Seeds of four L. virgata populations (S, R8, R14, and R15) collected in Persian lime groves from Veracruz, previously characterized with different levels of susceptibility to glyphosate by Alcántara-de la Cruz et al. (2016c), were used. Individuals of the L. virgata populations studied had a similar glyphosate response glyphosate within them. Glyphosate resistant R14 and R15 populations from Cuitláhuac municipality, were selected for being geographically close (18.75° N, 96.53° W), but genetically different. Also, the most glyphosate resistant population (R8) from Martínez de la Torre municipality (20.16° N, 97.07° W) was included in this study. The S population was used as control (18.79° N, 96.69° W).

The seeds were sown on trays with peat saturated at field capacity. The trays were covered with a layer of plastic until emergence and placed in a growth chamber at 26/18°C (day/night) with a photoperiod of 16 h at 850 mmol m^-2 s^-1 of light density and 60% relative humidity. Germinated seedlings were individually transplanted into plastic pots in 250 mL of substrate (sand and peat 1:1). Subsequently, the pots were placed in a growth chamber under the conditions described above and watered daily.

Plants of the four populations with 3–4 true leaves were treated with the following glyphosate rates: 0, 45, 90, 180, 360, 720, 1040, 1440, and 1800 g ae ha^-1. Glyphosate (Roundup Energy 45% w/v, Monsanto, Spain) applications were carried out in a treatment chamber (Devries Manufacturing, Hollandale, MN, USA) equipped with TeeJet 8002EVS flat fan nozzle calibrated at 200 kPa, a height of 50 cm and 200 L ha^-1 of application volume. The plants were harvested at ground level 21 days after treatment (DAT) and stored separately in paper envelopes. The samples were dried in an oven (JP Selecta S.A., Barcelona, Spain) at 60°C for 4 days and weighed to determine the dry weight. Data were expressed as percentage of dry weight compared to the untreated control plants. The experiment was repeated twice in a completely randomized design with 10 replicates per dose.

Samples of 50 mg (leaf disks 4 mm in diameter) of plant tissue were taken from young leaves of three plants with 3–4 true leaves of each L. virgata population according to Dayan et al. (2015). The disks were placed in 2 mL-Eppendorf tubes containing 999 μL of monoammonium phosphate (NH₄H₂PO₄ 10 mM, pH 4.4). Volumes of 1 μL of glyphosate solutions at different concentrations were added (0, 0.1, 1, 10, 50, 100, 200, 400, 600, and 1000 μM). The samples were incubated for 24 h in the growth chamber under growing conditions described above. Next, they were incubated at 60°C for 30 min. Volumes of 250 μL of HCl 1.25 N were added and incubated at 60°C for 15 min. Aliquots of 250 μL were transferred to new tubes adding 500 μL of periodic acid (0.25% w/v) and sodium metaperiodate (0.25 % w/v) in a 1:1 ratio. The samples were incubated at room temperature (25°C) during 90 min, and next, 500 μL of a mix of sodium hydroxide (NaOH 0.6 N) plus sodium sulfite (Na₂SO₃ 0.22 N) in a 1:1 ratio was added and mixed.

The experiment was arranged in a completely randomized design with three technical replications by sample of each population for each glyphosate concentration, and the study was repeated twice. A standard curve was done using known concentrations of shikimate. The absorbance of samples was measured at 382 nm in a spectrophotometer (DU-640, Beckman Coulter Inc., Fullerton, CA, USA). The absorbance values were converted into mg of shikimic acid per g of fresh weight.

Plants with 3–4 true leaves from the four L. virgata populations were treated with a solution of ¹⁴C-glyphosate [glycine-2-¹⁴C] (specific activity 273.8 MBq mmol^-1, American Radiolabeled Chemicals, Inc., Saint Louis, MO, USA) + commercial glyphosate. The solution applied contained a specific activity of 0.834 kBq μL^-1 and a glyphosate concentration of 1.8 g ea L^-1 (360 g ea ha^-1 in 200 L). One drop of 1 μL plant^-1 of solution was applied with a micropipette (Lab Mate HTL, Matosinhos, Portugal) on the adaxial surface of the first-second leaf. After treatment, the plants were maintained in the growth chamber at the growing conditions described above. At 24, 48, 72, and 96 HAT, the treated leaves were washed three times separately with 1 mL of water-acetone (1:1 v/v) to recover the non-absorbed ¹⁴C-glyphosate. The washing solution was mixed with 2 mL of scintillation liquid (Ultima Gold, Perkin-Elmer, BV BioScience Packard), and analyzed by liquid scintillation spectrometry (LSS) in a scintillation counter (LS 6500, Beckman Coulter Inc., Fullerton, CA, USA) during 10 min per sample. The whole plants were carefully removed from the pot and washed, mainly the roots. The plants were individually divided into treated leaf, remainder of the plant and root. The samples were stored in flexible combustion cones (Perkin-Elmer, BV BioScience Packard), dried in an oven at 60°C for 4 days. Next, the samples were combusted in a biological oxidizer (Packard Tri Carb 307, Packard Instrument Co., Downers Grove, IL, USA). The CO₂ released from the combustion was captured in 18 mL of a mix of Carbo-Sorb E and Permafluor (1:1 v/v) (Perkin-Elmer, BV BioScience Packard). The radioactivity of each individual sample was quantified by LSS during 10 min per sample. The experiment was arranged in a completely random design with five replicates per population at each time evaluated. The radioactive values were used to calculate recovery percentage as: [(kBq in treated leaf + kBq in plant + kBq in roots + kBq from washes)/kBq total applied] × 100. The average total recovery of ¹⁴C-glyphosate applied was >96% to the S, R8, and R14 populations, and <93% from the R15 population.

To visualize the ¹⁴C-glyphosate translocation, three whole plants per population at each visualization time (24, 48, 72, and 96 HAT) were treated under the same conditions as in the previous assay. The plants were washed individually, fixed on filter paper (25 cm × 12.5 cm) and dried at room temperature for 1 week. The samples were placed for 4 h beside a phosphor storage film (Storage Phosphor System: Cyclone, Perkin–Elmer Packard BioScience BV). A phosphor imager (Cyclon, Perkin-Elmer, Packard BioScience BV) was used to reveal the translocation.

Plants of the four L. virgata populations were grown in pots (25 cm in diameter × 15 cm high: four plants per pot) under greenhouse conditions, in temperatures ranging from 17 to 31°C, and a photoperiod of 16 h. The natural light was complemented by 900 μmol^-2 s^-1 photosynthetic photon flux density delivered by incandescent and fluorescent lights. Samples of 5 g of foliar tissue from each population were obtained from the second and third youngest totally expanded leaves.

The methodology described by Sammons et al. (2007) was used for EPSPS extraction. The total soluble protein (TPS) in the extract was measured using a Kit for Protein Determination (Sigma-Aldrich, Madrid, Spain) following the manufacturer’s instructions. The specific EPSPS activity in plants from L. virgata populations was studied in the presence and absence (basal activity) of glyphosate. The EPSPS activity was determined using a EnzChek Phosphate Assay Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The glyphosate concentrations used were: 0, 1, 10, 100, 1000, 10000 μM. Three replicates at each glyphosate concentration were analyzed. The release of phosphate on the bottom level was measured during 10 min at 360 nm in a spectrophotometer (DU-640, Beckman Coulter Inc., Fullerton, CA, USA).

Samples of young leaf tissue (≈100 mg) from 5 glyphosate-susceptible (S) and 15-resistant (R8, R14, and R15) L. virgata individuals were taken, and stored at -80°C for RNA extraction. Total mRNA was isolated following the methodology described by Pistón (2013). Integrity of RNA was verified in 0.8% agarose gel and quantified in a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Walthman, MA, USA). First strand complementary DNA (cDNA) synthesis was carried out using 1 μg of RNA in all the samples. An iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc. Hercules, CA, USA) was employed following the manufacturer’s instructions. Lv-F3 and Lv-R2 primers (Table 1) were designed using the software Primers3Plus¹, based on conserved regions of the EPSPS gene sequences from Eleusine indica (GenBank Accession: AY157642.1, HQ403647.1) and Lolium multiflorum (GenBank Accession: DQ153168.2). Individual PCR reactions were carried out using cDNA from each sample of the S and R15 populations. Each PCR reaction was performed using 50 ng of cDNA, 0.2 μM of each primer, 0.2 mM dNTP mix (PE Applied Biosystems; Life Technologies S.A., Madrid, Spain), 2 mM MgCl₂, 1X buffer, and 0.625 units of a 100:1 enzyme mixture of non-proofreading (Thermus thermophilus) and proofreading (Pyrococcus furiosus) polymerases (BIOTOOLS, Madrid, Spain) in a final volume of 25 μL. The PCR conditions were: 1 cycle of 94°C for 5 min; followed by35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and a final extension at 72°C for 10 min. PCR products (10 μL) were checked by 1% agarose gel to corroborate amplification. The PCR products were ligated using the pGEM-T Easy Vector System (Promega Biotech Ibérica, SL, Madrid, Spain) following the manufacturer’s instructions, and cloned into competent cells of E. coli DH5α. Positive transformants were selected and fragment insertion confirmed through PCR using the M13F and M13R primers (Table 1). A total volume of 15 μL per sample containing 0.2 μM of each primer, 0.2 mM dNTP mix (PE Applied Biosystems; Life Technologies S.A., Madrid, Spain), 2 mM MgCl₂, 1X buffer, and 0.625 units of non-proofreading (Thermus thermophilus) polymerase (BIOTOOLS, Madrid, Spain). The PCR conditions were: 1 cycle of 94°C for 5 min; followed by 28 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min; and a final extension at 72°C for 7 min. The plasmids were purified with the ilustra plasmidPrep Mini Spin kit (GE Healthcare, Buckinghamshire, UK), following the manufacturer’s instructions. Sanger sequencing was carried out by the STABVIDA sequencing service (Caparica, Portugal). A total of 30 clones from each population were sequenced. The assembly of the sequences was carried out by SeqMan Pro (Version 11, DNASTAR; Madison, WI, USA) and Geneious (Version 8.1.8, Biomatters Ltd, Auckland, New Zealand) software’s.

The L. virgata EPSPS cDNA sequences information can be found in GenBank with accession numbers KX425854 and KX425855.

Young leaf samples (≈100 mg) from six untreated plants of both S and R15 populations were taken before treatment. Plants were then treated with 360 g ae ha^-1 of glyphosate in the conditions used in the dose-response assays, and new samples were collected at 24 HAT. In both cases, the samples were stored at -80°C for RNA extraction and cDNA synthesis in the same conditions described in the previous section. The qLvFor1 and qLv-Rev1 primers (Table 1) to amplify a fragment of 114 bp were designed from the EPSPS gene sequences obtained in the previous section. The β-Actin and ADP-ribosylation factor genes were used as reference genes. Pairs of β-Actin primers (Table 1) were designed on the basis of the Agrostis stolonifera (JX644005.1), Avena sativa (KP257585.1), Lolium multiflorum (AJ585201.1), Triticum monococcum (AF326781.1) and Zea mays (U60510.1) sequences from GenBank. β-Actin and qLv primers were designed using Primers3Plus. The ADP-RF(a) primers designed by Giménez et al. (2011) were used to amplify the ADP-ribosylation factor gene (Table 1). For each quantitative RT-PCR reaction 40 ng of cDNA, 10 μL PerfeCTa SYBR Green FastMix ROX (Quanta Bioscience), and 0.2 μM of both forward and reverse primers in a 15 μL final reaction volume were used. The PCR conditions were: initial cycle at 94°C for 5 min; 40 cycles of 94°C 30 s and 62°C 1 min. The PCR reactions were carried out using an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA). Three to four technical replications per plant were carried out in a factorial design of two glyphosate treatments and two populations.

The PCR efficiency for each pair of primer and sample was determined by the program qPCR data analysis LinRegPCR (version 11) according to Ruijter et al. (2009) using raw fluorescence as input data. Expression level of both the reference and target genes for each sample was determined with the follow equation:

Where N₀ is expression of the gene, E is the PCR efficiency for each primer, Cq is the number of cycles needed to reach 0.2 arbitrary units of fluorescence. The mean PCR efficiency for each gene, population and treatment was determined according to Giménez et al. (2011). The stability of the expression of the reference genes (β-Actin and ADP-ribosylation factor) and Normalization Factor (NF) were determined using geNorm software for each sample according to Vandesompele et al. (2002).

The percentages data of dry weight reduction, survival and EPSPS enzyme activity were submitted to a non-linear regression analysis. The dose of glyphosate needed to reduce the weight of a population (ED₅₀), mortality (LD₅₀), and to inhibit EPSPS activity (I₅₀) by 50% were calculated. A log-logistic model of four parameters was conducted using the drc statistical package (Ritz et al., 2015) in the program R version 3.2.5. The statistical model is:

Where Y is the percentage of dry weight, survival and/or EPSPS-inhibiting with respect to the control, c and d are coefficients corresponding to the upper and lower asymptotic limits, b is the Hill slope, g is the glyphosate dose (ED₅₀, LD₅₀, or I₅₀) at the mean point of inflection between the upper and lower asymptote and x (independent variable) corresponds to the glyphosate dose. The data were plotted using SigmaPlot 11.0 (Systat Software, Inc., USA).

EPSPS normalized expression level was calculated for each qPCR reaction, the average and standard error from technical replicates were recorded for each plant and population.

Absorption, translocation and EPSPS expression results were subjected to ANOVA using Statistix version 9.0 from Analytical Software (Tallahassee, FL, USA). When necessary, the means were compared using Tukey’s test’s at the 95% probability level.

Glyphosate resistance was confirmed in the three L. virgata populations (R8, R14, and R15) collected in Persian lime groves from Veracruz. These glyphosate resistant populations were less sensitive to glyphosate than the S population (Figure 1). R8 and R14 populations had similar response to glyphosate (Figure 1A). The resistant index (RI) of the glyphosate resistant populations ranged between 2.9 to 5.2 with respect to S population (Table 2).

Based on 50% mortality (LD₅₀), the R8, R14 and R15 populations were, respectively, 2.5, 2.3, and 4.6 times more resistant than the S population (Figure 1B). A field dose of glyphosate (720 g ae ha^-1), used in Persian lime groves of Veracruz, was enough to achieve full control in the S population. However, with this dose of glyphosate, only 50% mortality was observed in R8 and R14 populations, and to the R15 for population, a dose 1.8 times more glyphosate than the field dose was required to obtain the same level of control. The ED₅₀ and LD₅₀ parameters showed the different resistance levels to glyphosate acquired by resistant L. virgata populations (Table 2).

The amounts of shikimic acid accumulated after glyphosate treatment is highly variable between species and populations. In this work, the shikimic accumulation was different in each L. virgata population. The S population presented the highest level of shikimic acid accumulation (Figure 2). This accumulation was exhibited at lower glyphosate concentrations (between 0.1 to 100 μM) reaching an average of 12.35 mg shikimic g^-1 fresh weight from 200 μM of glyphosate. The resistant populations only presented an appreciable accumulation from 100 μM of glyphosate. The averages of shikimic acid accumulated were 8.4, 7.9, and 2.6 mg shikimic g^-1 fresh weight for the R8, R14, and R15 populations, respectively, at the glyphosate concentration of 1000 μM.

The response patterns to glyphosate in shikimate accumulation assays were similar to that observed in the dose-response study. The lowest shikimate accumulation observed in the R15 population compared to the R8 and R14 populations was consistent with lower growth reduction and mortality observed in the plants (Figures 1 and 2).

The four L. virgata populations presented a high absorption index of ¹⁴C-glyphosate, absorbing amounts of over 50% from recovered herbicide at 96 HAT (Figure 3). At 24 HAT, the S population presented the highest ¹⁴C-glyphosate absorption rate. Between 48 to 96 HAT, glyphosate absorption was similar among populations, ranging from 47.7 to 67.7%. However, R15 population showed a clear reduced ¹⁴C-glyphosate absorption in comparison to the S population (Figure 3).

L. virgata populations showed similar ¹⁴C-glyphosate translocation patterns. In the aerial part of the plant (treated leaves and rest of plant). However, the greatest differences of ¹⁴C-glyphosate translocation were found in the roots. At 96 HAT, R15 population had the lowest rate of translocation to the root (21.9% of the absorbed herbicide), translocating ±10% less herbicide than the S population. The R8 and R14 populations translocated an average of 24.5 and 26.5%, respectively, of herbicide translocated to the roots at 96 HAT (Table 3). At this time, the S population had already reached a balanced distribution of herbicide between treated leaf, rest of plant and roots. The Phosphor Imager images confirmed the previous results of ¹⁴C-glyphosate translocation. At 96 HAT, the plants of resistant populations, mainly the R15 population ones, translocated smaller amounts of ¹⁴C-glyphosate from treated leaf to the root than the S population plants (Figure 4).

In the absence of glyphosate, significant differences (P = 0.035; DF = 3; n = 12) in the basal EPSPS activity between plants of L. virgata populations were found. R15 population presented an average of 0.41 μmol μg TSP^-1 min^-1, while the S, R8, and R14 populations an average of 0.29 μmol μg TPS^-1 min^-1. The EPSPS enzyme activity was inhibited by glyphosate in plants from susceptible and resistant populations while the concentrations increased. To inhibit EPSPS activity by 50% (I₅₀) for the S population was required 0.32 μM of glyphosate (Table 2). Plants from the R15 population showed an RI of 165.9 higher with respect to the S population plants (Figure 5). According to the confidence intervals (CI95%), R8, R14, and S population plants showed no significant differences in their EPSPS enzyme activity.

A fragment of 559 bp in length were amplified included the Thr-102 and Pro-106 positions in the protein sequence. The predicted amino acid sequence of S population presented as the same consensus of Lolium rigidum (GenBank: AF349754), and others grass glyphosate susceptible species. In the Thr-102 position no mutation was found. Only the R15 population showed a codon change from CCA to TCA resulting in an amino acid substitution from Proline to Serine at 106 position (Figure 6).

No significant differences (P = 0.6924; DF = 3; n = 12) were found in the EPSPS expression in untreated (0 HAT) plants used as a control between populations. The S and R15 populations showed an increased in the EPSPS expression level after glyphosate application at 24 HAT, but it was similar in both populations, with an average of 3.84 times more expression of the EPSPS (Table 4).