Retention, Uptake, and Translocation of Agrochemicals in Plants

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Chapter 8

Absorption and Penetration of Herbicides Viewed in Metabolism Studies: Case of Glufosinate and Imazamox in Wheat A. M. Rojano-Delgado,*,1 J. Menéndez,2 and R. De Prado1 Department of Agricultural Chemistry, C-3 Building, Campus of Rabanales, University of Córdoba, E-14071, Córdoba, Spain 2Department of Agroforestry Sciences, Campus La Rábida, University of Huelva, E-21819, Palos (Huelva), Spain *E-mail: [email protected].

1

The search for new alternatives for weed control in wheat has led to the creation of new resistant lines of wheat to herbicides that are not apt for this crop. This is the case of Clearfield wheat for the family of imidazolinone herbicides and genetically modified wheat to glufosinate. The behavior of these herbicides in contact with these lines has been studied, resulting in some very interesting ideas about the transformation of these herbicides within the plant as well as the way they take through them.

Wheat and Weeds The importance of the effect that each weed produces on the crop will depend on the type of weed management that is performed and the planting season of the wheat. In the case of wheat, there are three herbicide application times depending on the state of the development of wheat plants and weeds: before planting, in preemergence and in postemergence. Before sowing wheat the herbicides glyphosate, sulphosate, paraquat and / or diquat, and even mixtures with other non residual herbicides that increase their effectiveness are commonly used. In preemergence, i.e. before crop and weed emergence, it is common to use © 2014 American Chemical Society In Retention, Uptake, and Translocation of Agrochemicals in Plants; Satchivi, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


metsulfuron methyl and flurochloridone. Finally, in the case of postemergence, when the wheat and weeds have emerged, the herbicides used are clodinafop propargyl, chlortoluron, diclofop, isoproturon, pinoxaden, tralkoxydim, fenoxaprop-p-ethyl, bromoxynil and many others. However, despite the wide range of herbicides, some weeds are not controlled by their use and a search for new alternatives is therefore necessary. Some of these alternatives entails the "design" of crops resistant to non-selective herbicides, either by crossbreeding and biotechnology application or by transgenesis (integration of genes into the genome without crossbreedings). In the first case we refer to Clearfield® crops specific to imidazolinone (1), and in the second case to genetically modified organisms such as wheat resistant to glufosinate (2). Clearfield technology is considered as an integrated weed control (3) based on the development of varieties tolerant to imidazolinones using traditional induction mutations and conventional breeding. They are non-genetically modified seeds, and their performance has been widely assessed (4, 5). The advances in genetic engineering have promoted the application of recombinant DNA technology to obtain and commercialize new varieties of genetically modified crops in which two glufosinate resistance genes (designed as pat and bar) were introduced, encoding one of the phosphinothricin acetyltransferases (PAT) (6) widely applied in plant genetic engineering. This permits the following-up of glutamine synthetase (GS) activity in the presence of glufosinate. In both cases, the metabolism to its respective herbicides was also found (7, 8).

Importance of Absorption and Translocation Imazamox and Glufosinate in the Resistant Wheat Lines A necessary condition for achieving the effectiveness of a herbicide is that it reaches its site of action in a sufficient concentration to be lethal. The lack of movement of a herbicide will reduce its concentration at the site of action. These low concentrations can be occurred due to a reduction in the penetration, absorption or translocation or the existence of sequestration phenomena in metabolically inactive cell organelles as shown in Figure 1 as occurs in the glyphosate. Those mechanisms are difficult to study separately, and when it is done it is hard to tell them apart, because a differential absorption implies a differential translocation, and the latter may derive from the different herbicide degradation in the absorption site, resulting in more or fewer metabolites which can be translocated. The resistance/sensitivity to herbicides due to their lack of absorption in preemergence treatment can be associated with morphological factors (9) such as differences in depth or anatomical structure of the root system, or physiological factors such as the limited absorption of the herbicide active in susceptible species. Regarding the foliar path, the amount of herbicide penetrated in the plant tissue in postemergence applications is subject to the amount of it adhered to the plant. This depends on factors such as: weather conditions during the treatment, the surface tension of the herbicide solution, the volume of treatment, and foliar features such as area and leaf orientation and the amount of waxes present in 160 In Retention, Uptake, and Translocation of Agrochemicals in Plants; Satchivi, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


the leaves. Resistance to post-emergence treatments is often associated with the presence of differences in the leaf cuticle such as composition and epicuticular wax content (10).

Figure 1. Mechanisms known in which the concentration of herbicide does not reach a lethal dose in the target-site and by some of which can occur resistance to imazamox and glufosinate.

The same as in the absorption/penetration processes, those of herbicide translocation in plants can be classified according to the type of herbicide treatment that has been applied (11). In the case of absorption via root, the movement of the herbicide will depend on its chemical nature. This is due to three causes, namely: 1) the accumulation of the unmetabolized herbicide at the root, resulting in a lack of translocation of the active ingredient to the shoots; 2) the herbicide metabolism in the root and subsequent production of immobile forms which are generally polar conjugates; 3) the restriction of herbicide movement in the vascular system (primary and secondary vessels) making it impossible to reach its primary site of action. In the case of the foliar herbicide movement, after penetration, this can be classified according to whether its transport takes place in the xylem or the phloem. While the transport of the herbicides via the xylem path freely follows the water flow in this system to the leaf margins and to the inter-vascular spaces, the transport via phloem will depend on two distinctive processes: the concentration gradient of the herbicide from the phloem cells and the mesophilic, and the ability of the herbicide to be retained by the phloem cells during transport (11). 161 In Retention, Uptake, and Translocation of Agrochemicals in Plants; Satchivi, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.

Imazamox is a systemic herbicide and, hence, translocatable, but, glufosinate is mostly a contact herbicide but with a partial systemic action, so that it rarely translocates. In the case of glufosinate it has not been proved that, although the herbicide cannot be translocated, metabolites can do so.


Herbicides Metabolism: Transport of the Metabolites Most techniques used for the study of absorption and translocation are based on using radiolabeled herbicide. This is a help when visualizing the movement of the herbicide through the plant as long as there is a radioactivity detector type phophoimager, although the latter does not give any information about metabolite transport only about the radioactivity displaced as we can see in the studies realized in the references (10) and (11). In the case of glufosinate, the study does not have any meaning, as it hardly translocates, and, if it does, it takes a small proportion. In the case of imazamox, this makes more sense, because it is a systemic herbicide with a high translocation speed. Studies on Echinochloa crus-galli (12) or those made in red lentil and dry bean (13) demonstrate imazamox translocation through the plant and the speed at which it happens. The absorption and translocation of imazamox can also be studied by sampling the foliar part (leaves) and the root. Working in this way, it was observed in works made in two cultivars of Triticum aestivum with Clearfield technology (8) that the susceptible (S) cultivar, which is unable to metabolize the herbicide, translocates imazamox from the leaves to the root throughout the experiment; therefore, translocation of imazamox through the plant occurs. On the contrary, in the resistant (R) cultivar, the metabolites are the compounds translocated to the root, thus translocation took place proportionally to dose and time. The difference between the two cultivars is the compound translocated to the root, consisting of toxic or non-toxic forms determined by the metabolism. In both cultivars, and for the same amount of imazamox applied, the amount of herbicide penetrated is the same, but it appears as such or is metabolized, depending on the cultivar. In the resistant cultivar imazamox was metabolized about 67.74 % at 120 h after treatment with 200 g ai of herbicide ha–1 and about 7.70 % corresponding of the metabolized amount was translocated. Respect to the rest of imazamox unmetabolized, only the 9.32 % was translocated. While in the susceptible cultivar about 0.98 % was metabolized and 99.35 % of unmetabolized imazamox was translocated. The results obtained in this work indicated that the low translocation of imazamox to the root could be due to the metabolization to nontoxic forms of herbicide which are translocated, occurring when the enzyme is saturated with the herbicide, which reduces the amount of intracellular herbicide that can catalyze the ALS enzyme, thus increasing the tolerance to the herbicide as compared to other populations with a single resistance mechanism and (10). Figure 2 shows as the herbicides and the metabolites can be transported through the plant. 162 In Retention, Uptake, and Translocation of Agrochemicals in Plants; Satchivi, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


Figure 2. Illustration of distribution of imazamox and its metabolites (A) glufosinate and its metabolites (B) in plant as a result of foliar uptake.

Techniques Used To Study the Metabolism In the case of imazamox, some authors have studied the presence of metabolites by the measure of radioactivity in extracts of plants treated with 14C-imazamox (13, 14) as target metabolites have not been commercialized. The absence of commercial metabolites has delayed research on degradation of the herbicide, which has been restricted to studies involving radioactivity-based methods (13, 14) has required the synthesis of the target metabolites (15). Harir and his collegues (16), working with radioactivity-based methods, have measured the presence of metabolites through the amount of measured total radioactivity, which is not reliable information because the radioactivity can stem from a non-metabolic degradation occurring outside the plant by photolysis. The main problems in dealing with metabolite synthesis are its cost (both in reagents and time) and purity. The search for fast and effective procedures for the identification of metabolites without standards and without the use of radioactive compounds is a challenge in the agronomical field due to the need to find out the behavior of a plant against a given herbicide. In the case of resistant weeds, finding such a procedure would constitute a useful tool for developing new attack strategies. Capillary electrophoresis (CE) in its different modes has been the most widely used separation technique prior to determination of imidazolinones (17). Thus, Ohba, Minoura, Safarpour M., Picard, and Safarpour H. (15) described 163 In Retention, Uptake, and Translocation of Agrochemicals in Plants; Satchivi, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.


a method using reverse micellar electrokinetic chromatography (MEKC) with UV detection, which permitted the determination of imazamox and its hydroxy and glucoside metabolites after synthesis of both compounds. The capability of CE can be improved by coupling to a mass spectrometry (MS) detector, which has been the equipment used for several studies dealing with trace analysis of herbicides, including imazamox (18–21). In addition to the use of CE, separation of imazamox from mixtures with other herbicides and pesticides has involved gas chromatography (GC) (22) and, particularly, liquid chromatography (LC) (13, 14, 23) with MS detection in all instances. Despite the potential of MS in the identification of degradation products from herbicides (16), no MS-based studies have been reported to confirm the presence of imazamox metabolites in plants. In the case of glufosinate, the determination of the analytes has frequently been carried out after separation by liquid chromatography (LC) (24–26), gas chromatography (GC) (27, 28) or capillary electrophoresis (CE) (29, 30) by different detection techniques. In metabolism studies, the preferred methods for analysis of glufosinate and metabolites in plants are based on LC separation and radioactivity detection (31, 32), which require simple sample preparation protocols (usually precipitation of polysaccharides, protein and peptides and subsequent filtration or centrifugation), thus preventing or minimizing potential losses of the target analytes (particularly low concentrated metabolites) (32). Nevertheless, radioactivity-based methods have as major limitations the high cost of reagents, loss of radioactivity by quenching, difficulties in waste management and their inability to identify chemical structures of the metabolites. Apart from radioactivity-based methods, photometry by a diode array detector (DAD) (29, 30, 33), fluorescence detection (24, 25, 29) and mass spectrometry (MS) (27, 28) have also been used, but only for glufosinate determination. Derivatization is required prior to fluorescence detection, mostly using fluorenylmethoxycarbonyl chloride (FMOC–Cl) as a fluorogenic reagent (24, 25, 28) that reacts with the amino group. In short, studies on the metabolism of glufosinate have involved only some of its metabolites (mainly its major metabolite, 3-(methylphosphinic) propionic acid, also known as MPPA), developed in aqueous media.

References 1. 2. 3. 4. 5. 6. 7.

BASF Corporation. Clearfield production system; http://www.basf.com. León, E.; Marín, S.; Giménez, M. J.; Piston, F.; Rodríguez-Quijano, M.; Shewry, P. R.; Barro, F. J. Cereal Sci. 2009, 49, 148–156. Colquhoun, J.; Mallory-Smith, C.; Ball, D. Weed Management in Clearfield Wheat with Imazamox; Oregon State University: Corvallis, OR, 2003; p 4–4. Pfenning, M.; Palfay, G.; Guillet, T. J. Plant Dis. Prot. 2008, 21, 649–663. Tan, S.; Evans, R. R.; Dahmer, M. L.; Singh, B. K.; Shaner, D. L. Pest Manage. Sci. 2005, 61, 246–257. Wehrmann, A.; van Vliet, A.; Opsomer, C.; Botterman, J.; Schulz, A. Nat. Biotechnol. 1996, 14, 1274–1278. Rojano-Delgado, A.; Priego-Capote, F.; Barro, F.; Luque de Castro, M. D.; De Prado, R. Phytochemistry 2013 DOI:10.1016/j.phytochem.2013.10.008. 164 In Retention, Uptake, and Translocation of Agrochemicals in Plants; Satchivi, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.

8. 9. 10. 11.


12. 13. 14. 15. 16.

17. 18. 19. 20. 21.

22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

Rojano-Delgado, A.; Priego-Capote, F.; Luque de Castro, M. D.; De Prado, R. Agron. Sustainable Dev. 2014 DOI:10.1007/s13593-014-0232-7. Chachalis, D.; Reddy, K. N.; Elmore, C. D.; Steele, M. L. Weed Sci. 2001, 49, 628–634. Rojano-Delgado, A. M.; Cruz-Hipólito, H.; De Prado, R.; Luque de Castro, M. D.; Rodríguez Franco, A. Phytochemistry 2012, 73, 34–41. Cruz-Hipolito, H.; Osuna, M. D.; Vidal, R. A.; De Prado, R. J. Agric. Food Chem. 2009, 57, 4273–4278. Riar, D. S.; Norsworthy, J. K.; Srivastava, V.; Nandula, V.; Bond, J. A.; Scott, R. C. J. Agric. Food Chem. 2013, 61, 278–289. Bukun, B.; Nissen, S. J.; Shaner, D. L.; Vassios, J. D. Weed Sci. 2012, 60, 350–354. Pester, T. A.; Nissen, S. J.; Westra, P. Weed Sci. 2001, 49, 607–612. Ohba, K.; Minoura, M.; Safarpour, M. M.; Picard, G. L.; Safarpour, H. J. Pestic. Sci. 1997, 22, 277–281. Harir, M.; Frommberger, M.; Gaspar, A.; Martens, D.; Kettrup, A.; El Azzouzi, M.; Schmitt-Kopplin, Ph. Anal. Bioanal. Chem. 2007, 389, 1459–1467. Picó, Y.; Rodríguez, R.; Mañes, J. Trends Anal. Chem. 2003, 22, 133–151. Font, G.; Ruiz, M. J.; Fernández, M.; Picó, Y. Electrophoresis 2008, 29, 2059–2078. García, F.; Henion, J. J. Chromatogr. A 1992, 606, 237–247. Menzinger, F.; Schmitt-Kopplin, Ph.; Freitag, D.; Kettrup, A. J. Chromatogr. A 2000, 891, 45–67. Nejad, H.; Safarpour, M. M.; Cavalier, T.; Picard, G.; Souza, M.; Krynitsky, A. J.; Chiu, S.; Miller, P.; Stout, S. J. Capillary Electrophor. Microchip Technol. 1998, 5, 81–87. Stout, S. J.; DaCunha, A. R.; Allardice, D. G. Anal. Chem. 1996, 68, 653–658. Cessna, A. J.; Elliott, J. A.; Bailey, J. J. Environ. Qual. 2012, 41, 882–892. Sancho, J. V.; López, F. J.; Hernández, F.; Hogendoorn, E. A.; Van Zoonen, P. J. Chromatogr. A 1994, 678, 59–67. Akuzawa, H.; Akaiwa, H. Bunseki Kagaku 1997, 46, 69–74. Hori, Y.; Fujisawa, M.; Shimada, K.; Sato, M.; Kikuchi, M.; Honda, M.; Hirose, Y. J. Chromatogr. B 2002, 767, 255–262. Watanabe, S. J. Food Hyg. Soc. Jpn. 2002, 43, 169–172. Hori, Y.; Fujisawa, M.; Shimada, K.; Hirose, Y. J. Anal. Toxicol. 2001, 25, 680–684. Ishiwata, T. Bunseki Kagaku 2004, 53, 863–864. Asami, T.; Imura, H. Anal. Sci. 2006, 22, 1489–1493. Dröge, W.; Siemeling, U.; Pühler, A.; Broer, I. Plant Physiol. 1994, 105, 159–166. Jansen, C.; Schuphan, I.; Schmidt, B. Weed Sci. 2000, 48, 319–326. Suzuki, A.; Kawana, M. Bull. Environ. Contam. Toxicol. 1989, 43, 17–21.

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Retention, Uptake, and Translocation of Agrochemicals in Plants

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