Susceptible and Glyphosate-Resistant Palmer Amaranth ( Amaranthus palmeri ) Response to Glyphosate Using C 14 as a Tracer: Retention, Uptake, and Translocation

The foliar retention, absorption, translocation, and diffusion of glyphosate in glyphosate resistant-(R) and susceptible (S)-Palmer amaranth populations from seed collected in Georgia in 2007 were examined. The R population of Palmer amaranth had an elevated copy number of the EPSPS gene conferring the mechanism of resistance. When applications of 14 C-glyphosate to a single leaf followed entire plant treatment with glyphosate, the distribution per-centages were similar for R and S for the above and below treated leaves when harvested at 1, 6, 12, 24, and 48 hours after treatment (HAT). There were initially no differences between R and S at 1 HAT with an average of 8% absorption for both biotypes. However, data indicated that glyphosate absorption increased for R-Palmer amaranth reaching 41% within 6 HAT and was significantly different (P = 0.01) from the 28% absorbed by S-Palmer amaranth. Glyphosate resistant and susceptible Palmer amaranth averaged 44% 14 C-glyphosate absorption by 24 HAT. There were no differences for 14 C-glyphosate Bq/mg of plant tissue between R and S for the above the treated leaf and below the treated leaf portions of plants at 1, 6, 12, 24, or 48 HAT. However, root ac-cumulation of 14 C-glyphosate in plant tissue was significantly greater by were no biotype differences within bathing times. However, the rate of efflux (the slope of the curves) was greater for the R biotype. These data support the reported gene amplification non-target site glyphosate resistance mechanism in Palmer amaranth.


Introduction
The use of glyphosate as a tool for weed control has become a standard practice for large scale glyphosate resistant crop production and vegetation management around the world. But since 1996 the incidence of glyphosate resistant weed species worldwide has gone from a low of zero reported in 1995, to 42 total in 2018 [1]. Many of these weed resistances are associated with the overuse of glyphosate in non-crop areas and glyphosate resistant crops.
The extensive application of glyphosate for glyphosate-resistant crop weed control promoted selection pressure for the occurrence of glyphosate-resistant (R) Palmer amaranth to appear in Georgia in 2004 [2], [3], subsequently occurring throughout the Southern United States [4]. It became the most troublesome weed of cotton in the region by 2009 [5] and 2013 [6]. The glyphosate-resistant Palmer amaranth reported in Georgia possesses a different mechanism of resistance than glyphosate-resistant horseweed and rigid ryegrass biotypes [2]. The mechanism of resistance is novel and attributed to increased copies of the gene required for production of the enzyme 5-enol-pyruvylshikimate-3phosphate synthase (EPSPS), with reports of up to 100 copies occurring in this population of R-Palmer amaranth [7].
As noted by Gaines et al. [7], varying herbicide mechanism-of-action and using different agronomic practices such as crop rotation may reduce glyphosate selection intensity. The fitness penalty associated with EPSPS gene amplification could cause the frequency of resistance to decrease for the glyphosate R-Palmer amaranth populations over time. There were no differences for 14 C-glyphosate absorption or translocation by the glyphosate-resistant Georgia biotype of Palmer amaranth verses a susceptible biotype. However, absorption and translocation of 14 C-glyphosate for this study was measured only at 48 hours after treatment (HAT) [2]. Differential absorption and mobility can vary over time between biotypes. California rigid ryegrass (Lolium rigidum, Gaudin) exhibited no differences in absorption or distribution of 14 C-glyphosate between susceptible and glyphosate resistant biotypes 1 to 3 days after treatment [8]. However, more glyphosate was present in treated leaves.
In order to establish the differences in absorption and translocation of glyphosate in resistant and susceptible Palmer amaranth at the time of its identifi-American Journal of Plant Sciences cation, studies were conducted to examine differences between the biotypes. Experiments were conducted with glyphosate resistant and susceptible Palmer amaranth over time to compare glyphosate foliar retention, absorption, translocation in vivo and efflux in vitro using leaf discs assays.  [10]. Plants were cut at the soil line and sectioned into four parts: treated leaf, tissue above the treated leaf, tissue below the treated leaf, and roots. Soil was removed by washing the roots over a wire grid. Treated leaves were rinsed twice for 15 seconds with 5 ml of methanol:deionized water (1:1, v:v) to remove non-absorbed 14 C-glyphosate [10]. A 1-ml aliquot of the combined rinsates was added to 10 ml of scintillation fluid, and radioactivity was quantified by liquid scintillation spectrometry (LSC).

Materials and Methods
All plant parts were dried for 48 hours at 45˚C, weighed, and combusted with a biological sample oxidizer to recover absorbed 14 C-glyphosate as CO 2 . Radioactivity in the oxidized samples was quantified by LSC. The amount of herbicide absorbed was calculated as the total radioactivity recovered from oxidation of the four plant parts and expressed as a percent of the total radioactivity applied.
Distribution of 14 C-glyphosate in various plant parts was expressed as the percentage of total absorbed radioactivity, or as Bq mg −1 of tissue dry weight. Recovery of 14 CO 2 was 77 to 99% (Table 1). 14 C-glyphosate uptake and efflux by leaf discs. A leaf discs experiment was conducted to examine the efflux of glyphosate uptake by R and S Palmer amaranth, similar to Chase et al. [11]. All plants were grown in the same fashion as described in the absorption and translocation studies.
Leaf disc 14C-glyphosate loading. C-glyphosate uptake buffer was added to a beveled-edge watch-glass. Seven-millimeter wide leaf discs were used in uptake and efflux experiments. The discs were cut from fully expanded leaves using cork-borers, taking care to avoid the midrib and main veins. The discs were rinsed three times with distilled water to clean the surfaces and to remove debris from the cut edge. Discs were then allowed to float on distilled water until needed. The specific leaf disc weight was determined on 10 sets of three discs for each biotype prior to use in the uptake experiments. Leaf discs were blotted dry on filter paper, weight taken, and specific leaf weight was expressed as g/m 2 . Three leaf discs were plotted dry and then placed in the buffer, lower leaf surface down, and the watch-glass covered with a Petri-dish cover to reduce evaporation. The covered watch-glass was then transferred to the growth chamber for three hours, where lighting was provided by fluorescent and incandescent lamps at 450 μE m −2 s −1 at 30˚C. Since the mechanism of resistance is gene amplification, no glyphosate metabolism would occur for the S and R biotypes [7]. No sampling was done during the influx period.
Leaf discs efflux of 14 C-glyphosate. For the efflux study, a second buffer con-  was determined by LSC, and quantified based on a mass balance. Then each disc was oxidized as previously described to quantify any remaining 14 C-glyphosate.
The experimental design was repeated-measures and the treatments were replicated 5 times. The experiment was conducted twice and the data combined for analysis.
For the 14 C-glyphosate efflux experiments, regression analysis was performed using nonlinear regression. The intent was to determine if the response could be described by using the exponential decay equation [12] ( ) where y is 14 C-glyphosate concentration in Becquerel's, B 0 is the initial concentration in solution after 15 minutes, B 1 is efflux rate, and x is time in minutes after treatment. Data for the exponential decay equations were subjected to ANOVA using the general linear models procedures with mean separation of parameter estimates using 95% asymptotic confidence intervals. Data were graphed in Sigmaplot 14 (Systat Software, San Jose, CA) ( Figure 1).

Results and Discussion
Leaf disc study. There were no significant interactions when comparing 14 C-glyphosate in the rinses, bathing solutions, or retention by the leaf discs between R and S Palmer amaranth over the 150 minute study (Figure 1). Although there were no statistical differences between the R and S Palmer amaranth biotypes parameter estimates (Data not shown), time did effect the amount of Absorption and Translocation. Across experiments 77 to 99% of total applied radioactivity was recovered from leaf washes and oxidation of plant parts (Table   1). There were no plant weight differences between biotypes during the study; however, both biotypes continued to grow (Table 1). There were no biotypes differences in 14 C-glyphosate recovered from the washes for any of the exposure times. However, the percent of 14 [14]. The over expression of the target enzyme in the R Palmer amaranth biotype effectively decreases the concentration of glyphosate in the tissue due to herbicide/target enzyme interaction, thus maintaining a higher diffusion gradient in the R biotype compared to S biotype which facilitated additional absorption. Glyphosate that is interacting with the target enzyme is effectively not influencing the tissue concentration gradient. Consequently, because the S biotypes has orders of magnitude less available EPSPS synthases there is a higher concentration of free glyphosate in the tissue solution and a correspondently lower concentration gradient and less diffusion.
Although only 8% and 7% for the R and S Palmer amaranth biotypes of applied 14 C-glyphosate had been absorbed 1 HAT (Table 1), respectively, 82 and 90% remained in the treated leaf (Table 2). After 6 HAT, there was significantly greater mass of 14 C-glyphosate absorbed in the R treated leaf as compared to the S biotype: 13.5 verses 8.1 Bq, respectively with P = 0.0063 (Table 2). Although The only statistical differences in percent of absorbed 14 C-glyphosate re-distributed from treated to non-treated tissue (i.e., leaves above and below the treated leaf and roots) was at 48 HAT in shoot tissue and roots at 12, 24 and 48 HAT ( Table 2). Almost twice as much 14 C-glyphosate (28 verses 15% absorbed) had been acropetally translocated to tissue above the treat leaf in the R biotype.
In shoot tissue below the treated leaf, the slightly higher (25 verses 19% of absorbed) amount of 14  Differential translocation between the biotypes was also manifested using shoot/root ratios ( Figure 2

Conclusions
The data presented in this study support previous research demonstrating that the glyphosate resistance mechanism in Palmer Amaranth is gene amplification of  glyphosate will control the R biotype whereas with target site resistance even excessively high rates will not provide any control [2] [14]. This phenomena is because no matter how much glyphosate is applied to an altered site biotype, EPSPS is not inhibited. With EPSPS gene amplification, the wildtype form of EPSPS is inhibited by glyphosate so if enough is applied, some level of control will be achieved [2].
With gene amplification, the form of EPSPS is the same in both biotypes, but more is produced in the R biotype. Thus, absorbed glyphosate continues to inhibit EPSPS in both biotypes, but because the R biotype has orders of magnitude greater enzyme, treated plants are not susceptible. Gene amplification was first noted in tissue culture research examining glyphosate resistance in crop species [21]. The higher amount of wild type (i.e. normal inhibition by glyphosate) EPSPS in the R Palmer amaranth biotype was manifested in leaf discs as short term higher rates of retention. The lower short term efflux rates in the R biotype leaf discs (15 to 45 minute) could have resulted from more of the absorbed glyphosate complexing with EPSPS. The in vivo studies demonstrated that the R biotype had greater rates of absorption and translocation. Because of the R biotype complexes more of the absorbed glyphosate, a higher diffusion gradient is maintained resulting in higher initial influx. Over the study period, the R biotype gene amplification of EPSPS allowed for the continuation of glyphosate redistribution through normal acropetal and basipetal translocation. The over expression of EPSPS in the R biotype allows more glyphosate to be absorbed and translocated. As long as the stoichiometry favors significantly greater EPSPS than glyphosate molecules, normal aromatic amino acid biosynthesis of tryptophan, tyrosine, and phenylalanine will proceed and ultimately normal growth.