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      Growth-inducing effects of argon plasma on soybean sprouts via the regulation of demethylation levels of energy metabolism-related genes

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          Abstract

          This study was conducted to determine the effects of argon plasma on the growth of soybean [ Glycine max (L.) Merr.] sprouts and investigate the regulation mechanism of energy metabolism. The germination and growth characteristics were modified by argon plasma at different potentials and exposure durations. Upon investigation, plasma treatment at 22.1 kV for 12 s maximized the germination and seedling growth of soybean, increasing the concentrations of soluble protein, antioxidant enzymes, and adenosine triphosphate (ATP) as well as up-regulating ATP a1, ATP a2, ATP b1, ATP b2, ATP b3, target of rapamycin ( TOR), growth-regulating factor ( GRF) 1–6, down-regulating ATP MI25 mRNA expression, and increasing the demethylation levels of the sequenced region of ATP a1, ATP b1, TOR, GRF 5, and GRF 6 of 6-day-old soybean sprouts. These observations indicate that argon plasma promotes soybean seed germination and sprout growth by regulating the demethylation levels of ATP, TOR, and GRF.

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          Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer.

          Gene function in cancer can be disrupted either through genetic alterations, which directly mutate or delete genes, or epigenetic alterations, which alter the heritable state of gene expression. The latter events are mediated by formation of transcriptionally repressive chromatin states around gene transcription start sites and an associated gain of methylation in normally unmethylated CpG islands in these regions. The genes affected include over half of the tumor suppressor genes that cause familial cancers when mutated in the germline; the selective advantage for genetic and epigenetic dysfunction in these genes is very similar. The aberrant methylation can begin very early in tumor progression and mediate most of the important pathway abnormalities in cancer including loss of cell cycle control, altered function of transcription factors, altered receptor function, disruption of normal cell-cell and cell-substratum interaction, inactivation of signal transduction pathways, loss of apoptotic signals and genetic instability. The active role of the aberrant methylation in transcriptional silencing of genes is becoming increasingly understood and involves a synergy between the methylation and histone deacetylase (HDAC) activity. This synergy can be mediated directly by HDAC interaction with DNA methylating enzymes and by recruitment through complexes involving methyl-cytosine binding proteins. In the translational arena, the promoter hypermethylation changes hold great promise as DNA tumor markers and their potentially reversible state creates a target for cancer therapeutic strategies involving gene reactivation.
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            Targeted DNA Demethylation and Endogenous Gene Activation Using Programmable TALE-TET1 Fusions

            Recent genomewide studies have defined cell type-specific patterns of DNA methylation 1 , a modification known to be important for regulating gene expression in both normal development 2 and disease 3 states. However, determining the functional significance of specific methylation events remains a challenging problem due to the lack of targeted methodologies for removing such modifications. Here we describe an approach for efficient targeted demethylation of specific CpGs in human cells using fusions of engineered transcription activator-like effector (TALE) repeat arrays and the TET1 hydroxylase catalytic domain. Using these TALE-TET1 fusions, we demonstrate that modification of certain critical methylated promoter CpG positions can be associated with substantial increases in endogenous human gene expression. Our results delineate a general strategy for understanding the functional significance of specific CpG methylation marks in the context of endogenous gene loci and validate new programmable DNA demethylation reagents with broad potential utility for research and therapeutic applications. Methylation of DNA at cytosine bases is an important mechanism widely used to regulate gene expression and transposable elements in higher eukaryotic organisms 4 . Regions of hypermethylated DNA in mammalian cells are often associated with silenced, inactive chromatin whereas regions of hypomethylated DNA are often associated with expressed genes and open chromatin 1,5 . In mammalian cells, the generation of methylated cytosine (5mC) is catalyzed and maintained by DNA methyltransferases (DNMTs) primarily at CpG dinucleotides 6 . One pathway of active 5mC demethylation is initiated by the ten-eleven translocation (TET) family of proteins, enzymes that catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), a critical step that appears to be important for ultimate removal of the methyl mark 7–13 . Defining the causal effects of specific CpG methylation events has remained challenging due to the lack of targeted methods for converting 5mC to unmethylated cytosine in living cells. Currently, only non-specific approaches exist for removing methyl groups from CpGs. For example, the cytidine analog 5-aza-2’-deoxycytidine (decitabine), an inhibitor of DNMTs, has been widely used to study the effects of demethylation on specific gene promoters. However, decitabine leads to global demethylation of CpGs in cells, making it difficult to definitively establish causal effects. Here we sought to specifically demethylate CpGs in a targeted fashion at endogenous genes by fusing the hydroxylase activity of the human TET1 protein to engineered TALE repeat arrays with programmable DNA-binding specificities. Customized TALE repeat arrays make an attractive platform for directing TET1 activity because monomeric proteins that bind to nearly any target DNA sequence of interest can be robustly made by simple and rapid assembly of individual repeat domains with known single base specificities 14 . In initial experiments, we defined the architecture of a TALE-TET1 fusion protein that could mediate efficient targeted conversion of 5mC to 5hmC at specific CpGs with resulting subsequent demethylation in human cells. To do this, we fused TALE repeat arrays engineered to bind two different sites in the human KLF4 gene with either full-length human TET1 or its catalytic domain (CD) (Figs. 1a, 1b, 1c; Methods). We then tested whether these four proteins could demethylate CpGs adjacent to the TALE binding sites in human K562 cells using a bisulfite sequencing protocol that utilizes high-throughput next-generation sequencing to generate more than 10,000 sequencing reads per sample (Methods, Supplementary Results, and Supplementary Fig. 1). For both KLF4 target sites, we found that TALE fusions bearing the TET1 CD domain induced significantly greater decreases in methylation of CpGs proximal to the TALE binding site than those bearing the full-length TET1 protein (Fig. 1d and 1e; Methods). For example, one of the TALE-TET1CD fusion proteins reduced the methylation of CpGs located 10 and 16 bp from the 3’ boundary of the TALE binding site by 21% and 30%, respectively, with similar levels of demethylation observed on both DNA strands (Supplementary Fig. 2). Lengthening the linker between the TALE repeat array and the TET1 CD did not appreciably alter demethylation efficiencies observed (Supplementary Fig. 3). Therefore, all subsequent experiments used TALE-TET1CD proteins with a short GGGS linker (hereafter referred to as simply “TALE-TET1” fusion proteins). Control fusion proteins bearing a TALE repeat array targeted to an unrelated EGFP reporter gene sequence did not demethylate CpGs in the KLF4 intron (Figs. 1d and 1e), demonstrating that demethylation requires specific binding to the target locus by the TALE repeats and is not due simply to overexpression of proteins harboring TET1 hydroxylase activity. Based on a dose-response experiment, which showed increased levels of demethylation in cells transfected with greater amounts of plasmid encoding a TALE-TET1 protein, we identified optimal transfection conditions that maximized both CpG demethylation and cell viability (Supplementary Fig. 4). We next determined whether TALE-TET1-induced demethylation of CpGs in human promoters might induce expression changes in proximal endogenous genes. The RHOXF2 /2B homeobox gene (hereafter referred to as simply RHOXF2) is expressed primarily in male germ cells 15 . Plasmid-based reporter gene studies using decitabine have demonstrated that RHOXF2 expression in non-germ cells is strongly repressed by DNA methylation (ref. 16 and M. Richardson et al., manuscript submitted). We engineered eleven TALE-TET1 proteins (hereafter referred to as RH-1 through RH-11) targeted to sites that lie in close proximity to a total of 18 different CpGs in the RHOXF2 promoter (Fig. 2a). We transfected plasmids encoding each of these 11 TALE-TET1 proteins into both 293 and HeLa cells and then assayed RHOXF2 expression and promoter methylation status using quantitative RT-PCR and high-throughput bisulfite sequencing, respectively (Supplementary Fig. 5). We successfully identified three out of six fusions that induced significant demethylation (greater than 15%) at the −250 to +1 region in HeLa and 293 cells and another three out of six that induced significant demethylation (greater than 15%) at the −650 to −850 region in 293 cells. Two of the 11 TALE-TET1 proteins we tested (RH-3 and RH-4) induced high levels of RHOXF2 mRNA expression in both the 293 and HeLa cell lines and also demethylated proximal CpGs in the −200 to +1 region of the RHOXF2 promoter (Figs. 2b-2d). The RH-3 fusion also binds to an additional site in the −650 to −850 region of the RHOXF2 promoter but demethylation of CpGs in this region can only be observed in 293 cells because cytosines in this region are not methylated in HeLa cells (Supplementary Figs. 5 and 6 and data not shown). Interestingly, we found that even greater increases in RHOXF2 expression could be induced by combined expression of both the RH-3 and RH-4 TALE-TET1 proteins in 293 cells (Supplementary Fig. 7). Although we do not know the mechanism for this dramatic increase, understanding this phenomenon will be an important focus of future studies. To assess whether the enzymatic activity of the TET1 domain is important for the gene activation observed with the RH-3 and RH-4 proteins, we tested variant fusions bearing mutations (H1671Y, D1673A) known to inactivate TET1 catalytic activity. 7 We found that these catalytically inactive RH-3 and RH-4 mutants neither demethylated their proximal CpGs nor activated RHOXF2 gene expression in either 293 or HeLa cells (Figs. 2b, c and e). Western blots also confirmed that the observed inactivity of these RH-3 and RH-4 mutant proteins is not due to their decreased expression in 293 cells (Supplementary Fig. 8). These results strongly suggest that activation of RHOXF2 expression is mediated by TALE-TET1-induced modification (either hydroxylation and/or demethylation) of specific methylated CpGs in the promoter and not simply by competitive binding of TALE-TET1 fusions with endogenous transcription factors or the presence of a fortuitous transcriptional activation function within the fusion protein. To further generalize these results, we next sought to demethylate CpGs in an additional locus, the human beta-globin (HBB) gene promoter. Previous work has suggested that four CpGs, which are differentially methylated in erythroid cells isolated from fetal liver and adult bone marrow, may play a role in regulating HBB gene expression 17 . To test this hypothesis, we constructed ten TALE-TET1 proteins targeted to various sites proximal to these four CpGs (Fig. 3a). Although all ten TALE-TET1 fusions (termed HB-1 through HB-10) induced significant demethylation of CpGs near their respective binding sites in human K562 cells (Fig. 3b and 3c), significant increases in HBB gene expression as measured by quantitative RT-PCR were observed with only four of these proteins (HB-3, HB-4, HB-5, and HB-6) (Fig. 3d). Of note, the three proteins (HB-4, HB-5, and HB-6) that induced the greatest fold-activation of the promoter were the fusions that induced the greatest demethylation of the CpG at position −266 (numbered relative to the transcription start site; Fig. 3d). HB-4, HB-5, and HB-6 proteins bearing the H1671Y/D1673A mutations (which inactivate TET1 catalytic domain activity) failed to demethylate the −266 CpG and also failed to efficiently activate HBB gene expression in K562 cells (Figs. 3e and 3f). Western blot experiments confirm that the loss of demethylation and gene activation activities observed with the catalytically inactive mutants of HB-5 and HB-6 is not due to decreased protein expression in K562 cells (Supplementary Fig. 9). Furthermore, time-course experiments show that both demethylation of the −266 CpG and activated expression of HBB diminish as transfected cells continue to be cultured (Supplementary Results and Supplementary Fig. 10), suggesting that continued expression of TALE-TET1 is required to maintain these effects. Taken together, our findings strongly suggest that either hydroxylation and/or demethylation of this particular methylated CpG is required for the observed activation of HBB gene expression. Our results define a generalizable approach for targeting 5-methylcytosine hydroxylase activity with subsequent cytosine demethylation to any endogenous genomic locus of interest in living cells. While the majority of cytosines that are converted to uracil in our bisulfite experiments are most likely to be unmethylated cytosines, it is possible that a very small percentage of these might also represent 5-formylcytosine (5-fC) or 5-carboxylcytosine (5-caC), further oxidation products of 5-hmC catalyzed by TET1 9 . However, given that 5-fC and 5-caC are rapidly removed from DNA via TDG–mediated excision 18 , these oxidation products are likely to be short-lived in our cells. Therefore, we presume that these species will be present at very low levels, as has been previously observed in other mammalian cells 9 . The TALE-TET1 framework described here can be easily programmed to target essentially any DNA sequence using the simple TALE repeat code and we used this platform to induce locus-specific demethylation at three endogenous genes (KLF4, RHOXF2, and HBB) in three different human cell lines. Although we frequently observed the greatest degree of demethylation within 30 bps of either end of the TALE target binding site, some fusions also induced demethylation of CpGs approximately 150–200 bps away from the target site, suggesting that the TET1-CD might also access regions of open chromatin located at least one nucleosome distance away. In this study, we only examined CpGs proximal to the TALE-TET1 binding site but it is also possible that other sites elsewhere in the genome might also be modified due to higher-order interactions in the three-dimensional structure of nuclear DNA. Our success rate for making TALE-TET1 fusions capable of modifying methylated cytosines was high but varied by target gene. For the KLF4 and HBB genes, all 12 fusions we made (two at KLF4 and 10 at HBB) induced significant demethylation greater than 15% at CpGs adjacent to their target binding sites; however, for the RHOXF2 promoter, only ~50% of the fusions we made induced significant demethylation (greater than 15%). The inability of some fusions to mediate significant demethylation might be caused by locus-dependent effects that affect target site occupancy such as chromatin structure, nucleosome occupancy, DNA methylation, or other parameters that could affect DNA-binding by the TALE repeat arrays and/or the efficiency of hydroxylation by the TET1 catalytic domain. Our experiments provide a framework for using TALE-TET1 proteins to evaluate the functional significance of specific CpG (and possibly non-CpG cytosine) methylation events. In this report, we successfully identified several CpGs within the RHOXF2 promoter and a single CpG within the HBB promoter that, when modified by either hydroxylation and/or demethylation, are associated with an increase in gene expression. Even modest levels of methylated CpG modification in the population of cells can, in some cases, be associated with high levels of gene activation. We hypothesize that modification of these methylated CpGs might allow endogenous transcription factors present in the 293, HeLa, or K562 cell lines to bind the promoter and activate expression of the endogenous gene. Although the particular CpGs we identified in our transformed cancer cell-based experiments may or may not be involved in normal physiologic regulation of the RHOXF2 or HBB genes, our proof-of-principle experiments nonetheless illustrate a general strategy that could be used in other more physiologically relevant cell types (e.g.--primordial germ or erythroid cells) to define critical methylation events involved in the regulation of these genes. Additionally, our TALE-TET1 fusions should provide important tools for performing other, more detailed mechanistic studies that define how the loss of methylation marks in turn leads to increases in promoter activity (e.g.—by enhancing or reducing the binding of particular transcription factors). An important and as-yet unanswered issue for future studies will be to define the genome-wide specificities of our TALE-TET1 proteins. All of the proteins we constructed for the RHOXF2 and HBB promoters were designed to bind 20 bp sites, sequences sufficiently long enough to be potentially unique in the human genome. However, although previously published in vitro SELEX experiments suggest that monomeric TALE repeat arrays are specific for their intended target sites 19 , to our knowledge the genome-wide specificities of such proteins in human cells have not been described. Engineered zinc finger (ZF) proteins might provide a potential alternative to TALE repeat arrays for targeting TET1 activity and at least one published report has suggested that monomeric six-finger proteins can be highly specific in human cells 20 . We have also engineered multiple six-finger ZF-TET1 fusion proteins targeted to 18 bp sequences in the KLF4 and HBB genes (Supplementary Methods) and demonstrated that these can induce targeted demethylation with efficiencies that appear to be comparable to those induced by TALE-TET1 proteins (Supplementary Results and Supplementary Figs. 11 and 12). Regardless of which platform ultimately proves to be more specific, potential off-target effects can be readily accounted for by constructing and testing multiple targeted TALE-TET1 or ZF-TET1 fusion proteins for each CpG or cluster of CpGs to be demethylated. For example, our finding that three different TALE-TET1 proteins can all demethylate a common CpG and induce changes in HBB gene expression strongly suggests that the observed phenotype is due to binding at the intended target sequence and not at an off-target site elsewhere in the genome. In addition to off-target effects resulting from unintended binding elsewhere in the genome, it is also possible that TALE-TET1 fusions could induce demethylation that is not dependent upon binding by the TALE repeat array portion of the protein. This can be seen in some of our experiments with fusions targeted to an off-target site in the EGFP reporter gene causing some level of non-specific demethylation at endogenous loci (see, for example, Fig. 3c and 3d), presumably caused by non-DNA-bound proteins acting from solution. Until such non-specific effects can be minimized (perhaps by decreasing or controlling the expression level of TALE-TET1 proteins), these results highlight the need to always perform controls with fusions targeted to other sites. These controls will be crucial for interpreting whether phenotypic effects induced by a particular TALE-TET1 protein depend upon the TALE repeat array-mediated sequence-specific localization of TET1 activity We also do not yet understand why some CpGs are more efficiently demethylated than others by our TALE-TET1 fusions. For example, in our experiments we were able to demethylate some CpGs in the HBB locus very efficiently (as high as 84%) whereas other CpGs in the RHOXF2 locus were less efficiently demethylated (maximum of 42% and 25% in HeLa and 293 cells, respectively). As noted above, this could be partly due to locus-specific effects that affect the DNA-binding and/or hydroxylase activities of the fusions. However, it is also possible that various factors in the cells may be actively re-methylating CpGs and thus the extent of methylation observed may represent a steady-state between de- and re-methylation. Our time-course results at the HBB locus (Supplementary Fig. 10) are consistent with the idea that re-methylation may be occurring in K562 cells because demethylated CpGs appear to become re-methylated as the TALE-TET1 encoding plasmid is lost from the cells. Delineating the parameters that affect the ultimate efficiency of demethylation will be important to further optimize the effects of our TALE-TET1 proteins. The TALE-TET1 platform described here and other fusions proteins recently described by our group 27 and others 28 represent novel and important additions to the growing toolbox of reagents for performing targeted editing of epigenomic modifications. Previously described reagents that target histone methyltransferases (SUV39H1 and G9A 21 ) or DNA methyltransferases (bacterial enzymes 22–26 and human DNMT3a and 3b subunits 27–29 ) used engineered zinc finger proteins, which can be more challenging to construct than TALEs. Continued construction and characterization of tools to modify histones and DNA methylation might in the future enable the stable, heritable changes in the expression of any gene of interest. Development of such a capability in the longer-term would enable numerous research applications as well as potential therapeutic strategies for diseases caused by dysregulated gene expression. Online Methods TALE-TET1 Fusion Protein Design and Construction The full TET1 coding sequence was synthesized as gBlocks (Integrated DNA Technologies) and assembled by standard restriction enzyme digest and ligation to construct TET1-FL and TET1-CD expression vectors. All TALEs were assembled using the FLASH method and were cloned into TALE-TET1 expression vectors (pJA344C7, pJA345D4, pJA344E9 and pJA247) containing one of four 0.5 C-terminal TALE repeats, an N-terminal nuclear localization signal, the Δ152 TALE N-terminal domain and the +95 TALE C-terminal domain as previously described 19,30 . In all expression plasmids used, either TET1-FL or TET1-CD was fused to the C-terminal end of the TALE-derived DNA-binding domain via a GlyGlyGlySer linker and expression of this fusion was driven by an EF1alpha promoter. For the Western blot experiments, expression vectors had an additional triple-FLAG tag cloned upstream of the nuclear localization signal. Sequences of all constructs are shown in Supplementary Fig. 13. Cell Culture and Transfection FlpIn-TRex HEK293 (Life Technologies) and HeLa (ATCC) cells were cultured in Advanced DMEM supplemented with 10% FBS, 1% Glutamax, and 1% penicillin-streptomycin (Invitrogen). K562 (ATCC) cells were maintained in RPMI supplemented with 10% FBS, 1% Glutamax, and 1% penicillin-streptomycin. All cell lines were tested for mycoplasma every two weeks. Plasmids encoding TALE-TET1 fusions targeted to the KLF4 and HBB loci were transfected into K562 cells by Nucleofection. Briefly, 10 ug of TALE-TET1-encoding plasmid (or 2, 5, 10, 20 or 50ug in the dose-response experiment) and 500 ng pmaxGFP plasmid were Nucleofected into 1×106 K562 cells using Kit V (Lonza) and program T-016. Control transfections used 500 ng pmaxGFP plasmid (Lonza). Fluorescent microscopy was used to ensure consistent and high levels of transfection efficiency in all K562 experiments and FACS analysis showed ~80–90% of transfected cells are GFP+ under these conditions. Plasmids encoding TALE-TET1 fusions targeted to human RHOXF2 were transfected into 293 or HeLa cells using Lipofectamine LTX according to the manufacturer’s instructions (Life Technologies). Briefly, 3.2 × 105 293 cells or 1 × 105 HeLa cells were seeded into 12-well plates and transfected the following day with 1.2 µg TALE-TET1-encoding plasmid, 60 ng pmaxGFP plasmid, 1 µl Plus reagent, and 3.3 µl Lipofectamine LTX. Fluorescent microscopy was used to ensure consistent and high levels of transfection efficiency. Cell viability in K562 cells was assayed by resuspending cells in PBS with 10% FBS and 1 ug/ml propidium iodide and analyzing by FACS. Genomic DNA and Total RNA isolation Four days post-transfection, genomic DNA (gDNA) was isolated using the QIAamp DNA Blood Mini Kit (Qiagen) according to manufacturer’s protocol. Total RNA was isolated from cells transfected with plasmids encoding TALE-TET1 fusions targeting HBB or RHOXF2 using the PureLink RNA Mini Kit (Ambion) according to the manufacturer’s instructions. RNA was treated with TurboDNA-Free (Ambion). High-Throughput Bisulfite Sequencing 500 ng of genomic DNA isolated from transfected cells was bisulfite treated using the EZ DNA methylation, EZ DNA Methylation-Gold or EZ DNA Methylation-Lightning Kit (Zymo Research) according to the manufacturer’s instructions. All samples underwent bisulfite conversion with an efficiency of at least 98.5% as judged by conversion of unmethylated, non-CpG cytosines. Genomic DNA sites in KLF4, HBB, and RHOXF2 were amplified by PCR using bisulfite-converted gDNA as a template with Kapa HiFi HotStart Uracil+ ReadyMix (Kapa Biosystems) (for KLF4 and HBB sites) or Qiagen’s PyroMark PCR Kit (for RHOXF2 sites). Standard Illumina adaptors were added by either ligation or PCR and Illumina multiplex barcodes were added by PCR. For details of PCR reactions, see Supplementary Methods. Pooled amplicons were sequenced using an Illumina MiSeq with 150 bp paired-end reads (Dana Farber MBCF Genomics Core). For each experimental sample assayed, we analyzed between 10,000 and 375,000 reads. Note that because RHOXF2 and RHOXF2B sequences are identical in the region examined, the bisulfite PCR analysis does not distinguish between these two loci. For Sanger sequencing of KLF4 samples, initial bisulfite PCR products were cloned using the TOPO Zero-Blunt cloning kit (Life Technologies) and transformed into E. coli. Plasmid DNA was purified from the resulting colonies and sequenced by the MGH DNA Core facility. qRT-PCR assays For assay of HBB gene expression, RNA was reverse transcribed using the SuperScript III First-Strand Synthesis SuperMix and oligo-dT (Life Technologies). Quantitative PCR was performed with Taqman Universal PCR Mastermix (Applied Biosystems) on an ABI 7500 Fast Real-Time PCR system with the following primer/probe sets: Forward HBB primer 5’-CAAGGGCACCTTTGCCACAC-3’; Reverse HBB Primer 5’-TTTGCCAAAGTGATGGGCCA-3’; HBB Taqman Probe 5'-/56-FAM/CCTGGGCAA/ZEN/CGTGCTGGTCTGTGT/3IABkFQ/-3'; Forward β-actin (ACTB) Primer: 5’-GGCACCCAGCACAATGAAG-3’; Reverse ACTB primer 5’-GCCGATCCACACGGAGTACT-3’; ACTB Taqman Probe 5’-/5MAX550-Y/TCAAGATCA/ZEN/TTGCTCCTCCTGAGCGC/3IABlk_FQ/-3’. For assay of RHOXF2 gene expression, RNA was reverse transcribed using iScript cDNA Synthesis Kit (BioRad) according to manufacturer’s protocol. qPCR was performed with SsoAdvanced SYBRGreen Supermix (BioRad) on an ABI StepOnePlus instrument with the following primers: RHOXF2 Forward primer 5’-GGCAAGAAGCATGAATGTGA-3’; RHOXF2 Reverse primer 5’-TGTCTCCTCCATTTGGCTCT-3’; M/H Actin Forward primer 5’-GTCCACACCCRCCGCCAG-3’; M/HActin Reverse primer 5’-CCCACGATGGAGGGGAA-3’. Note that this assay does not distinguish between RHOXF2 and RHOXF2B. All transfections were performed in triplicate and for each biological replicate at least three technical replicates of the qPCR assay were performed. Statistical significance was determined by comparing experimental samples against the off-target control using a one-sided t-test after confirming that data sets exhibited a normal distribution as determined by a Shapiro-Wilk test for normality (p<0.05). The similarity of variance between groups was determined using an f-test. When variance was equal between data sets, a two-sample equal variance t-test was used and when variance was unequal, a Welch’s t-test was used. Accession Codes All raw sequenced reads and BSMAP processed data files have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE50761 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE50761) GSE50761. Supplementary Material 1 2
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              Plant responses to stresses: Role of ascorbate peroxidase in the antioxidant protection

              When plants are exposed to stressful environmental conditions, the production of Reactive Oxygen Species (ROS) increases and can cause significant damage to the cells. Antioxidant defenses, which can detoxify ROS, are present in plants. A major hydrogen peroxide detoxifying system in plant cells is the ascorbate-glutathione cycle, in which, ascorbate peroxidase (APX) enzymes play a key role catalyzing the conversion of H2O2 into H2O, using ascorbate as a specific electron donor. Different APX isoforms are present in distinct subcellular compartments, such as chloroplasts, mitochondria, peroxisome, and cytosol. The expression of APX genes is regulated in response to biotic and abiotic stresses as well as during plant development. The APX responses are directly involved in the protection of plant cells against adverse environmental conditions. Furthermore, mutant plants APX genes showed alterations in growth, physiology and antioxidant metabolism revealing those enzymes involvement in the normal plant development.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                07 February 2017
                2017
                : 7
                : 41917
                Affiliations
                [1 ]Laboratory of Animal Genetic Engineering and Stem Cell Biology, Department of Animal Biotechnology and Advance Next Generation Convergence Technology, Jeju National University , Jeju 690756, South Korea
                [2 ]Department of Chemical and Biological Engineering, Jeju National University , Jeju 690756, South Korea
                [3 ]Intellectual Property Law Firm PCR , Seoul 06194, South Korea
                Author notes
                [*]

                These authors contributed equally to this work.

                Article
                srep41917
                10.1038/srep41917
                5294452
                28167819
                48347d59-f29e-4466-8759-61f4e10d8fce
                Copyright © 2017, The Author(s)

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 29 April 2016
                : 04 January 2017
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