Tolerance of Brazil pusley biotypes to glyphosate: role of absorption and translocation and behavior of shikimic acid pathway metabolites

Abstract

Background:  Concerns about the tolerance of weeds to herbicides have increased in recent decades. Within soybean cultivation areas in Southern Brazil, differences have been observed in terms of glyphosate tolerance in Brazil pusley (Richardia brasiliensis Gomes) biotypes.

Objectives:  The experiment was carried out to determine the response of two Brazil pusley biotypes to glyphosate and investigate possible reasons responsible for their differences in tolerance to glyphosate.

Methods:  The response to glyphosate doses was investigated. Also, experiments with ¹⁴C-glyphosate were performed to determine the absorption and translocation of the herbicide in both biotypes. Chromatographic analyses were also performed to assess concentrations of shikimic acid, and aromatic amino acids in both plant shoots and roots.

Results:  The results of the dose-response study showed a higher tolerance to glyphosate in the Paraná biotype comparison with the São Paulo biotype. The Paraná biotype showed lower ¹⁴C-glyphosate absorption than the São Paulo biotype, although only minor differences were detected. The two Brazil pusley biotypes did not differ in terms of ¹⁴C-glyphosate translocation. Chromatographic analyses by HPLC detected lower shikimic acid accumulations in the Paraná biotype. Overall, higher concentrations of the aromatic amino acids phenylalanine, tyrosine, and tryptophan indicated a possible pattern for the biotype with lower tolerance to glyphosate.

Conclusions:  Higher tolerance to glyphosate in Brazil pusley biotype Paraná is caused by the lower leaf absorption of the herbicide, although this might not be the only mechanism involved.

Keywords:
Richardia brasiliensis Gomes; Weed Tolerance; 5-enolpyruvylshikimate-3-Phosphate synthase; Aromatic amino acids

1. Introduction

The introduction of genetically modified glyphosate-resistant crops (GR crops) worldwide has intensified the selection of weed species tolerant to this herbicide. Examples include dayflower (Commeiina spp.), morningglory (Ipomoea spp.), Brazil pusley (Richardia brasiliensis Gomes), broadleaf buttonweed (Borreria latifolia (Aubl.) K. Schum), and hemp sesbania (Sesbania exaltata ((Mill.) McVaugh)) (Trezzi et al., 2020).

Brazil pusley is recognized for its tolerance to the herbicide glyphosate (Galon et al., 2013; Lucio et al., 2019; Bottcher et al., 2022). This herbaceous plant belongs to the Rubiaceae family and is an annual species that thrives in areas cultivated with annual and/or perennial crops, particularly soybean (Diesel et al., 2018). Brazil pusley is distributed across various regions worldwide and infests approximately 5,861,895 hectares of soybean crops in Brazil (Lucio et al., 2019).

Studies indicate that each Brazil pusley plant per square meter can reduce crop productivity by 2% to 2.6% when competing with soybean plants (Diesel et al., 2020). Naturally tolerant plants can survive and reproduce after herbicide application, making tolerance an innate characteristic of the species (Cruz-Hipolito et al., 2009). Herbicide tolerance can be associated with the selection pressure exerted on a weed community, potentially leading to an increased prevalence of one species over others (Christofoletti et al., 2008).

The study of variation associated with herbicide tolerance, both between and within geographically separated populations, provides valuable insights into the evolutionary processes underlying this trait (Baucom, Mauricio, 2004). Genetic variation in glyphosate tolerance has been documented in populations of tall morningglory (Ipomoea purpurea (L. Roth)) (Baucom, Mauricio, 2004). Similarly, studies on Brazil pusley biotypes collected from soybean fields in Brazil have revealed substantial variation in glyphosate tolerance (Diesel et al., 2018). While identifying tolerant biotypes is essential, it is equally important to investigate the mechanisms responsible for tolerance and their role in mitigating the herbicide's detrimental effects.

The absorption and translocation of glyphosate are influenced by various factors, including the physicochemical properties of the molecule, herbicide formulation, concentration, leaf surface characteristics, and environmental conditions such as temperature and relative humidity. Glyphosate has a low octanol-water partition coefficient (Kow), indicating low lipid affinity and high water solubility (Franz et al., 1997). This characteristic can result in differential uptake behavior among weed species. Within plants, glyphosate translocates primarily through the phloem via symplastic movement, although apoplastic transport can also occur. The herbicide's efficacy may be reduced in more mature plants possessing cuticle which is rich in waxes and other poorly soluble compounds. These compounds act as significant barriers to absorption and subsequent translocation (Hatterman-Valenti et al., 2011).

The effect of glyphosate on the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) can be assessed through the presence of metabolites associated with the shikimic acid pathway. Among these, shikimic acid is the most extensively studied compound, serving as a sensitive biomarker for glyphosate poisoning in susceptible plants, which accumulate this substance upon exposure to the herbicide (Schrübbers et al., 2014).

The accumulation of shikimic acid indicates that the EPSPs enzyme is inhibited by glyphosate, as it is the first upstream metabolite to accumulate when enzyme activity is disrupted. This accumulation also suggests that glyphosate encounters no significant barriers, such as limitations in absorption, translocation, or metabolization, allowing it to effectively reach and inhibit the EPSPs enzyme. The inhibition of EPSPs disrupts the biosynthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. The resulting deficiency in these amino acids and their downstream metabolites often leads to plant death (Rojano-Delgado et al., 2012).

Studies on the tolerance mechanisms of Brazil pusley remain limited, highlighting the need for further research to elucidate the processes underlying glyphosate tolerance. Such knowledge is essential for developing effective chemical management strategies for crops affected by infestations of this species.

Considering the hypothesis that specific mechanisms contribute to the differential tolerance of Brazil pusley biotypes to glyphosate, this research aimed to: (i) evaluate the response of two Brazil pusley biotypes to varying rates of glyphosate, (ii) investigate leaf absorption and translocation of ¹⁴C-glyphosate in these biotypes, and (iii) analyze the concentrations of metabolites associated with the shikimic acid pathway in plants treated with glyphosate.

2. Materials and Methods

2.1 Dose-Response Curves

The first stage was conducted in a greenhouse at the Federal Technological University of Paraná (UTFPR), Pato Branco Campus, in the state of Paraná, Brazil. A previous characterization of biotypes from the state of Paraná (8 biotypes), Santa Catarina (two biotypes) and São Paulo (one biotype) was carried out (Diesel et al., 2020). Two contrasting biotypes were selected for this study. Each biotype has been subjected to distinct glyphosate selection pressures in agricultural environments. Therefore, the biotypes were designated as "São Paulo" and "Paraná". The "São Paulo" biotype was collected in the city of São Paulo, São Paulo State, Brazil (23°33'01''S 46°38'02''W). The "Paraná" biotype is considered more tolerant to glyphosate than São Paulo biotype. Paraná biotype was collected in the municipality of Itapejara D'Oeste, state of Paraná, Brazil (25°58'43"S 52°48'47"W), from an area cultivated for nine consecutive years with Roundup Ready (RR) soybean, and, according to information from the soybean producer, has been reported as difficult to control by glyphosate.

The "dose-response" experiment was conducted in a completely randomized design with four replications, arranged in a 2 x 6 factorial scheme in which the first factor was represented by the biotypes "São Paulo" and "Paraná" and the second factor by the rates of the glyphosate herbicide (0, 74, 163, 360, 792, and 1742 g a.e. ha^-1).

The seeds of Brazil pusley were placed to germinate in Gerbox boxes with a double layer of germination paper moistened with distilled water in a growth chamber at 25°C with a photoperiod of 12/12 h. Approximately 15 days after germination, two seedlings of Brazil pusley were transplanted into polyethylene pots with a capacity of 5 dm³ of the Oxisol. The soil was collected in the Experimental Area of UTFPR, in an area free of rubiaceous spp. propagules. The soil had been previously sifted, and 20 days after transplantation, the plants were thinned, leaving only one uniform plant per pot. Irrigation was performed twice a day to maintain soil moisture close to field capacity.

Treatments with glyphosate were applied when the plants had 6 to 8 fully expanded leaves (Burgos et al., 2001; Norsworthy et al., 2008; Resende et al., 2022), using a CO₂ pressurized backpack sprayer at 2.96 bar, containing three fan type 110.02 nozzles, 0.50 m apart, totaling a mixing volume of 200 L ha^-1. The meteorological conditions at the beginning and end of the applications were as follows: air temperature: 24 and 21.8°C; relative humidity: 84 and 88.6%.

At 14 and 28 days after the application (DAA) of the treatments, visual control evaluations were performed based on the scale proposed by (Frans et al., 1986), where 0% represents the null effect of the herbicide symptoms on the plants and 100% represents the death of the plants. After the last evaluation (28 DAA), the shoots were collected to quantify the green mass of shoots (GSM). The visual control results were transformed to 100 - % control, and the GSM was expressed as a percentage of the check.

2.2 Absorption and Translocation of ¹⁴C-Glyphosate

The seeds of Brazil pusley were allocated in Gerboxes and placed to germinate in a growth chamber at 25°C with a photoperiod of 12 h in the Laboratory of Weed Science of UTFPR, Campus Pato Branco, State of Paraná, Brazil. The seedlings were transplanted into pots with a capacity of 500 mL, containing sifted soil, and left in a greenhouse with controlled conditions (minimum temperature of 10^oC and maximum of 35^oC). When the plants reached the 4 to 6 leaf, they were transported to the Ecotoxicology Laboratory at Center for Nuclear Energy in Agriculture (CENA), University of São Paulo (USP), State of São Paulo, Brazil, for application of ¹⁴C-glyphosate.

The study was developed in a completely randomized design with three replications, with the treatments being the biotypes "São Paulo" and "Paraná". Each plant of Brazil pusley was considered an experimental unit. The evaluation periods were 2, 8, 24, 48, and 72 h after application (HAA) of herbicide. When the plants reached 4 to 6 fully expanded leaves, ¹⁴C-glyphosate (P-methylene-¹⁴C) on the phosphonomethyl radical presenting specific activity of 537.4 MBq mmol^-1 and 96.81% radiochemistry (Institute of Isotopes Co., Ltd., Budapest, Hungary) purity was applied. The herbicide solution was composed of a mixture of ¹⁴C-glyphosate + analytical standard (non-radiolabeled glyphosate) at a concentration of 360 g a.e. ha^-1 of glyphosate with a mixed volume of 200 L ha^-1. Ten drops of 1.0 μL of this mixture were applied to the adaxial face of the last pair of fully expanded leaves, with the aid of a microapplicator (Hamilton PB6000 Dispenser, Hamilton Co., Reno, NV, USA), totaling 20 drops per plant.

In each evaluation period the treated leaves were cut to interrupt the translocation process for the rest of the plant. The treated leaves were placed inside a funnel arranged on a scintillation bottle and washed with 1 mL (washed three times with the aid of a pipette calibrated to 1,000 μL) of an 80% methanol solution in order to quantify the unabsorbed herbicide. An aliquot of 500 | L was removed from the liquid from this washing and mixed with 10 mL of scintillation liquid to later perform the analysis of the radioactivity of this mixture through liquid scintillation spectrometry (LSS) (Packard 1900 TR, PerkinElmer, Waltham, MA, USA) by 5 min. After each evaluation period, the plants were washed to remove the soil from its roots and dried at 40°C until reaching constant mass. Subsequently, they were submitted to combustion in a biological oxidizer (OX 600 Harvey Instruments, Tappan, NY, USA) by 3 min for subsequent quantification of radioactivity through LSS and determination of translocation in each part of the plant.

This methodology made it possible to determine the absorption of the herbicide through the percentage of radioactivity present inside the plant (treated leaves, leaves above, leaves below the treated, stem, and root) about the total radioactivity recovered (unabsorbed + radioactivity inside the plant).

To visualize the ¹⁴C-glyphosate translocation, the Paraná and São Paulo biotypes were treated under the same conditions as described in the previous assay. Whole plants were gently rinsed, pressed on filter paper, dried at room temperature (≈6 days) and then placed next to a phosphor storage film (25 × 12.5 cm) for 6 h. The radiolabel dispersion trapped in the film was scanned in a storage phosphor system (Cyclone, PerkinElmer).

2.3 Distribution of Shikimic Acid and Amino Acids in the Shoot and Roots

An experiment was conducted using a completely randomized design with three replications. Treatments were arranged in a 2 × 5 factorial scheme, with the first factor comprising the Paraná and São Paulo biotypes and the second factor representing four evaluation periods after glyphosate application (0, 2, 8, 24, and 72 hours). Plants of both biotypes were cultivated in pots, as described in the absorption and translocation study.

For glyphosate treatments, 360 g a.e. ha^-1 of the herbicide (Zapp QI 620™, Syngenta Crop Protection) was applied to plants with six fully expanded leaves. Applications were carried out using a sprayer with constant CO₂ pressure and XR110.02 fan nozzles, delivering a spray volume of 200 L ha^-1. At the designated intervals after herbicide application, plants were removed from the pots, and their shoots and roots were separated, immediately weighed, and washed with distilled water. The samples were then frozen at -50°C for further analysis.

The analysis of the contents shikimic acid, phenylalanine, tyrosine, and tryptophan of plant tissues was made according to (Gomes et al., 2015) using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system composed of a high-performance liquid chromatography (Prominence UFLC, Shimadzu, Kyoto, Japan) equipped with two LC-20AD pumps, a SIL-20AC autoinjector, a DGU-20A5 degasser, a CBM-20A controller system, and a CTO-20AC oven. The chromatograph was coupled to a Triple Quad 4500 mass spectrometer (Applied Biosystems, Foster City, USA). Chromatographic analyses were performed with a C18 column (Phenomenex Gemini 5μ C18RP 110Å) using an injection volume of 20 μL, with five mM ammonium acetate (Avantor Performance Materials, Inc., Center Valley, PA) in water and five mM ammonium acetate in methanol (Merck KGaA, Darmstadt, Germany). The flow rate used was 0.8 mL min^-1, and the ratio of the solvents was gradually increased from 90:10 (water/ methanol) to 5:95 at 4 min and returned to the initial condition at 10 min. Detection and separation of the compounds were performed in one run, with ionization of the compounds in positive and negative mode with a total time of 12 min. The retention time in the chromatographic column of each compound was: glyphosate (2.89 min) and shikimic acid (2.95 min).

2.4 Statistical Analysis

The dose-response, absorption and translocation assays were fitted using nonlinear regression models implemented in the drc package of the R software, which enabled the estimation of ED₅₀ values (Ritz et al., 2015). The selection of models was based on the AIC criterion and parameter standard errors. For the analysis of the distribution of ¹⁴C-glyphosate, data were subjected to analysis of variance (ANOVA) using the F-test at a 5% significance level. Analysis of amino acids was performed by comparing biotypes at each time using the Student's t-test, with a 5% probability of error. All statistical analyses and plotting were conducted using R software (R Core Team, 2023).

3. Results

3.1 Dose-Response Curves

The biotypes exhibited significantly different responses to glyphosate rates (p<0.05). At 14 DAA, the Paraná biotype demonstrated a lower level of control compared to the São Paulo biotype (Figure 1a). In the Paraná biotype, even at the highest dose (1,742 g a.e. ha^-1), 50% control (ED₅₀) was not achieved. In contrast, the ED₅₀ for the São Paulo biotype was only 405 g a.e. ha^-1 (Table 1). At 28 DAA, although control levels decreased in both biotypes, the differences in response to glyphosate rates between the tolerant and susceptible biotypes remained evident (Figure 1b). The ED₅₀ values for the Paraná and São Paulo biotypes were 769 and 290 g a.e. ha^-1, respectively (Table 1), further emphasizing the greater tolerance of the Paraná biotype to glyphosate.

Figure 1
Control (100 - control) of two Brazil pusley biotypes at 14 (a) and 28 days after application (DAA) (b) in response to glyphosate rates of 0, 74, 163, 360, 792 and 1742 g a.e. ha^-1. Vertical bars represent the standard error of the mean of each treatment (n = 4)

Table 1
Coefficients of the Weibull type I and Log-Logistic equation parameters and their respective standard errors

Variable/Figure	Biotype	Model 1	Coefficients				ED50 *
			b	c	d	e
100 - control/ Figure 1a	Paraná	Weibull type I -3 parameter	0.58 ± 0.21	-	100.86 ± 3.40	16804.00 ± 15011.00	>1742
	São Paulo		92.51 ± 3.97	-	2.42 ± 0.55	406.48 ± 38.25	405.432 ± 32.44
100 - control/ Figure 1b	Paraná	Weibull type I -3 parameter	1.07 ± 0.13	-	100.89 ± 3.30	1084.91 ± 90.31	769.04 ± 71.79
	São Paulo		1.45 ± 0.22	-	96.34 ± 3.91	373.65 ± 26.84	290.32 ± 25.81
Reduction of shoot growth/ Figure 2	Paraná	Weibull type II -3 parameter	-1.74 ± 0.67	-	100.24 ± 7.70	284.65 ± 48.67	351.14 ± 65.61
	São Paulo	Weibull type I -3 parameter	0.34 ± 0.03	-	99.92 ± 2.09	79.65 ± 11.45	27.79 ± 6.26
Total absorption/ Figure 3	Paraná	Weibull type I -4 parameter	-3.78 ± 0.08	10.22 ± 0.06	30.66 ± 0.17	95.75 ± 0.13	11.26 ± 0.09
	São Paulo		-4.21 ± 0.13	9.92 ± 0.08	37.59 ± 0.17	98.49 ± 0.12	10.83 ± 0.12
Total translocation/Figure 5	-	Log-Logistic -3 parameter	-0.11 ± 0.02	-	17.46 ± 0.80	16.70 ± 2.07	16.70 ± 2.07

• Numbers after the ± symbol correspond to the standard error of the parameters. In Figure 1, ED₅₀ was used, and in Figure 2, GR₅₀ . b: slope parameter; c: lower limit; d: upper limit; e: inflection-point parameter (inflection at exp(e))
• * ED₅₀ is the parameter that provides a 50% reduction in the value of the y-axis; only for the variable "reduction of shoot growth" does this parameter refer to GR₅₀.¹ Model available in (Ritz et al., 2010)

Increasing glyphosate rates significantly reduced the green shoot mass (GSM) of both biotypes (Figure 2). However, the Paraná biotype exhibited a 12.6-fold lower reduction in GSM as glyphosate rates increased (from 0 to 1,742 g a.e. ha^-1), with a GR₅₀ of 351 g a.e. ha^-1. In contrast, the São Paulo biotype required only 28 g a.e. ha^-1 to achieve a 50% reduction in GSM (Table 1).

Figure 2
Reduction of shoot growth of two Brazil pusley biotypes at 28 days after application (DAA) in response to glyphosate rates of 0, 74, 163, 360, 792 and 1742 g a.e. ha^-1. Vertical bars represent the standard error of the mean of each treatment (n = 4)

3.2 Absorption and Translocation of ¹⁴C-Glyphosate

The analysis of variance for absorption showed an interaction between time and biotype (p<0.05). Within the biotypes, an analysis over time revealed an increase in ¹⁴C-glyphosate uptake (Figure 3). The São Paulo biotype showed higher absorption of ¹⁴C-glyphosate than the Paraná biotype in all time intervals. The absorption of glyphosate labeled with ¹⁴C reached 43% and 35% for the São Paulo and Paraná biotypes, respectively, 8 HAA, and both biotypes achieved 90-95% absorption starting 24 HAA (Figure 3). Specifically, the time to reach 50% absorption was 25 minutes shorter for biotype São Paulo (Table 1).

Figure 3
Total absorption of ¹⁴C-glyphosate by two Brazil pusley biotypes (Paraná and São Paulo) and times after application (2, 8, 24, 48, and 72 HAA). Vertical bars represent the standard error of the mean of each treatment (n = 4)

The São Paulo and Paraná biotypes showed no significant differences in ¹⁴C-glyphosate translocation (p>0.05) across any of the evaluated compartments (treated leaf, above the treated leaf, below the treated leaf, stem, and root), at any time after application (Figure 4). Regarding total ¹⁴C-glyphosate translocation in Brazil pusley plants, differences were observed only between times after application, and the biotypes were statistically similar in this regard (Figures 5 and 6). This trend was also evident in the analysis of individual plant compartments (Figure 4), where pairwise comparisons of times showed no significant differences between the biotypes. Translocation of 50% of the total ¹⁴C-glyphosate was observed at 16h:42min after application (Table 1).

Figure 4
Distribution of ¹⁴C-glyphosate in plant parts of Brazil pusley biotypes (Paraná and São Paulo) in 2 (a), 8 (b), 24 (c), 48 (d), and 72 h after application (HAA) (e). Bars followed by the same letter do not differ from each other, according to Tukey's test (p<0.05). Vertical bars represent the standard error of the mean of each treatment (n = 4)

Figure 5
Total translocation of ¹⁴C-glyphosate of Brazil pusley biotypes (Paraná and São Paulo) in periods of 2, 8, 24, 48, and 72 h after application (HAA). Vertical bars represent the standard error of the mean of each treatment (n = 4)

Figure 6
Autoradiography of the total translocation of ¹⁴C-glyphosate of Brazil pusley biotypes (São Paulo and Paraná) in periods of 2, 8, 24, 48, and 72 h after application (HAA)

3.3 Concentration of Shikimic Acid, and Amino Acids in the Shoot and Roots

A more pronounced accumulation of shikimic acid was observed in the roots and shoots of the São Paulo biotype, compared to that of the Paraná biotype (Figures 7a and 7b). The concentration of shikimic acid in the roots differed between the biotypes at times 0, 8, 24, and 72 HAA (Figure 7a), while in the shoots, the differences were significant at times 2, 8, 24, and 72 HAA (Figure 7b).

Figure 7
Concentration of shikimic acid (μg g^-1) in roots (a) and shoots (b) among the biotypes of pusley from Brazil (Paraná and São Paulo) as a function of time after application (0, 2, 8, 24, and 72 HAA), analyzed by LC-MS/MS. Vertical bars represent the standard error of the mean of each treatment (n = 4)

The concentrations of amino acids varied significantly between biotypes and across time points, with no consistent pattern of differences between the factors biotype and time. In general, the concentration of phenylalanine in the roots was higher in the São Paulo biotype than in the Paraná biotype, although this difference was not consistent at all times, occurring only at 0, 8, and 24 HAA (Figure 8a). A similar trend was observed in shoots, where phenylalanine concentration differed significantly at times 0, 8, and 24 HAA (Figure 8b).

Figure 8
Phenylalanine in roots (a) and shoots (b), tyrosine in roots (c) and shoots (d) and tryptophan in roots (e) and shoots (f) concentration in plant parts (μg g^-1) in biotypes of Brazil pusley biotypes (Paraná and São Paulo) and periods (2, 8, 24, and 72 HAA) after application. Vertical bars represent the standard error of the mean of each treatment (n = 4)

Tyrosine concentration in roots was higher in the São Paulo biotype than in the Paraná biotype. However, this difference was significant only in 8 and 24 HAA (Figure 8c). In the shoots, the results were inconsistent. The tyrosine concentration was 41%, 66%, and 44% higher in the São Paulo biotype, at 0, 8, and 24 HAA, respectively, but 56% and 79% lower than that observed in the Paraná biotype, at 2 and 72 HAA, respectively (Figure 8d).

Overall, the tryptophan concentration in the roots was 60% higher in the São Paulo biotype compared to the Paraná biotype. However, this difference was significant at 8, 24, and 72 HAA following glyphosate treatment (Figure 8e). In the shoots, the São Paulo biotype exhibited tryptophan concentrations 39% higher than those in the Paraná biotype, except at 2 HAA, when tryptophan levels were 40% higher in the Paraná biotype (Figure 8f).

4. Discussion

4.1 Dose-Response Curves

The tolerance to glyphosate found in the biotype Paraná is in agreement with other studies, which detect low levels of Brazil pusley control by glyphosate. Glyphosate at a dose of 770 g a.e. ha^-1 showed low efficiency for controlling Brazil pusley reaching a maximum of 14% of control (Sharma, Singh, 2000). A range of glyphosate rates between 480 and 1200 g a.e. ha^-1 also did not result in a satisfactory control of Brazil pusley (Correia, Durigan, 2010). It was observed in another study that the Brazil pusley biotypes were not well controlled by glyphosate even at rate of 2160 g a.e. ha^-1, reaching a control level of close to 75% at 28 days after treatment (Monquero et al., 2005).

The difference between the biotypes may be due to the selection process of tolerant biotypes through the intense use of glyphosate in the crop fields. This process was intensified at the end of the 90s by introducing genetically modified crops with resistance to glyphosate in Brazil (Benbrook, 2016). The area where biotype Paraná seeds were collected was cultivated for nine consecutive years with RR soybean. It might be suggested, therefore, that the biotype Paraná is in a more advanced degree of the process of evolution of tolerance to glyphosate compared with the biotype São Paulo. This might explain the reduced efficacy of glyphosate on the Paraná biotype. In other words, the São Paulo biotype was in an initial selection process at the collection time, which justifies its lower tolerance to glyphosate.

4.2 Absorption and Translocation of ¹⁴C-Glyphosate

The effective action of glyphosate is attributed to its excellent uptake by the plant, efficient translocation to meristems, partial degradation, and slow mode of action (Nguyen et al., 2016). In both biotypes, higher percentages of herbicide absorption were observed at 24 hours after application (HAA), compared to a study conducted with barnyardgrass (Echinochloa colona (L.) Link.), prickly sida (Sida spinosa L.), hemp sesbania, and pitted morningglory (Ipomoea lacunosa L.) (Norsworthy et al., 2001). Although significant, we believe that the lower ¹⁴C-glyphosate absorption in the more tolerant biotype (Paraná) is insufficient to characterize it as the sole mechanism of tolerance to glyphosate.

The differences in absorption between the two biotypes were greater in the first evaluations, i.e. at 2 and 8 HAA. Glyphosate penetration into leaves encounters barriers at the cuticle and plasma membrane regions. The high water solubility of glyphosate (Franz et al., 1997) may pose an unfavorable characteristic for its penetration through the cuticle and the phospholipid bilayer of the plasma membrane. The interaction between the herbicide and the components of these barriers varies across species (Travlos et al., 2017) and biotypes, determining the degree of foliar absorption capacity.

Most of the herbicide applied remained in the treated leaf of Brazil pusley; however, a significant percentage, 8.73%, was translocated after 16h:42 min. Compared to other species considered tolerant to glyphosate, this percentage are higher than the total translocation of ¹⁴C-glyphosate in coat buttons (7.5%) and broadleaf buttonweed (7.9%) at 72 HAA (Galon et al., 2013), but lower than the total translocation observed in two biotypes of Japanese morningglory (Ipomoea nil (L.) Roth) (38-53%) at 48 HAA.

Glyphosate translocation from the treated leaf requires the herbicide to enter the phloem, where it is retained and transported along with sugars from the source to the sink regions of the plant (Singh et al., 2020). In some weed species, resistant biotypes present cellular-level barriers that reduce glyphosate absorption, resulting in lower herbicide concentrations and, consequently, decreased translocation (Shaner, 2009). In the present study, the results do not show any differences in translocation between the most tolerant (Paraná) and the least tolerant (São Paulo) biotypes of Brazil pusley, despite observing lower absorption in the herbicide-tolerant biotype. Therefore, differential translocation cannot be identified as the factor responsible for the observed tolerance differences in this study.

There is evidence supporting the involvement of additional mechanisms in the process of glyphosate tolerance. These mechanisms include target-site mutations, target-site gene duplication (Patterson et al., 2018), active vacuolar sequestration (Ge et al., 2013), and a rapid necrotic response (Moretti et al., 2018). Notably, glyphosate resistance tends to exhibit lower magnitude and less frequent reliance on target-site and metabolic mechanisms compared to herbicides that inhibit acetolactate synthase (ALS) or acetyl-CoA carboxylase (ACCase). At least one study identified an aldo-keto reductase (AKR) enzyme as being responsible for the oxidation of glyphosate into AMPA and glyoxylate (Pan et al., 2019).

Mutations in the EPSPs enzyme and gene duplication stand out as target site mechanisms capable of increasing plant tolerance to glyphosate. Several studies confirm the importance of substitutions in conserved positions of EPSPs. Chen et al. (2020) identified amino acid alterations and substitutions in populations of the Indian goosegrass (Eleusine indica (L.) Gaertn.). In addition, combined mutations may occur, further reducing the herbicide's affinity for the active site (Li et al., 2022). These findings reinforce the notion that target-site mutations can alter enzyme dynamics, thereby reducing herbicide efficacy.

Gene duplication leads to the presence of one or more additional copies of the gene within the genome, which can increase the amount of EPSPs enzyme in resistant biotypes than that in susceptible biotypes. This surplus requires proportionally higher glyphosate concentrations to achieve complete inhibition (Patterson et al., 2018). Gene duplication has been reported in glyphosate-resistant E. indica species (Chen et al., 2020) and in palmer amaranth (Amaranthus palmeri S. Watson) (Barco-Antoñanzas et al., 2022). It is important to note that both EPSPs mutations and amplifications can occur simultaneously (Chen et al., 2020; Li et al., 2022). Although these mechanisms have been reported in the literature, in R. brasiliensis they remain unknown. Therefore, future research on these mechanisms is important, as they can range from the complete absence of mutations to extreme gene overexpression (Aulakh et al., 2024).

4.3 Concentration of Shikimic Acid, and Amino Acids in the Shoot and Roots

By inhibiting EPSPs, glyphosate induces elevated levels of shikimic acid in cells, a response that serves as a sensitive bioindicator of the herbicide's toxic effects on susceptible tissues (Schrübbers et al., 2014). In tolerant plants, the lack of EPSPs inhibition prevents the accumulation of shikimic acid. Notably, shikimate accumulation was significantly lower in both the roots and shoots of the Paraná biotype compared to the biotype São Paulo. The reduced accumulation of shikimic acid in the Paraná biotype suggests the presence of mechanisms that either limit the amount of glyphosate reaching the EPSPs enzyme or alter the enzyme's site of action, as previously discussed.

When glyphosate binds to the EPSPs enzyme, it inhibits the biosynthesis of 5-enolpyruvyl-shikimate-3-phosphate, leading to the accumulation of shikimic acid and disrupting the production of essential aromatic amino acids (phenylalanine, tryptophan, and tyrosine), proteins, and secondary metabolites (Velini et al., 2009).

The analysis of aromatic amino acid profiles revealed, with some temporal variation, higher concentrations of phenylalanine, tyrosine, and tryptophan in the plant tissues (roots and shoots) of the São Paulo biotype compared to those from the Paraná biotype. Glyphosate exposure is expected to inhibit the production of aromatic amino acids and, therefore, lower concentrations of these amino acids would be expected in the São Paulo biotype compared to the Paraná biotype; however, this was not observed. Similar findings were reported by Vivancos et al. (2011), who, in comparing a glyphosate-resistant (RR) soybean cultivar with a glyphosate-sensitive cultivar, noted minimal changes in the amino acid profile and no significant variation in free amino acid content in the RR cultivar. In contrast, the sensitive cultivar exhibited increased levels of free amino acids, including tryptophan and threonine. These discrepancies may be explained, as suggested by Velini et al. (2009), by the regulatory systems of the shikimic acid pathway, which do not follow a universal pattern across all species and may partially compensate for the production of certain intermediate compounds.

Another explanation for the increased level of amino acids in the present study might be protein hydrolysis (Zulet et al., 2013). This has also been suggested by Wang (2001) in purple nutsedge (Cyperus rotundus L.) and soybean plants (Moldes et al., 2017) exposed to glyphosate. However, according to Maroli et al. (2016), de novo synthesis also contributed to increased amino acid levels in A. paimeri treated with glyphosate. Thus, further studies are required to clarify this issue.

5. Conclusions

The dose-response study revealed differences in glyphosate tolerance between Brazil pusley biotypes, providing critical information for farmers and technicians developing management plans for this species.

The greater tolerance to glyphosate in Brazil pusley biotype Paraná is caused by the lower leaf absorption of the herbicide; however, the differences in glyphosate absorption between biotypes can be considered relatively small, suggesting the involvement of other mechanism(s) responsible for the observed differences in tolerance. The results did not show significant differences in ¹⁴C-glyphosate translocation between the biotypes.

Shikimic acid levels following glyphosate application strongly correlated with the sensitivity of the Brazil pusley biotypes to the herbicide. Additionally, higher concentrations of aromatic amino acids were generally observed in the susceptible biotype, suggesting a potential pattern specific to this biotype. These elevated levels suggest that shikimic acid is a highly reliable biomarker for detecting glyphosate susceptibility in biotypes of Brazil pusley.

Further investigation into possible mechanisms, such as glyphosate transport to vacuoles or alterations in the EPSPS enzyme (e.g., gene mutations or duplications), is necessary to better understand the mechanisms of tolerance.

Acknowledgements

The authors thank CNPq for awarding the research productivity grant process 312487/2021-5; to the UNIEDU/FUMDES Graduate Program to grant scholarship No. 17491.They also thank LabSolos/UTFPR for carrying out the chemical and textural analyses of the soil. They also acknowledge the infrastructure and financial support from the Federal Technological University of Paraná.

Funding

The authors declare that there was no specific funding for this work.

Data Availability

The data generated or analyzed during this study are available upon reasonable request to the corresponding author.

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* Corresponding author:

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Editor in Chief:

Carol Ann Mallory-Smith

Associate Editor:

Sheing Qiang

Conflict of Interest:

The authors declare that there is no conflict of interest regarding the publication of this manuscript.

Publication Dates

• Publication in this collection
  10 Aug 2026
• Date of issue
  2026

History

• Received
  14 Aug 2025
• Accepted
  11 June 2026