A glutathione s-transferase confers herbicide tolerance in rice

Plant glutathione S-transferases (GSTs) have been a focus of attention due to their role in herbicide detoxification. OsGSTL2 is a glutathione S-transferase, lambda class gene from rice (Oryza sativa L.). Transgenic rice plants over-expressing OsGSTL2 were generated from rice calli by the use of an Agrobacterium transformation system, and were screened by a combination of hygromycin resistance, PCR and Southern blot analysis. In the vegetative tissues of transgenic rice plants, the over-expression of OsGSTL2 not only increased levels of OsGSTL2 transcripts, but also GST and GPX expression, while reduced superoxide. Transgenic rice plants also showed higher tolerance to glyphosate and chlorsulfuron, which often contaminate agricultural fields. The findings demonstrate the detoxification role of OsGSTL2 in the growth and development of rice plants. It should be possible to apply the present results to crops for developing herbicide tolerance and for limiting herbicide contamination in the food chain.


INTRODUCTION
GSTs are an ancient and multifunctional protein family encoded by a large gene family found ubiquitously in bacteria, fungi, animals and plants (Frova 2006).Plant GSTs have been the focus of attention due to their role in herbicide detoxification, which have been actively investigated during the last decades (Chi et al. 2011).Intensive studies of plant GSTs are required due to the considerable agronomic potential of these enzymes with regard to herbicide selectivity, tolerance and environmental safety (Edwards and Dixon 2005).
The complete identification in a genome-wide level revealed the presence of at least 79 GST genes in rice genome (Soranzo et al. 2004).Sequence analysis, and the organization of putative motifs indicated the potential diverse functions of GST gene family members in rice.Microarray data analysis revealed tissue-/organ-, and developmental stage-specific expression patterns of some rice GST genes.At least 31 GST genes responded to auxin and cytokinin plant hormones, 20 to abiotic stress, 32 to arsenate stress, and 48 to biotic stress.Many of GST genes in rice were commonly controlled by developmental processes, hormones, abiotic and biotic stresses (Jain et al. 2010).
GSTLs are one of the smallest clades of plant GST superfamily.Several GSTL features make them important targets for functional characterization.In particular, specific GSTLs are strongly upregulated in response to exposure to xenobiotic compounds, including herbicides, herbicide safeners and pharmaceuticals (Hershey and Stoner 1991, Dixon et al. 2002, Theodoulou et al. 2003).As with other GST classes, this correlation provides circumstantial evidence for a role in stress tolerance (Dixon et al. 2011).OsGSTL2 is a glutathione S-transferase, lambda class gene from rice, which showed response to chlorsulfuron.OsGSTL2 protein has a specific activity of GST (Hu et al. 2011).The purpose of this study was to over-express OsGSTL2 in rice and investigate the role of OsGSTL2 in protecting plants from the injury caused by herbicides.

Construction of transgene vector and plant transformation
To construct OsGSTL2 over-expression vector, OsGSTL2 was cloned first into pENTR/D-TOPO vector, and then recombined into pHOS vector by LR reaction (Figure 1).The construction was confirmed by restriction digestion analysis, and then by sequencing.After transferring to Agrobacterium tumefaciens strain AGL0 through the freeze-thaw method (Höfgen and Willmitzer 1988), the construct was used to transform embryogenic calli derived from Oryza sativa cv.Zhonghua through the Agrobacterium-mediated co-cultivation method (Hu 2008).Transgenic plants that rooted on hygromycin were transferred to vermiculitemixed soil (rich soil: vermiculite=1:3, V/V) in small pots, and grown at 28 °C under a 16/8 h light/dark photoperiod at an intensity of approximately 250 µE m -2 s -1 .

Confirmation of positive transgenic rice plants
The positive transgenic plants were selected by PCR and Southern blot analysis.Genomic DNA of transgenic rice plants and non-transformed rice plants was extracted using Plant Genomic DNA Extraction kit (VOK-Bio, Beijing, China), and was used as models.Since HPT is closely adjacent to the target gene in the transformation constructs and not present in the non-transformed rice genome, it can be used to indicate the presence of the transgene.The primers hyg-f (5'-ATGAAAAAGCCTGAACTCACC-3') and hyg-r (5'-CCGGTCGGCATCTACTCT-3') were designed according to the HPT gene sequence.Amplification was carried out by initial denaturation at 94 ºC for 2 min followed by 35 cycles of 94 ºC denaturation for 1 min, 58 ºC annealing for 1 min, and 72 ºC elongation for 1 min.
About 15 μg of genomic DNA of transgenic and nontransformed rice plants were digested by EcoRI.The digested genomic DNA was separated on a 0.8% agarose gel electrophoresis and was transferred to a Hybond nylon membrane (Amersham, Piscataway, USA).DNA was hybridized with a labeled DNA probe, which was generated from HPT gene located within the T-DNA borders of pHOS vector and radiolabeled with [α-32 P]dCTP, using random primer system.Standard procedures of Southern blot analysis were performed.

Expression analysis of OsGSTL2 gene
Real-time PCR for analysis of OsGSTL2 expression was performed using total RNA from rice plant tissues.Total RNA samples were isolated using Trizol reagent (Gibco-BRL, USA), and subsequently treated with DNase I. RNAs were reverse-transcribed using the Superscript TM III RNase H-Reverse Transcriptase kit (Invitrogen, USA).Real-time PCR DNA amplification and analysis were carried out as described by He et al. (2012).DNA amplification and analysis were carried out using the CFX96 TM Real-Time System (C1000 TM Thermal Cycler).Using the quant Sybr green PCR Kit (Tianwei, China), and following the manufacturer's instructions, amplifications were performed with OsGSTL2-qf (5'-CGTTCAACAAAGCATCGTAC-3'), and OsGST-qr (5'-GCAAAAACTGTGGGTCCTGT-3').Data were normalized to OsEF1α, which was amplified with primers OsEF1α-f (5'-AGGGATGGGTCAAAAGGATGC-3') and OsEF1α-r (5'-GAGACAACACCGCCTGAATAGC-3').Standard curves were constructed using serial cDNA dilutions.RT-PCR data were normalized with the relative efficiency of each primer pair.PCR amplification was performed using two-step cycling conditions of 98 °C for 3 min, followed by 40 cycles of 98 °C for 2 s, and 58 °C for 10 s.Amplification was followed by a melting curve analysis with continual fluorescence data acquisition from the 55-95 °C melt.Melt curve analysis of qPCR samples revealed that there was only one product for each gene primer reaction.Relative gene expression was determined using the Pfaffl method (Pfaffl 2001), and the non-transformed plant product was set as 1.0.

Activity assay of enzyme and detection of superoxide
OsGSTL2 over-expression transgenic seedlings and nontransformed rice seedlings cultured for 15 d were used to assay GST activity and glutathione peroxidase (GPX) activity.Crude protein extracts were prepared from rice plants (Hu 2008).Protein concentration was determined using the Bradford method (Bradford 1976).GST activity was measured spectrophotometrically (Takesawa et al. 2002).One unit of activity was defined as the amount of the enzyme that T Hu catalyzes the conversion of 1 µM 2,4-dinitrochlorobenzene (CDNB) per minute at 25°C.Using the GSH-PX Kit (NJBI, China), and following the manufacturer's instructions, GPX activity was determined with H 2 O 2 as substrate.One unit of activity was defined as the amount of the enzyme that catalyzes the consumption of 1 µM H 2 O 2 per minute.Transgenic seedlings and non-transformed rice seedlings cultured for a month were used to detect superoxide.Superoxide levels were visually detected with nitro blue tetrazolium (NBT) described previously (Yang et al. 2004).

Assay for herbicide tolerance of transgenic rice plants
In order to measure responses to herbicide, transgenic seedlings and non-transformed rice seedlings cultured for 12 d were treated with 100 μmol L -1 glyphosate and 0.02% chlorsulfuron for 24 h, respectively.Seedlings were subsequently transferred into vermiculite-mixed soil and grown as described above.

Generation and confirmation of transgenic rice plants
Transgenic rice plants were generated to test OsGSTL2 physiological function in rice.Plant expression vector was introduced to Agrobacterium tumefaciens AGL0, and transformed into Oryza sativa cv.Zhonghua.Ten transgenic lines were obtained and the transformants were verified by PCR analysis.HPT gene fragments (1,042 bp) were detected in selected independent lines and in positive control OsGSTL2 over-expression vector, whereas they were not found in non-transformed plants (Figure 2A).Southern blot analysis was used to further confirm the presence of transgene, and determine insertion copy number.Genomic DNA samples of transgene rice lines and non-transformed rice plants were hybridized with a radiolabeled HPT gene fragment as probe.Since HPT is adjacent to the target gene OsGSTL2 in the transformation constructs, and is not present in the wildtype rice genome, its presence in the blot should indicate the copy number of the transgene.The result showed that the HPT hybridizing fragments were detected in all ten of the independent transgenic lines, but they were not found in non-transformed rice (Figure 2B).Three independent transgenic lines were selected for further analyses.

Expression of OsGSTL2 in transgenic rice plants
To investigate the in vivo role of OsGSTL2 in rice, OsGSTL2 was over-expressed in transgenic rice plants.
Independent transgenic lines were selected based on PCR analysis.Real time RT-PCR revealed that expression level of OsGSTL2 was higher in the transformants than in the non-transformed control.OsGSTL2 transcripts in transgenic lines 1, 2, and 3 were 4.29, 3.16, and 5.65-fold of that observed in non-transformed plants (Figure 3), respectively.These findings suggest that OsGSTL2 gene introduced in transgenic rice plants was indeed over-expressed.

Transgenic rice has higher activities of GST and GPX and lower superoxide
GST activity of crude extracts from transgenic seedlings and non-transformed rice seedlings was measured with CDNB as substrate.Transformants contained higher levels of GST activities than non-transformed rice.GST activity of lines 1, 2 and 3 were 2.07, 1.79 and 2.40-fold of that observed in non-transformed plants, respectively (Figure 4A).GPX activities in OsGSTL2 transgenic lines were measured with H 2 O 2 as substrate.GPX activity in transgenic lines was higher than the non-transformed rice as well.GPX activity in transgenic lines 1, 2 and 3 were 1.61, 1.50, and 1.71-fold of that detected in non-transformed plants, respectively (Figure 4B).GPX activities, which could degrade superoxide levels, were higher in transgenic plants as well.
Superoxide detection was performed during the vegetative growth phase of plants.Leaves of rice seedlings were stained with NBT for visually detect superoxide.Leaves of OsGSTL2 transgenic rice seedlings contain lower superoxide than those of non-transformed rice plants (Figure 5).
GSTs are known to protect plants against oxidative stress induced by biotic and abiotic agents.Some GSTs also have secondary activities as glutathione peroxidase and can protect the cells/organisms from oxidative damage.GSTs can eliminate membrane lipid peroxides, as well as products derived from oxidative DNA degradation via GSH conjugation (Berhane et al. 1994).

Transgenic rice has higher tolerance to herbicide
OsGSTL2 transgenic rice plants grew normally, and phenotypes were indistinguishable with non-transformed rice plants.To examine the role of OsGSTL2 in plants, 12-day-old seedlings of OsGSTL2 transgenic rice and non-transformed plants were incubated with 100 μmol L -1 glyphosate or 0.02% chlorsulfuron for 24 h, respectively.After glyphosate treatment, seedlings were also cultivated for 10 d; although all treated plants grew more slowly than the non-treated ones, the non-transformed plants grew even more slowly in comparison to the transgenic plants (Figure 6A).After chlorsulfuron treatment, seedlings were also cultivated for 10 d, all the treated plants grew slowly and became yellow.However, the phenotypic difference of transgenic rice seedlings and non-transformed seedlings was obvious.The non-transformed rice plants showed more severe yellowing and grew more slowly compared with the transgenic rice plants (Figure 6B).These results suggest that  Earlier work had shown that over-expression of some GSTs in plants improved herbicide tolerance.For example, transgenic wheat plants expressing the maize GST-27 gene were resistant to chloroacetanilide herbicide alachlor and dimethenamid, and thiocarbamate herbicide S-ethyldipropylthio-carbamate (Milligan et al. 2001).Transgenic tobacco plants expressing the cotton Gst-cr1 gene showed much higher expression levels of GST and GPX activities, and showed an enhanced resistance to oxidative stress induced by a low concentration of methyl viologen (Yu et al. 2003).Transgenic tobacco plants expressing maize glutathione S-transferase I presented substantially higher tolerance to alachlor compared to non-transgenic plants (Karavangeli et al. 2005).Over-expression of a specific soybean GmG-STU4 gene improved diphenyl ether and chloroacetanilide herbicide tolerance of transgenic tobacco plants (Benekos et al. 2010).

CONCLUSIONS
Transgenic rice plants over-expressing OsGSTL2 gene showed higher levels of OsGSTL2 gene expression in the absence of any treatment, increased levels of GST and GPX enzyme activities, and showed lower level of superoxide compared to wild type plants.Transgenic rice seedlings had higher tolerance to glyphosate and chlorsulfuron than nontransformed rice seedlings.Transgenic plants detoxifying herbicide are potentially useful biotechnological tools for the development of phytoremediation system for the degradation of herbicide pollutants in agricultural fields.The present results are potentially extendable to other crops in programs aiming at developing herbicides tolerance and at limiting herbicide contamination in the food chain.

Figure 3 .
Figure 3. Real time RT-PCR analysis expression of OsGSTL2 in transgenic rice plant.NT, non-transformed plants; Line 1-3, independent transgenic lines.Data are representative of three separate experiments performed in triplicates.Results are mean ± SD.Asterisks above the bars indicate differences between mean values measured in the indicated transgenic line compared to non-transformed plants is P < 0.05 (Student's t-test).

Figure 4 .
Figure 4. Activities of GST and GPX in transgenic rice plant.A. GST activity; B. GPX activity.NT, non-transformed plants; Line 1-3, independent transgenic lines.Data are representative of three separate experiments performed in triplicates.Results are mean ± SD. * and ** above the bars indicate differences between mean values measured in the indicated transgenic line compared to non-transformed plants is P < 0.05 and P < 0.001, respectively (Student's t-test).

Figure 5 .
Figure 5. Detection of superoxide level by NBT staining in rice leaves.NT, non-transformed plants; Lines 1 to 3, independent transgenic lines.

Figure 6 .
Figure6.The effect of glyphosate and chlorsulfuron treatment in transgenic lines.A. After 100 μmol L -1 glyphosate treatment for 24 h and then the seedling was cultivated for 10 d; B. After 0.02% chlorsulfuron treatment for 24 h and then the seedling was cultivated for 10 d.NT, nontransformed plants; Line 1-3, independent transgenic lines.