact function of Trx is still largely unknown. There are more and more reports, however, on the participation of plant Trx in the response to oxidative stress (Rey et al., 2005 2005 ; Vieira Dos Santos and Rey, 2006 2006 ; Shao et al., 2007a 2007a ; 2007b 2007b ; 2008 2008 ; 2009 2009 ). The development of molecular biology has provided new insights into plant Trx functions in the oxidative stress response. ROS induces the expression of plant Trx genes. The amount of Arabidopsis Trxh5 expression, for example, increases remarkably under oxidative stress conditions (Laloi et al., 2004 2004 ), and methyl viologen induces an increase in Trxh transcript abundance in rice seedlings (Tsukamoto et al., 2005 2005 ; Vieira Dos Santos and Rey, 2006 2006 ). As well, target proteins of plant Trx are involved in antioxidative mechanisms. One of the 2-Cys peroxiredoxins (Prxs) and B-type methionine sulfoxide reductase (MsrB), for example, have been identified as targets of CDSP32 (the chloroplastic drought-induced stress protein, composed of two Trx modules) (Rey et al., 2005 2005 ). Several plant GPXs display peroxidase activity preferentially in the presence of Trx (Vieira Dos Santos and Rey, 2006 2006 ). In addition, there are some Trx-regulated enzymes, such as CAT, SOD, and germin-like proteins (Balmer et al., 2004 2004 ). Finally, Trx may protect plant from oxidative damage as antioxidants in vivo. The sensitivity and lipid peroxidation levels of transformed plants lacking CDSP32 are higher, for example, than those of wild-type plants upon photo-oxidative stress exposure (Broin and Rey, 2003 2003 ). These results indicate that plant Trx fulfils an important function in protecting the plant from oxidative damage, and raises the possibility that transgenic barley with overexpressed PTrx will improve tolerance of aluminum stress. The PTrx gene belongs to the thioredoxin family because it shares nearly 94% sequence identity with Trxh , and its expression product has the same bio-functions and active site as Trxh (Li et al., 1995 1995 ). In the present study, we measured the change of the growth, protein oxidation, cell viability, and antioxidase activity in roots of germinating barley seeds treated with 2 mmol/L AlCl 3 on filter paper. The main aim was to determine whether the transgenic barley with overexpressed PTrx activates protective responses to aluminum stress through increasing antioxidase activity during germination. Materials and methods 1 Materials 2 Non-transgenic barley variety ( Hordeum vulgare L. cv. LSY) and its third generation transgenic lines (LSY-11-1-1) transferred with exogenous PTrx (isolated from Phalaris coerulescens , mRNA No. GB: AF159388) by particle bombardment, were used in this study. Polymerase chain reaction (PCR) analysis 2 Genomic DNA was extracted from leaf tissue according to cetyltrimethylammonium bromide (CTAB) method described by Wang and Fang ( 1998 1998 ). Specific primers were designed according to the coding sequence of PTrx gene. The sequences of the primers were P1 (5′-TTCTGTGCCAGCCATGCTTAT-3′) to target the endosperm-specific α-gliadin promoter region and P2 (5′-GTCCCAGTCCTCTTTGGTAGTTATG-3′) to target the PTrx sequence. PCR products with loading dye were separated on a 1.5% (w/v) agarose gel by electrophoresis and scanned using a gel scanner JEDA801E (Jieda Science and Technology, Jiangsu, China). Reverse transcriptase PCR (RT-PCR) analysis and sulfhydryl content in seeds 2 Seed samples of transgenic and non-transgenic lines were prepared and total RNA was isolated from the samples using the RNeasy kit (Sangon, Shanghai, China) according to the manufacturer’s instructions. RT-PCR analysis was used to determine transcript levels both in transgenic and non-transgenic lines according to the method of Li et al. ( 2009 2009 ). Specific primers P3 (5′-CTCCCGATCCCAGGGCCTTCA-3′) and P4 (5′-GGCGGAAAAGACACGGAAACTG-3′) for PTrx were used in the RT-PCR analysis. The reaction included an initial 3 min of denaturation at 94 °C, followed by 27 cycles of 30 s at 94 °C, 30 s at 59 °C, and 40 s at 72 °C, a final extension of 10 min at 72 °C. As a control, a 415-bp PCR fragment of the barley constitutively-expressed actin gene Hvpro1 (AK248710) (Sato et al., 2009 2009 ) was amplified from the above samples. The primers for Hvpro1 were HvPr-1 (5′-CAGCATTGTAGGAAGGCCACG-3′) and HvPr-2 (5′-CCACCACTGAGAACAACATTACCG-3′). The PCR parameters for amplifying Hvpro1 fragments were the same as those for PTrx . PCR products were separated and photographed as described in PCR analysis. Two bands, 415 and 285 bp, were detected for the Hvpro1 and PTrx genes, respectively. The sulfhydryl content was measured by the method of Ellman ( 1959 1959 ). Flours (0.15 g) were homogenized in 10 ml of Tris-glycine buffer (0.08 mol/L, pH 8.0) containing 3 mmol/L ethylene diamine tetraacetic acid (EDTA), and then 4 ml denaturation reagent (8 mol/L urea) and 0.1 ml Ellman’s reagent [Tris-glycine buffer containing 0.4% (w/v) 2,2′-dinitro-5,5′-dithiodibenzoate (DTNB)] were added in 1 ml samples. Thionitrobenzoate, released by sulfhydryl groups of proteins reacted with DTNB, was measured at 412 nm, and the results were expressed as µmol sulfhydryl/g dry weight (DW). Germination conditions 2 Germination and growth conditions were conducted according to the method of Tamás et al. ( 2006 2006 ). Seeds were surface sterilized with 12% H 2 O 2 for 10 min and then rinsed three times with sterilized distilled water. After 4 h of incubation in 8.2 mmol/L CaCl 2 solution (pH 4.0, untreated) or in 6.2 mmol/L CaCl 2 containing 2 mmol/L AlCl 3 solution (pH 4.0, Al-treated) at 25 °C in darkness, the seeds were germinated between two layers of filter paper fully moistened with the same solutions, and the germinating seeds were transferred on the freshly moistened filter papers with appropriate solutions every 20 h. Root growth was measured by the length of the main roots. Protein oxidation and cell viability 2 The degree of protein oxidation was measured by the reaction of carbonyls with 2,4-dinitrophenylhydrazine (DNPH), according to the method of Levine et al. ( 1990 1990 ). Fresh root tips (0.2 g) were homogenized in 2 ml phosphate buffer solution (25 mmol/L, pH 7.0) containing 0.1 mmol/L EDTA and 1 mmol/L phenylmethanesulfonyl fluoride (PMSF), and then centrifuged at 15 000× g for 15 min. After the supernatant reaction with DNPH, the carbonyl content was calculated by absorbance of aliphatic hydrazones [extinction coefficient: 22.1 L/(mmol·cm)] at 374 nm and expressed as nmol carbonyl/mg protein. The cell viability loss was investigated though Evans blue staining method as described by Yamamoto et al. ( 2001 2001 ) and Boscolo et al. ( 2003 2003 ), with minor modifications. Fresh roots were washed three times with deionized water and stained with 0.25% (w/v) Evans blue aqueous solution for 15 min. After rinsing with deionized water for 30 min, 20 root tips (5 mm) were excised and the accumulated Evans blue was released by dipping the root sections in 3 ml of N , N -dimethylformamide reagent for 1 h at 37 °C, and then centrifuged at 3000× g for 5 min. The optical density (OD) of the supernatant was determined spectrophotometrically at 600 nm. Soluble protein concentrations were measured using the method of Bradford ( 1976 1976 ). Enzyme activity 2 Enzyme activity was measured spectrophotometrically, and temperature was controlled to (25±0.5) °C. CAT activity was determined as outlined by Cakmak and Horst ( 1991 1991 ), and the activity was analyzed by measuring the degradation of H 2 O 2 , which was monitored at 240 nm. One unit of CAT decomposed 1 μmol/L of H 2 O 2 [extinction coefficient: 39.4 L/(mmol·cm)] per minute. GR activity was assayed following the procedure of Schaedle and Bassham ( 1977 1977 ), measuring the decrease of optical density at 340 nm, with the extinction coefficient of 6.22 L/(mmol·cm). One unit of activity is defined as the amount of enzyme that oxidizes 1 µmol of nicotinamide adenine dinucleotide phosphate (NADPH) per minute. GPX activity was determined following the method of Livingstone et al. ( 1992 1992 ). The assay was monitored at 340 nm and one unit of activity is defined as the amount of enzyme that oxidizes 1 µmol of NADPH [extinction coefficient: 6.22 L/(mmol·cm)] per minute. APX activity was measured according to Asada ( 1984 1984 ) and the activity was recorded by the decrease of optical density at 290 nm, taking 2.8 L/(mmol·cm) as the extinction coefficient, and one unit of activity is defined as the amount of enzyme that oxidizes 1 µmol of ascorbate per minute. Statistical analysis 2 Each experiment was repeated at least five times. Data were expressed as mean±standard error (SE). Variance analysis between different treatments was carried out with the SPSS 11.5 program using Duncan’s multiple range test at P < 0.01. Results 1 PCR analysis 2 The presence of exogenous PTrx gene in transgenic barley was examined with PCR analysis. DNA from leaves of the transgenic barley lines (LSY-11-1-1) was amplified by PCR reaction using primers specific for exogenous PTrx . The transgenic plants exhibited the polymorphic band 810 bp in length, the same size as the positive control (plasmid), but non-transgenic plant DNA was negative (Fig. 1 1 ). The results showed that exogenous PTrx gene had been transferred in barley genome. 1–13: transgenic plants of LSY-11-1-1 (the next generation from T 5 in Figs. 2 2 and 3 3 ); CK: non-transgenic plants; P: positive control (plasmid); M: marker RT-PCR analysis and sulfhydryl content 2 To investigate the expression of exogenous PTrx gene in LSY-11-1-1, the template cDNAs from transgenic and non-transgenic barley seeds 15 d after anthesis were assayed by RT-PCR and the sulfhydryl content in the mature seeds was measured. The specific band of 215 bp was exhibited in transgenic seeds, while non-transgenic seeds were negative (Fig. 2 2 ). These results indicate that exogenous PTrx gene can be transcribed and expressed normally in transgenic barley seeds. CK: non-transgenic plants; T 1 –T 5 : seeds of different transgenic lines of the third generation (T 5 is the plants of LSY-11-1-1) The sulfhydryl content in seeds of transgenic barley was generally higher than that in non-transgenic (Fig. 3 3 , P < 0.01), and, in LSY-11-1-1, was 1.3 times that of non-transgenic barley. Data are expressed as mean±SE ( n =5); Bars (i.e. means) with different letters are significantly different ( P < 0.01). CK: non-transgenic; T 1 –T 5 : seeds of different transgenic lines of the third generation (T 5 is the seeds of LSY-11-1-1) Effect of Al-treatment on root growth 2 As seen in Fig. 4 4 , the root growth of germinating barley seeds was obviously inhibited between 72 and 96 h after the onset of seed imbibition treated by 2 mmol/L AlCl 3 . Although a continuous decrease of the root growth of both transgenic and non-transgenic germinating barley seeds was observed, the root length of LSY-11-1-1 was obviously longer than that of non-transgenic barley. Data are expressed as mean±SE ( n =5) Protein oxidation and cell viability 2 Carbonyl group content in the transgenic and non-transgenic barley root tips were presented in Fig. 5a 5a . The result showed that protein oxidation of LSY-11-1-1 was clearly lower than that of non-transgenic barley. The carbonyl content of LSY-11-1-1 was decreased, for example, by 37.0%, 20.8%, and 23.3% after 2 mmol/L AlCl 3 stress for 72, 84, and 96 h, respectively, compared with the results of non-transgenic barley. Data are expressed as mean±SE ( n =5); Bars (i.e., means) with different letters are significantly different ( P < 0.01) The Evans blue uptakes in root tips were significantly increased after AlCl 3 treatment (Fig. 5b 5b ). The non-transgenic barley showed the highest value after 96 h treatment, and it was 2.9-fold higher than that of the untreated sample. Compared to the non-transgenic barley, the Evans blue uptakes in LSY-11-1-1 were significantly lower ( P < 0.01), only 66.0%, 68.5%, and 56.1% that of non-transgenic barley after 48, 72, and 96 h treatments, respectively. Effect of Al-treatment on antioxidase activity 2 The changes in antioxidase activities are shown in Table 1 1 . Our experimental results demonstrated that the activities of CAT, GPX, GR, and APX were significantly increased after aluminum stress, except that of GR at 48 h ( P < 0.01). Data are expressed as mean±SE ( n =5). Means followed by different letters are significantly different ( P < 0.01) Time (h) Group CAT activity (U/mg protein) GPX activity (U/mg protein) APX activity (mU/mg protein) GR activity (mU/mg protein) 48 Non-transgenic 25.65±0.66 a 41.46±1.19 a 398.77±3.38 a 151.44±0.83 a LSY-11-1-1 24.47±0.68 a 39.93±0.81 a 418.66±9.84 ab 145.48±1.83 a Al–non-transgenic 50.69±1.23 b 68.14±1.39 b 455.27±6.89 b 148.62±1.38 a Al–LSY-11-1-1 50.04±0.74 b 70.63±0.65 b 453.01±9.55 b 150.58±1.92 a 6 60 Non-transgenic 35.04±0.75 a 39.99±1.32 a 340.17±4.11 a 130.69±1.72 a LSY-11-1-1 38.96±0.93 a 39.11±0.90 a 321.33±2.40 a 139.93±1.09 b Al–non-transgenic 47.56±0.92 b 76.77±1.91 b 377.33±5.24 b 203.44±1.99 c Al–LSY-11-1-1 53.19±1.69 c 77.85±1.01 b 407.33±4.67 c 209.59±2.11 c 6 72 Non-transgenic 30.48±1.46 a 36.88±1.38 a 381.67±5.24 b 134.60±2.56 a LSY-11-1-1 32.59±1.80 a 41.85±1.06 a 344.00±3.21 a 123.79±1.63 a Al–non-transgenic 46.30±1.10 b 56.96±1.48 b 493.65±5.04 c 198.72±1.29 b Al–LSY-11-1-1 55.07±1.03 c 65.26±0.63 c 562.66±3.38 d 208.18±2.39 b 6 84 Non-transgenic 32.18±1.21 a 44.00±1.74 a 452.78±5.59 b 161.14±1.71 a LSY-11-1-1 31.38±1.14 a 44.30±2.09 a 402.66±6.84 a 153.59±2.00 a Al–non-transgenic 55.00±1.61 b 68.99±0.71 b 693.66±8.84 c 239.11±1.74 b Al–LSY-11-1-1 72.92±1.72 c 96.66±2.17 c 764.43±10.49 d 275.01±2.08 c 6 96 Non-transgenic 38.11±1.49 a 46.29±0.99 a 410.00±6.43 a 136.63±0.38 a LSY-11-1-1 34.40±1.41 a 39.79±0.76 a 441.63±4.94 a 140.01±1.07 a Al–non-transgenic 52.24±1.26 b 70.87±1.15 c 677.25±3.76 b 234.40±2.41 b Al–LSY-11-1-1 61.44±2.03 c 92.78±1.45 d 753.66±18.22 c 269.41±1.23 c Compared to non-transgenic barley, the activities of these antioxidases were enhanced significantly in LSY-11-1-1 root tips ( P < 0.01). In the presence of 2 mmol/L Al 3+ treatment, the CAT activity of LSY-11-1-1 was 11.8%, 18.9%, 32.6%, and 17.6% higher than that of non-transgenic barley at 60, 72, 84, and 96 h after treatment, respectively. PTrx overexpressing induced similar activity changes of GPX, the maximal value of GPX in LSY-11-1-1 was 96.7 U/mg protein at the activity peak, while it was 76.8 U/mg protein in non-transgenic barley. Compared with non-transgenic barley, the GPX activity of LSY-11-1-1 was significantly increased by 14.6%, 40.1%, and 30.9% after 72, 84, and 96 h treatments, respectively. APX activity of LSY-11-1-1 was 7.9%, 13.9%, 10.2%, and 11.3% greater than that of non-transgenic barley at 60, 72, 84, and 96 h after treatment, respectively. Although the GR activity was not significantly different between LSY-11-1-1 and non-transgenic barley from 48 to 72 h after Al 3+ stress, it was significantly higher in LSY-11-1-1 at the other time sampled. The GR activity of LSY-11-1-1 was 1.2 and 1.1 times of that of non-transgenic barley at 84 and 96 h, respectively. Discussion 1 We transferred exogenous PTrx gene into malting barley variety and obtained transgenic plants by the detection of PCR analysis. In order to confirm the expression of PTrx , the specific band of 215 bp in the transgenic line was lifted by RT-PCR, while the non-transgenic barley was negative. This revealed a positive result, indicating that exogenous PTrx gene had been successfully transferred and expressed in transgenic barley. The active site Trp-Cys-Gly(Pro)-Pro-Cys of Trx contains a highly reactive dithiol, which may reduce disulfide bridges of many proteins by thiol-disulfide interchange (Lemaire et al., 2003 2003 ), so overexpressed PTrx will enhance the reducing ability in the transgenic barley seeds. Our experimental data demonstrate that the sulfhydryl content in the seeds of transgenic barley is higher than that in the non-transgenic barley, which also explains the PTrx expression. Root growth of both transgenic barley and non-transgenic barley was inhibited seriously in the presence of Al 3+ , consistent with the results reported by Kochian ( 1995 1995 ) and Boscolo et al. ( 2003 2003 ), but the transgenic barley showed a stronger root growth than non-transgenic barley on filter paper with AlCl 3 treatment during germination, as reflected by the greater root length. These results demonstrate that the phytotoxic effects of Al 3+ on root growth are less severe in transgenic barley root growth than in non-transgenic barley root growth. Oxidative stress is possibly an important component of the plant’s reaction to aluminum toxicity (Pan et al., 2001 2001 ). Several studies have shown that aluminum treatment causes excessive ROS formation (Cakmak and Horst, 1991 1991 ; Boscolo et al., 2003 2003 ). In our experiment, the increased activities of SOD, POD, CAT, APX, GPX, and GR induced by aluminum treatment suggest that Al 3+ induces ROS excessive production. The result of excessive ROS formation may cause biomolecule oxidation or even cell death (Cakmak and Horst, 1991 1991 ; Delisle et al., 2001 2001 ; Pan et al., 2001 2001 ; Yamamoto et al., 2001 2001 ). We investigated protein oxidation through a determination of the amount of carbonyl, an indicator that describes protein oxidation degree caused by ROS (Levine et al., 1990 1990 ; Boscolo et al., 2003 2003 ), and cell death through changes of Evans blue uptakes, a parameter of loss of cell viability, another parameter of oxidative stress damage in plant (Yamamoto et al., 2001 2001 ; Boscolo et al., 2003 2003 ). The lower protein carbonyl contents and Evans blue uptakes in roots of LSY-11-1-1 indicate that excessive ROS, caused by AlCl 3 stress, was eliminated more efficiently in the transgenic barley roots than in the non-transgenic barley roots during germination, so the degree of the damage caused by AlCl 3 stress in transgenic plants was less than that in non-transgenic plants, which may be due to its effective antioxidase system. In general, the major ROS-scavenging enzyme pathways of plants include SOD [Eq. ( 1 1 )], CAT [Eq. ( 2 2 )], the ascorbate-glutathion cycle [Eq. ( 3 3 )], and GPX cycle [Eq. ( 4 4 )] (Mittler, 2002 2002 ). $${\text{O}}_2^ - \xrightarrow{{{\text{SOD}}}}{{\text{H}}_2}{{\text{O}}_2}$$ ; $${{\text{H}}_2}{{\text{O}}_2}\xrightarrow{{{\text{CAT}}}}{{\text{H}}_2}{\text{O}} + 1/2{{\text{O}}_2}$$ ; $$\begin{gathered}{{\text{H}}_2}{{\text{O}}_2} + {\text{ascrobate}}\xrightarrow{{{\text{APX}}}}{{\text{H}}_2}{\text{O}} + {\text{MDA}}, \\{\text{MDA}} + {\text{NAD(P)H}}\xrightarrow{{{\text{MDAR}}}}{\text{ascrobate}} + {\text{NAD(P}}{{\text{)}}^ + }, \\{\text{Dehydroascrobate}} + {\text{GSH}}\xrightarrow{{{\text{DHAR}}}}{\text{ascrobate}} + {\text{GSSG}}, \\{\text{GSSG}} + {\text{NAD(P)H}}\xrightarrow{{{\text{GR}}}}{\text{GSH}} + {\text{NAD(P}}{{\text{)}}^ + } \\\end{gathered}$$ ; $$\begin{gathered}{\text{GSSG}} + {\text{NAD(P)H}}\xrightarrow{{{\text{GR}}}}{\text{GSH}} + {\text{NAD(P}}{{\text{)}}^ + }, \\{{\text{H}}_2}{{\text{O}}_2} + {\text{GSH}}\xrightarrow{{{\text{GPX}}}}{{\text{H}}_2}{\text{O}} + {\text{GSSG}} \\\end{gathered}$$ . In our experiment, AlCl 3 treatment induced a significant increase in SOD, POD, CAT, GPX, APX, and GR activities, although the activities of SOD and POD were similar changes in both LSY-11-1-1 and non-transgenic barley (data not shown). It is apparent that all the ROS-scavenging enzyme pathways of plants were indispensable for ROS detoxification in roots during barley seed germination in the presence of 2 mmol/L AlCl 3 , but the CAT pathway, ascorbate glutathione cycle, and GPX cycle played a more important role on the progress of quenching ROS caused by Al 3+ stress in transgenic barley, and the increased antioxidase activity contributed to a more effective reduction in the oxidative damage in roots during germination. It is suggested that the redox state of proteins, including enzymes and regulatory components, is controlled by cellular redox agents, the paramount one among which is Trx (Wong et al., 2004 2004 ). Several antioxidases, protecting plant cells from oxidative stress, are potential targets of Trxh, such as CAT, SOD (Cu-Zn), and GR (Jung et al., 2002 2002 ), almost all of the which contain conserved cysteine, e.g., the numbers of conserved cysteine of CAT, GPX, POD, SOD (Cu-Zn), APX, 2-Cys Prxs, and 1-Cys Prxs are 5, 1, 8, 2, 2, 1, and 1, respectively (Wong et al., 2004 2004 ). Trx might be involved in modulating redox-dependent signaling cascades, as shown by Schenk et al. ( 1994 1994 ), or reduced Trx transfers its electrons to a target disulfide (Meyer et al., 1999 1999 ; Wong et al., 2004 2004 ), as a result providing reducing equivalents for regulating activity and increasing the antioxidase activity (Wong et al., 2004 2004 ). Moreover, the thioredoxin gene and several genes encoding Cys-rich proteins were previously shown to be induced by aluminum stress (Maron et al., 2008 2008 ; Goodwin and Sutter, 2009 2009 ), suggesting that thioredoxins could play similar role as antioxidants. Aluminum is the most abundant metal in the earth’s crust, and aluminum toxicity is a very important factor limiting crop growth in acidic soil (Kochian, 1995 1995 ). Thus, it is necessary to enhance the aluminum endurance of plants. Increased activities of antioxidases endow the transgenic barley with more effective elimination of excessive ROS induced by aluminum stress. This decreases the damage of biomolecules by oxidation and contributes to the integrity of membrane structure. In turn, aluminum is prevented from exerting its full physiological toxicity in the roots, as reflected by the fact that the roots of transgenic barley geminating seeds showed better growth than those of non-transgenic barley. 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Lipid peroxidation is an early symptom triggered by aluminum, but not the primary cause of elongation inhibition in pea roots 10.1631/jzus.B1000048 Project supported by the National Natural Science Foundation of China (No. 30871530), the Science and Technology Transformation Plan of Henan Province (No. 0636000005), One Hundred-Talent Plan of Chinese Academy of Sciences (CAS), CAS-local Government Cooperative Project, the CAS/SAFEA International Partnership Program for Creative Research Teams, and CAS Young Scientists Fellowship (No. 2009Y2B211), China 2010-02-10 2010-10-15 2010-04-05 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2010 2010 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 11 11