The development of herbicide-resistant edger populations is a aloft crisis adverse avant-garde agronomics (Bonny, 2016). Herbicide attrition has acquired in populations of at atomic 470 adapted weeds, including 35 breed that accept developed attrition to the herbicide glyphosate (Heap, 2016), the best broadly acclimated herbicide in the history of agronomics (Duke & Powles, 2008). Mechanisms of herbicide attrition are calmly explained in some cases, as back a alteration occurs in the protein ambition of the herbicide that reduces herbicide bounden (González-Torralva et al., 2012) or alike the archetype cardinal of herbicide ambition genes (Gaines et al., 2010). Added cases, termed non-target armpit resistance, are ailing understood, but accept been attributed to quantitative accession of accessory attrition alleles alleged from continuing abiogenetic aberration at assorted gene loci (Busi, Neve & Powles, 2013; Délye, 2013). Non-target armpit attrition may absorb assorted mechanisms that affect herbicide metaism or about-face (González-Torralva et al., 2012). Attrition to glyphosate is absorbing in that it generally appears to absorb non-target armpit mechanisms, which can arise and boss a citizenry afterwards as few as three ancestors of sub-lethal acknowledgment to the herbicide (Busi & Powles, 2009), and can advance through a citizenry faster than predicted by gene breeze or propagule breakdown (Escorial et al., 2011; Espeby, Fogelfors & Milberg, 2011; Okada et al., 2013). Given that herbicides abet a able abiotic stress, it is acceptable that weeds acknowledge by activating stress-signaling networks that reprogram gene announcement (Busi, Neve & Powles, 2013), and we accept that this involves epigenetic acclimation of gene action that could accord to herbicide attrition as active in edger acclimation to added stresses (Verhoeven et al., 2010) and in the acknowledgment of rice to the pesticide atrazine (Lu et al., 2016).
A arresting apparatus of epigenetic acclimation of gene announcement is through cytosine methylation. Methylated cytosines (mCs) action in three adapted arrangement contexts in plants: CG methylation, which is the absolute anatomy of methylation in gene-rich regions, and CHH or CHG (H = A, T, or C) methylation predominantly in transposable elements and repetitive sequences (Cokus et al., 2008). CHH and CHG mCs are abounding added accepted in plants than animals (Cokus et al., 2008). Assorted RNA-dependent methylation pathways ascendancy de novo methylation in the alacrity edger Arabidopsis thaliana (Matzke & Mosher, 2014) with methylation at the adapted arrangement contexts adapted and maintained through both overlapping and sequence-context-specific mechanisms. Mutations in the genes of these pathways advance to aberrations in the methylome (Stroud et al., 2013). Additionally, wildtype A. thaliana plants accrue epimutations (i.e., changes in the methylome) over ancestors of greenhouse advancement (Becker et al., 2011; Schmitz et al., 2011). Accent acknowledgment can advance to abounding methylome reprogramming as empiric in A. thaliana afterward antibody advance or salicylic acerbic assay (Dowen et al., 2012), phosphate starvation (Secco et al., 2015; Yong-Villalobos et al., 2015), and salinity accent (Jiang et al., 2014; Wibowo et al., 2016). The majority of methylation changes afterward these stresses were advised transient—not transgenerationally stable—but a subset of stress-responsive methylations is durably anchored in populations over several consecutive ancestors of acknowledgment to accent (Jiang et al., 2014). Thus, transgenerational epigenetic changes in gene announcement could advance to added and adaptive accent tolerance. However, the adeptness of epigenetic changes to be transgenerationally abiding and affect the acclimation of plants in their ambiance charcoal advancing (Hagmann et al., 2015; Kawakatsu et al., 2016).
Because the specificity of methylome reprogramming afterward accent may arise inexact [e.g., analyze Secco et al. (2015) to Yong-Villalobos et al. (2015)] and the aftereffect of alterations in methylation levels on gene announcement may assume inconsistent (Karan et al., 2012; Kawakatsu et al., 2016; Li et al., 2012), we activated whether there was a dosage aftereffect by glyphosate accent on specific alterations of the bulb methylome. Glyphosate has been ahead apparent to adapt DNA methylation in aureate (Nardemir et al., 2015) appliance almost low resolution techniques. In addition, the herbicide atrazine was afresh apparent to abet all-around changes in methylation in rice (Lu et al., 2016). We acclimated bisulfite sequencing to actuate the aftereffect of glyphosate on the Arabidopsis methylome at audible abject brace resolution to analyze specific abiogenetic loci that accept adapted methylation patterns afterward glyphosate exposure.
Seeds of A. thaliana ecotype Columbia were sown in Sunshine Cardinal 1 media and stratified for three canicule in the aphotic at 4 °C. The flats were again transferred to a Conviron advance alcove with a 12 h ablaze aeon and ablaze acuteness of 90 μmol m−2 s−1 and accustomed to abound to a absolutely developed rosette, pre-floral shoot stage. Blocks of four plants were again about assigned assay of glyphosate at 0%, 5%, 10%, or 15% of the characterization bulk (0.9 kg acerbic adequation (ae) ha−1 of RoundUp Pro Concentrate). Glyphosate-treated plants were sprayed at 187 L ha−1 in a aerosol booth. Afterward glyphosate treatment, plants were transferred to a advance shelf with a 12 h ablaze aeon and ablaze acuteness of 90 μmol m−2 s−1 and developed until absolutely developed siliques were formed (approximately two weeks for the 0% and 5% glyphosate-treated plants and eight weeks for the 10% glyphosate-treated plants). Thus, tissues were harvested at agnate adorning stages, admitting slower advance bulk of glyphosate-treated plants.
Genomic DNA was abandoned from two to three cauline leaves formed afterward glyphosate acknowledgment from alone plants in quadruplicate for anniversary of the three assay levels (0%, 5%, and 10%) appliance the Biosprint-15 bulb DNA abstraction kit (Qiagen, Hilden, Germany). The 12 samples were beatific to Genomics Research Laboratory at Biocomplexity Institute of Virginia Tech (Blacksburg, VA, USA) for library alertness and bisulfite sequencing. 100 ng of complete DNA was bisulfite adapted appliance EZ DNA Methylation-Gold Kit (#D5005; Zymo Research, Irvine, CA, USA), afterward the manufacturers protocol, except eluting into 9 ul. The absolute bulk of the antiseptic bisulfite-treated DNA was adapted to Illumina DNA libraries appliance EpiGnome Methyl-Seq kit (Epicentre, Madison, WI, USA). Six samples anniversary were alone barcoded, quantitated by qPCR, and affiliated to arrangement on the absolute Illumina HiSeq Rapid Run flowcell. Libraries were amassed on-board at a absorption of 8.5 pM with 3% phiX, assimilate a breeze corpuscle appliance Illumina’s HiSeq Rapid Commutual End Cluster Kit V2 (PE-402-4002), and sequenced 2× 101 cycles appliance HiSeq Rapid SBS Kit (200-cycles) (FC-402-4021).
The sequencing reads were accountable to pre-processing affection ascendancy appliance FastQC to annihilate adapter sequences and barcodes appliance Trimmomatic (http://www.usadellab.org/cms/) and FastX Tookit (http://hannonlab.cshl.edu/fastx_toolkit/). Low affection reads (quality annual Q < 30) were alone and alone reads casual the affection assay were mapped to Col-0 A. thaliana (TAIR 10) advertence genome appliance Bismark aligner (v 0.14.5) beneath absence ambit (−n 1 −l 50) (Krueger & Andrews, 2011). Cytosine methylation advice was extracted from accumbent reads appliance Bismark methylation extractor and methylation calls for CG, CHH, and CHG contexts were generated. The about-face ability of bisulfite assay (methylation cachet of cytosine) was estimated from reads mapped to the chloroplast genome, which is accepted to be unmethylated.
Differentially methylated regions (DMRs) amid advised and ascendancy plants were articular appliance the R (v3.0.3) amalgamation methylkit/eDMR (Li et al., 2013). Methylkit allows constant acclimation to analyze DmCs based on q-value, percent methylation difference, and types of methylation (hyper or hypo) appliance statistical tests such as logistic corruption and Fisher’s exact test. Pairwise Pearson’s alternation accessory and Hierarchical absorption (Ward’s method, alternation ambit metric) were afflicted based on percent methylation ethics for all 12 samples. Cogwheel methylation amid advised (5% and 10%) and ascendancy groups were bent appliance Fisher’s exact assay with a minimum 25% aberration in methylation arrangement amid groups and q-value <0.01. DMRs were complete appliance abounding access algorithm eDMR.
Differential methylation amid glyphosate-treated and ascendancy libraries was additionally bent in R appliance bsseq amalgamation (bioconductor). The advantage (.cov) files generated by Bismark were acclimated to run methylation cutting which generates per-CG, CHH, and CHG methylation ethics based on at atomic one biological carbon for ascendancy and advised samples appliance beggarly t-statistics. DMRs were filtered by areaStat which was abounding by the cardinal of methylation sites for anniversary ambience (Hansen, Langmead & Irizarry, 2012). We articular the overlapping DMR regions for anniversary ambience amid percent of glyphosate acknowledgment appliance the intersectBed action aural the bedtools apartment with absence ambit (Quinlan & Hall, 2010). The acerbity of the ambit to use back calling DMRs was optimized through allegory the aftereffect of accretion the cardinal of analyzed replicates on the cardinal of articular DMRs by calling DMRs appliance all accessible combinations of two replicates, three replicates, and four replicates from our methylC-seq data. For the beneath acrimonious ambit (see Fig. S1 legend) relying on two replicates acceptable leads to abounding apocryphal positives because of the aciculate abatement in the cardinal of DMRs back accretion the cardinal of replicates (Fig. S1). Back appliance the added acrimonious parameters, the all-embracing cardinal of DMR calls is lower and accretion replicates has no aftereffect on raw cardinal of alleged DMRs suggesting beneath apocryphal positives. We, therefore, acclimated the DMRs alleged appliance the added acrimonious ambit for afterwards analyses.
Annotations from the TAIR10 database were assigned to DMRs appliance a custom Perl calligraphy (script provided in added materials) in which genomic appearance were associated with DMRs that overlapped aural 2Kb in either direction. Arabidopsis thaliana ID lists from associated DMRs were candy and analyzed in VirtualPlant 1.3 software (http://virtualplant.bio.nyu.edu/cgi-bin/vpweb/) appliance the BioMaps bore (p ≤ 0.05) to acquisition decidedly over-represented gene aesthetics (GO) categories. To baddest cogent anatomic categories, we set a blow point in a normalized affluence (relative affluence of ascribe gene list/relative affluence of reference) that was greater than 1.5-fold.
Statistical antecedent tests appliance the Student’s t-test with 5% acceptation levels were conducted to actuate if the beggarly aberration in 5% glyphosate vs. ascendancy accumulation (Δ5) is decidedly adapted from the beggarly aberration in 10% glyphosate vs. ascendancy accumulation (Δ10) for anniversary DMR. We again adapted one nonlinear ambit for sites in which Δ5 is decidedly greater than Δ10 and all DMRs beneath the adapted dejected ambit in hypermethylation case and aloft the adapted dejected ambit in hypomethylation case were classified as afflicted dose-dependent (see Fig. S2). Accession nonlinear ambit for sites in which Δ5 is decidedly beneath than Δ10 was again fitted. All DMRs aloft the adapted blooming ambit in hypermethylation case and beneath the adapted blooming ambit in hypomethylation case were classified as absolute dose-dependent. Nonlinear curves are adapted for applicable the abstracts based on both Akaike advice archetype (AIC) and Bayesian advice archetype (BIC). The nonlinear ambit applicable AIC and BIC was abate compared to beeline fitting.
The cardinal of DMR-associated genes afterward glyphosate treatment, phosphate starvation and amoebic accent are 3,680, 712, and 884, appropriately (Dataset S7). Alone 13 genes were articular as actuality differentially methylated afterward all these stresses (Dataset S7). We advised the achievability that the abridgement of aggregate DMR-associated genes amid all three stresses is affirmation for DMRs actuality about broadcast beyond the genome as against to careful in acknowledgment to stress. The absolute cardinal of genes in the TAIR10 comment of the A. thaliana genome is 33,602. We included the absolute cardinal of A. thaliana genes as the baseline rather than ahead arise lists of methylated A. thaliana genes because a majority of our genic DMRs (73%) were articular in genes ahead characterized as unmethlyated. The glyphosate-induced DMR-associated genes annual for 11% of all accessible genes. If these 3,680 genes are about broadcast beyond the genome, and the 884 amoebic accent DMR-associated genes are additionally random, again the accepted cardinal of overlapping genes can be afflicted by 0.11 × 884 = 97. Appliance the aforementioned blueprint for the phosphate starvation DMR-associated genes, the accepted cardinal of overlaps with the glyphosate DMR-associated genes is 78 if the DMRs are about distributed. Beneath the aforementioned assumptions, the accepted cardinal of DMR-associated genes affiliated to all three accent responses is 2 [(97/33602) × 712], abounding abate than the empiric 13 aggregate genes. In fact, χ2 assay of ability of the three stresses is awful cogent (χ2 = 179.39, df = 4, p = 1.01e−37), suggesting that the three stresses absolutely abet accepted epigenetic changes in A. thaliana. Moreover, all pairwise allegory tests of ability appearance statistical acceptation (glyphosate and phosphate: χ2 = 62.21, df = 1, p = 3.088e−15; glyphosate and biotic: χ2 = 7.557, df = 1, p = 0.005977; phosphate and biotic: χ2 = 100.08, df = 1, p = 1.463e−23). Taken together, the allegory aftereffect shows that DMR-associated genes are not about broadcast in the Arabidopsis genome and DMR-associated genes in acknowledgment to one accent are not mutually absolute of those complex in acknowledgment to accession stress.
To actuate whether glyphosate abrasion induces changes in bulb methylation, we advised A. thaliana with low absorption glyphosate sprays adumbrative of real-world doses a edger on the allowance of a advised acreage could receive. Because a bulb charge survive herbicide abrasion and aftermath applicable berry to be a architect of an herbicide aggressive population, we aboriginal articular the adapted sub-lethal doses for glyphosate assay of A. thaliana. Four-week-old A. thaliana rosettes were apparent to 0%, 5%, 10%, or 15% of a archetypal acreage bulk of 0.9 kg acerbic adequation (ae) ha−1 glyphosate, with the 5% and 10% ante causing arresting herbicide injury, but acceptance for bulb adjustment and reproduction (Fig. 1A). gDNA was calm from anew formed cauline leaves at silique maturation of four individuals from anniversary assay (Figs. 1B–1D). MethylC-seq of 12 absolute libraries (four replicates from anniversary treatment) produced a absolute of 530,042,668 accumbent reads constant in genome coverages from 48× to 76× for anniversary carbon (Table S1).
The absolute bulk of methylated cytosines (mCs) ranged from 95 to 143 actor for all replicates (Table S1) and were the best abounding in the CG arrangement ambience (Fig. 2A), which is constant with antecedent analyses of the A. thaliana methylome (Cokus et al., 2008). Alone an boilerplate of 0.2% mCs was articular on the chloroplast genome, which is not methylated in A. thaliana (Cokus et al., 2008), demonstrating the allegiance of the methylC-seq agreement and abstracts filtering. mCs were broadcast beyond all bristles nuclear chromosomes. In addition, a baby cardinal of mCs were articular in the mitochondrial genome admitting these mCs are acceptable apocryphal positives because of the admittance of a arena of the mitochondrial genome on chromosome 2 of A. thaliana eco. Columbia (Lin et al., 1999). Neither the affluence of mCs nor the about affluence of mCs in the three adapted analyzed arrangement contexts (CG, CHG, and CHH) decidedly afflicted due to herbicide abrasion (Fig. 2A). Absorption the Pearson’s alternation accessory of the mCs did not aftereffect in treatment-specific monophyletic clades, demonstrating the about affinity of the methylomes beyond all 12 individuals (Fig. S3). Taken together, these after-effects advance that glyphosate-induced accent did not advance to changes in all-embracing levels of methylation in any of the three arrangement contexts.
Because glyphosate did not abet all-around accouterment in methylation levels, differences in specific area and ambience of DNA methylation can be potentially classified as careful responses to herbicide injury. We acclimated Methylkit (Akalin et al., 2012) to analyze mCs in the 5% or 10% glyphosate treatments to the ascendancy (0% treatment) to analyze differentially methylated cytosines (DmCs). This access abounding 17,017 DmCs afterward 5% glyphosate assay and 23,341 DmCs afterward 10% glyphosate assay afterwards normalizing all four replicates and applying a blow to alone accede the top 25% accomplished aplomb DmCs (Dataset S1). Interestingly, 10% glyphosate assay led to essentially added DmCs than 5% glyphosate assay in the two best abounding arrangement contexts CG and CHG (Figs. 2B–2D), suggesting that the amount of herbicide abrasion is activated with the ambience and consequence of methylation changes. However, it is important to agenda that the 10% glyphosate-treated plants appropriate an added six weeks to ability silique maturity. Therefore, a subset of the methylation differences could be due to the added growing time and not the herbicide treatment.
Differentially methylated regions are added acerb associated with authoritative changes in gene announcement than DmCs. We alleged DMRs induced by both 5% and 10% glyphosate assay appliance two absolute approaches: a bimodal DmC administration clay access appliance eDMR (Li et al., 2013), and a ambit cutting access of DmCs appliance bsseq (Hansen, Langmead & Irizarry, 2012). All DMRs were authentic as CG, CHH, or CHG based on the advantage of the arrangement ambience of DmCs absolute the DMR. The eDMR algorithm articular 1,949 hypomethylated and 1,229 hypermethylated non-redundant DMRs afterward 5% and 10% glyphosate treatments (Dataset S2). Added than 95% of the DMRs alleged by eDMR were authentic by the CG arrangement ambience (Fig. S4). Appliance the bsseq algorithm, 4,053 hypomethylated DMRs and 5,082 hypermethylated DMRs were articular afterward glyphosate herbicide abrasion beyond all three arrangement contexts (Dataset S3). We afar 70 DMRs articular in both the 5% and 10% assay groups but adverse in directional change of methylation (i.e., bang in one assay and aggressive in the added treatment) and accordingly absurd to represent biologically accordant DMRs. In adverse to the eDMR data, alone 78% and 51% of hypomethylated and hypermethylated bsseq DMRs, respectively, were categorized as CG (Fig. 3A). The abridgement of accordance amid the bsseq and eDMR datasets suggests that the best of algorithm charcoal a primary disciplinarian for the specificity of DMRs articular as ahead appropriate (Yu & Sun, 2016). We accede the 545 DMRs articular by both eDMR and bsseq as our accomplished aplomb DMRs (Dataset S3). We focused on the bsseq DMRs for consecutive analyses because of the about affluence of non-CG DMRs in this dataset and the ascertainment that non-CG DMRs are added accepted in plants than they are in animals.
We articular 1,964 DMRs (counting both hypo- and hyper-methylated DMRs) altered to the 5% glyphosate-treated samples, and 5,614 DMRs altered to the 10% samples (Fig. 3A), which are constant with the trend empiric for DmCs in which the severity of herbicide abrasion correlates with the consequence of methylome shifts. The 5,614 DMRs present alone in the 10% glyphosate-treated samples were tentatively classified as absolute dose-dependent DMRs (i.e., the consequence of changes in methylation levels is absolutely activated with severity of herbicide injury) and the 1,964 DMRs altered to the 5% glyphosate-treated samples as afflicted dose-dependent DMRs (i.e., added astringent herbicide abrasion activated with decreases in the consequence of changes in methylation levels). The 1,557 DMRs present in both the 5% and 10% advised samples were added analyzed for dose-dependency constant in 224, 300, and 209 DMRs classified with 95% aplomb as dose-independent, absolute dose-dependent, and afflicted dose-dependent respectively, beyond both hypomethylated and hypermethylated DMRs (Fig. 3B; Dataset S4; Fig. S2; see Methods). We, therefore, achieve that for about two-thirds (5,914 out of 9,205) of DMRs that are absolute dose-dependent, the consequence of changes in methylation is abased on the severity of the stress. We adduce that these absolute dose-dependent DMRs are the best candidates for anecdotic biologically accordant genomic loci that are adapted in acknowledgment to glyphosate stress. However, it is important to agenda that the 10% glyphosate-treated plants were harvested several weeks afterwards the 5% glyphosate-treated plants to atone for the adorning adjournment induced in the 10% glyphosate-treated plants (see Methods). It is accessible that a cardinal of the articular cogwheel DMRs amid the two assay levels are due to aliment of these DMRs actuality bachelor from development and instead affiliated to the age of the plants.
We tentatively accept for the abate cardinal of the afflicted dose-dependent DMRs that the severity of herbicide abrasion afterward 10% glyphosate assay prevented the bulb from responding to the accent through specific alterations in DNA methylation. Interestingly, the CHH ambience had the best arresting afflicted dose-dependent trend, with added DmCs in the 5% assay accumulation than the 10% assay accumulation (Fig. 2), in abrupt adverse to the CG and CHG contexts. While some atomic mechanisms that ascendancy methylation act on all three arrangement contexts [e.g., DRM 1/2 (Cao & Jacobsen, 2002b)], added mechanisms accept audible furnishings beyond adapted arrangement contexts with the best arresting differences amid absurd and balanced Cs (Cao & Jacobsen, 2002a; Matzke & Mosher, 2014). CHH sites are absurd (no C in the antisense strand) in adverse to CG and CHG sites. Additionally, CHH sites were ahead articular as responding abnormally to amoebic accent in adverse to CG and CHG sites (Dowen et al., 2012). However, it is absurd to achieve that glyphosate herbicide abrasion alters adapted atomic methylation mechanisms accurately until the furnishings of glyphosate on methylation patterns in accepted methylation aberrant bulb ambit are empirically assessed.
To annotate which genomic regions are responding to the glyphosate accent through changes in methylation, we annotated all DMRs. A advantage of DMRs were articular in gene coding sequences and in the CG ambience (Fig. 4A) as accepted (Cokus et al., 2008). The DMRs categorized as CHG or CHH were predominantly associated with transposable elements, agnate to antecedent studies in plants (Cokus et al., 2008; Li et al., 2012). In total, 1,818 transposable elements or transposable aspect (TE) genes were hypermethylated and 936 were hypomethylated (Fig. 4A; Dataset S3). Transposable element-associated DMRs occurred at capricious affluence beyond superfamily groups in acknowledgment to both the 5% and 10% glyphosate treatments (Fig. S5; Dataset S5). Alike admitting hypermethylated TEs are alert as accepted as hypomethylated TEs, the 936 hypomethylated TEs may accord to genome destabilization due to added transposon advancement associated with bargain transposon anatomy methylation (Chan, Henderson & Jacobsen, 2005; Mirouze & Vitte, 2014). The cardinal of hypomethylated TEs is beneath than the cardinal of activated TEs in the DNA methylation A. thaliana aberrant ddm1 (Zemach et al., 2013), but still greater than the cardinal of TEs activated in A. thaliana afterward assay with DNA demethylating abettor azacytidine based on announcement abstracts (Griffin, Niederhuth & Schmitz, 2016). We, therefore, achieve that glyphosate induces hypomethylation of TEs on a calibration commensurable to added accepted treatments able of destabilizing the A. thaliana genome through TE hypomethylation. The hypomethylated TEs are absorbing to analyze in agreement of their abeyant for accidental to the abnormality of gene addition empiric in several glyphosate-resistant weeds (Gaines et al., 2010).
An advantage to belief bulb DNA methylation changes in acknowledgment to glyphosate is that the biochemical apparatus of action of this herbicide has been abundantly characterized. Glyphosate stops the breeze of carbon through the shikimate alleyway by inhibiting action of the agitator 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase (Amrhein et al., 1980). Two of the seven genes in the shikimate alleyway of Arabidopsis (Tzin & Galili, 2010) were differentially methylated in the CG ambience in acknowledgment to glyphosate stress: 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase (At4g33510), which encodes the aboriginal agitator (and aloft authoritative footfall for the pathway), and both forms of shikimate kinase (SK1, At2g21940 and SK2, At4g39540) were all hypermethylated in the 10% glyphosate-treated plants. Notably, all three of these genes were ahead classified as unmethylated in A. thaliana (Niederhuth et al., 2016). EPSP synthase (At2g45300), which is additionally advised unmethylated in A. thaliana populations (Niederhuth et al., 2016), was not differentially methylated due to glyphosate abrasion (Dataset S3). Several biological processes and atomic functions were accomplished in the GO agreement affiliated to the DMR-associated genes (genes that accommodate DMRs in their coding sequence) (Figs. 4B and 4C; Dataset S6). The accessory of genes associated with phosphate (including phosphorylation, phosphotransferase, protein kinase, and pyrophosphatase) was striking. This could be accompanying to glyphosate consecration of microRNAs that adapt phosphate carriage pathways empiric in maize (Żywicki et al., 2015). The glyphosate atom is accustomed by phosphate transporters (Denis & Delrot, 1993) and some cases of glyphosate attrition accept been attributed to alterations in herbicide carriage and aloofness (Sammons & Gaines, 2014).
As expected, the majority of the accomplished GO agreement were associated with genic DMRs in the CG ambience (Fig. S6; Dataset S6), and these included abounding of the agreement accompanying to phosphate. The GO agreement accomplished in the CHG and CHH contexts were distinct. In the CHG context, glyphosate herbicide abrasion was associated with alterations in methylation of corpuscle wall-associated and hydrolase genes. Genes associated with arresting accession and transduction were accurately accomplished in this assay for genic DMRs in the CHH context.
Formation and aliment of DMRs is apprenticed by changes in gene announcement (Secco et al., 2015), and DMRs are sometimes associated with afterwards changes in gene announcement (Li et al., 2012) admitting generally accept no aftereffect on gene announcement (Kawakatsu et al., 2016). We, therefore, cannot achieve whether glyphosate is anon altering the methylation patterns of the genes associated with phosphate metaism and the shikimate alleyway or if glyphosate is altering archetype of associated genes that in about-face adapt the methylation states of the basal genomic loci. Antecedent assignment showed that glyphosate acquired alone accessory changes in actual transcriptional action in A. thaliana eco. Columbia (Das et al., 2010), but induced aloft furnishings on the transcriptome of A. thaliana eco. Lansberg erecta (Faus et al., 2015). Future assignment including commutual transcriptome analyses are appropriate to associate methylation patterns with gene expression.
Next, we asked whether the DMRs induced by glyphosate assay are apocalyptic of a accepted accent acknowledgment or potentially at atomic partially glyphosate specific. We compared the 3,680 non-redundant DMR-associated genes in either the 5% or 10% assay accumulation to DMR-associated genes that were ahead articular afterward salicylic acerbic (SA) treatment, which mimics amoebic accent (Dowen et al., 2012), or phosphate starvation accent (Yong-Villalobos et al., 2015). Alike because the use of adapted approaches and cutoffs for calling DMRs, we accepted that the three stresses would abet overlapping methylome alterations as a aftereffect of the plants’ accepted acknowledgment to ecology stresses and would accordingly allotment some DMR-associated genes. Alone 13 genes were articular as actuality differentially methylated afterward all three accent treatments (Fig. 4D; Dataset S7). These 13 DMR-associated genes are top candidates for genes that are epigenetically adapted as allotment of a accepted accent response. An added 109 and 130 glyphosate-induced DMRs overlapped with DMRs associated with amoebic accent and phosphate starvation stress, respectively. In total, 3,428 (93%) of the glyphosate-induced DMR-associated genes were altered to glyphosate exposure. While the cardinal of overlapping DMR-associated genes amid the three analyzed stresses seems small, they represent decidedly added than would action by accidental alternative from all A. thaliana genes (see Methods). Moreover, χ2 tests of ability of DMR-associated genes in three stresses appearance that accent responses are not absolute of one accession (χ2 = 179.39, df = 4, p = 1.01e−37), and there is absolutely a college than accepted cardinal of genes complex in acknowledgment to all three stresses, advertence the actuality of a accepted methylome reprogramming alleyway in Arabidopsis behindhand of the stressors. Nevertheless, the majority of articular DMRs arise specific to glyphosate injury.
This assignment identifies a ample set of genes and added genomic regions epigenetically adapted in acknowledgment to glyphosate herbicide injury. Determining the admeasurement to which stresses abet blooming against accidental epimutations is analytical for compassionate the role of DNA methylation in bulb acclimation to stresses. We favor the antecedent that the methylome changes are, at atomic partially, stress-specific over the antecedent that methylome alterations are universally accidental because: (1) A majority of the DMRs display dose-sensitive acknowledgment patterns; (2) all analyzed glyphosate-induced DMRs were articular in four absolute biological replicates; and (3) DMRs were accomplished in gene pathways accepted to be afflicted by glyphosate exposure. Identification of transgenerationally abiding DMRs and acceptance of specific DMRs anon activated with glyphosate attrition will added analyze the role of methylome reprogramming in the change of herbicide resistance.
Overview of sequencing results, beyond of coverage, and identification of methylated cytosines (mCs) afterward bisulfite sequencing of gDNA calm from anew formed cauline leaves at silique maturation of four A. thaliana individuals from anniversary assay area four-week-old rosettes were apparent to 0, 5, or 10% of a archetypal acreage bulk of 0.9 kg acerbic adequation ha−1 glyphosate.
Effect of accretion the cardinal of replicates in attached the cardinal of DMRs alleged by the BSmooth/bsseq R amalgamation (Bioconductor) appliance beneath acrimonious qcutoff of 0.1 (left panel) and added acrimonious qcutoff of 0.01 (right panel) parameters. The added acrimonious ambit and all four replicates were acclimated to analyze the DMRs in Dataset S3. Colored ambit announce 5 and 10% of a 0.9 kg acerbic ha−1 glyphosate rate.
Categorization of overlapping DMRs articular afterward assay at 5 and 10% of a 0.9 kg acerbic ha−1 glyphosate bulk based on dose-dependency of methylation response. Appliance a 95% aplomb breach cutoff, overlapping DMRs were classified as either doseindependent (red points), absolute dose-dependent (i.e. beyond methylation differences in the 10% glyphosate-treated plants than the 5% glyphosate-treated plants–green points), afflicted dose-dependent (i.e. beyond methylation differences in the 5% glyphosate-treated plants than the 10% glyphosate advised plants–blue points) or bereft abutment for assay (grey points). See Dataset S4 for account of DMRs categorized by this analysis.
Treatments comprised percentages of a 0.9 kg acerbic adequation ha−1 glyphosate bulk activated to four-week-old A. thaliana rosettes.
Number of DMRs articular appliance eDMR beyond the three arrangement contexts and afar by hypomethylation vs hypermethylation and whether or not the DMR was articular in the 5% glyphosate-treated samples only, the 10% glyphosate-treated samples only, or in both assay groups. See Dataset S2 for abundant account of all DMRs articular appliance eDMR. Glyphosate percentages based on an appliance bulk of 0.9 kg acerbic adequation ha−1.
Frequency of DMRs associated with transposable elements superfamilies, articular from the TAIR10 genome release, beyond all contexts (CG, CHG, CHH). Panels abstracted hypomethylation vs hypermethylation and colors differentiate the 5% (orange) and 10% (maroon) glyphosate-treated samples. Methylation contest are assorted beyond superfamily, treatment, and context. See Dataset S5 for a abundant account of all transposable aspect families and superfamilies associated with the articular DMRs. Glyphosate percentages based on an appliance bulk of 0.9 kg acerbic adequation ha−1.
Gene aesthetics (GO) agreement for atomic action accomplished (p<0.05) in DMR associated genes for anniversary of the three arrangement contexts. Rfi/Rfr represents the arrangement of the about affluence of GO agreement in the ascribe (glyphosate DMRs) to the advertence (TAIR10 Arabidopsis genome) datasets. * indicates p<0.01. See Dataset S6 for abounding account of GO agreement with Rfi/Rfr >1.5 in anniversary of the three arrangement contexts.
All differentially methylated positions (DMPs) articular by methylkit.
All differentially methylated regions (DMRs) articular appliance eDMR algorithm.
All differentially methylated regions (DMRs) articular appliance bsseq algorithm.
DMRs categorized by dosage dependency.
DMR-associated transposable elements.
GO appellation accessory in DMR-associated genes.
All DMR-associated genes afterward glyphosate accent (this work), amoebic accent actor (1), or phosphate starvation (2).
1. Dowen RH, Pelizzola M, Schmitz RJ, Lister R, Dowen JM, Nery JR, et al. Widespread activating DNA methylation in acknowledgment to amoebic stress. Proceedings of the National Academy of Sciences. 2012;109(32):E2183–E91. 2. Yong-Villalobos L, González-Morales SI, Wrobel K, Gutiérrez-Alanis D, Cervantes-Peréz SA, Hayano-Kanashiro C, et al. Methylome assay reveals an important role for epigenetic changes in the acclimation of the Arabidopsis acknowledgment to phosphate starvation. Proceedings of the National Academy of Sciences. 2015;112(52):E7293–E302.
Annotation of genomic appearance with DMRs. This custom Perl calligraphy identifies differentially methylated regions (DMRs) and provides genomic appearance comment with TAIR10 gff file.
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