The aim of this study was to test the hypothesis that pathogenesis induced in sensitive plants by ALS- and ACC-inhibiting herbicides is an active process which involves programmed cell death (PCD). A reliable marker of PCD is DNA fragmentation, which is carried out by endogenous nucleases. The root meristems from herbicide-sensitive plants were analyzed by the TUNEL assay to estimate DNA fragmentation. It was shown that the highest level of DNA fragmentation in root meristem cells under pathogenesis, induced on maize seedlings by the action of ACC-inhibiting herbicide propaquizafop, and on pea seedlings by the action of ALS-inhibiting herbicide tribenuron-methyl, was associated with increased total nuclease activity in root meristem lysates comparing to the corresponding control. In response to tribenuron-methyl application, the level of total nuclease activity elevated due to high activity of both Zn2+-dependent («acidic») and Ca2+/Mg 2+-dependent («alkaline») nucleases, while in a case of propaquizafop, the increased activity was detected only for Zn2+-dependent nucleases. These differences may be due to different mechanisms of PCD initiation, depending on the mode of herbicides action. The facts, that TUNEL method recorded DNA fragmentation in plant cells induced by ACC- and ALS-inhibiting herbicides, and this fragmentation is associated with an increased activity of endogenous nucleases, represent evidence for PCD, that occurs during pathogenesis. The significance of data obtained is that they emphasize the fact that herbicide-induced pathogenesis is a complex, multi-stage, active process. The discovery of pathogenesis distinct stages mechanisms nature opens up new possibilities for regulating the herbicides selective phytotoxicity by physiologically active substances and genetically engineered manipulations.
Keywords: DNA fragmentation, endonucleases, programmed cell death, herbicides, propaquizafop, tribenuron-methyl
Full text and supplemented materialsFree full text: PDF
1. Sychuk, A.M., Radchenko, M.P. & Morderer, Ye.Yu. (2013). Programmed cell death in pathogenesis induced by ACC-inhibiting herbicides. Biological Studies Studia Biologica, 7, pp. 101-106 [in Ukrainian]. https://doi.org/10.30970/sbi.0702.294
2. Morderer, Ye.Yu., Radchenko, M.P. & Sychuk, A.M. (2013). Programmed cell death in the pathogenesis, induced by herbicides in plants. Fiziol. rast. genet., 45, No. 6, pp. 517-526 [in Ukrainian].
3. Chen, S. & Dickman, M. (2004). Bcl-2 family members localize to tobacco chloroplasts and inhibit programmed cell death induced by chloroplast-targeted herbicides. J. Exp. Bot., 55, pp. 2617-2623. https://doi.org/10.1093/jxb/erh275
4. Chichkova, N.V., Shaw, J., Galiullina, R.A., Drury, G.E., Tuzhikov, A.I., Kim, S.H., Kalkum, M., Hong, T.B., Gorshkova, E.N., Torrance, L., Vartapetian, A.B. & Taliansky, M. (2010). Phytaspase, a relocalisable cell death promoting plant protease with caspase specificity. EMBO J., 29, pp. 1149-1161. https://doi.org/10.1038/emboj.2010.1
5. de Freitas, D., Coelho, M. & Souza, M. (2007). Introduction of anti-apoptotic baculovirus p35 gene in passion fruit induces herbicide tolerance, reduced bacterial lesions, but does not inhibit passion fruit woodiness disease progress induced by cowpea aphid-borne mosaic virus (CABMV). Biotechnol. Lett., 29, pp. 79-87. https://doi.org/10.1007/s10529-006-9201-9
6. Caverzan, A., Piasecki, C. & Chavarria, G. (2019). Defenses against ROS in crops and weeds: the effects of interference and herbicides. Int. J. Mol. Sci., 20, p. 1086. https://doi.org/10.3390/ijms20051086
7. Das, M., Reichman, J.R. & Haberer, G. (2010). A composite transcriptional signature differentiates responses towards closely related herbicides in Arabidopsis thaliana and Brassica napus. Plant Mol. Biol., 72, pp. 545-556. https://doi.org/10.1007/s11103-009-9590-y
8. Ozheredow, S.P., Emec, A.I., Litvin, D.I., Britsun, V.N., Shvartau, V.V., Lozinskii, M.O. & Blium, I.B. (2010). Antimitotic activity of new 2,6-dinitroaniline derivatives and their synergistic activity in compositions with graminicides. Tsitol. Genet., 44, No. 5, pp. 54-59 [in Russian]. https://doi.org/10.3103/S0095452710050087
9. Sychuk, A.M. (2015). The participation of programmed cell death in herbicide-induced pathogenesis (Unpublished candidate thesis). Institute of Plant Physiology and Genetics NAS of Ukraine, Kyiv, Ukraine [in Ukrainian].
10. Kacprzyk, J., Gunawardena, A.H.L.A.N., Bouteau, F. & McCabe, P.F. (2021). Editorial: plant programmed cell death revisited. Front. Plant. Sci., 255, No. 3, pp.785-802. https://doi.org/10.3389/fpls.2021.672465
11. Reape, T.J., Molony, E.M. & McCabe, P.F. (2008). Programmed cell death in plants: distinguishing between different modes. J. Exp. Bot., 59, No. 3, pp. 435-444. https://doi.org/10.1093/jxb/erm258
12. Gordeziani, M., Adamia, G., Khatisashvili, G. & Gigolashvili, G. (2017). Programmed cell self-liquidation (apoptosis). Ann. Agr. Sci., 15, pp. 148-154. https://doi.org/10.1016/j.aasci.2016.11.001
13. Mondal, R., Antony, S., Roy, S. & Chattopadhyay, S.K. (2021). Programmed cell death (PCD) in Plant: Molecular mechanism, regulation, and cellular dysfunction in response to development and stress. Regulation and Dysfunction of Apoptosis. London: IntechOpen. Available from: https://www.intechopen.com/chapters/77139 https://doi.org/10.5772/intechopen.97940
14. Sychuk, A.M. & Morderer, Ye.Yu. (2017). The influence of NADPH-oxidase inhibitor and calcium antagonists on phytotoxic action of Acetyl-CoA-carboxylase and acetolactate synthase-inhibiting herbicides. Fiziol. rast. genet., 49, No. 1, pp. 64-70. https://doi.org/10.15407/frg2017.01.064
15. Gavrieli, Y., Sherman, Y. & Ben-Sasson, S. (1992). Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol., 119, pp. 493-501. https://doi.org/10.1083/jcb.119.3.493
16. Tripathi, A.K., Pareek, A. & Singla-Pareek, S. (2017). TUNEL Assay to assess extent of DNA fragmentation and programmed cell death in root cells under various stress conditions. Bio. Protocol., 7, No. 16, pp. 1-10. https://doi.org/10.21769/BioProtoc.2502
17. Thelen, H.P. & Northcote, D.M. (1989). Identification and purification of a nuclease from Zinnia elegans L: A potential molecular marker for xylogenesis. Planta, 179, No. 2, pp. 181-195. https://doi.org/10.1007/BF00393688
18. Sibirtsev Y.T. & Rasskazov V.A. (2000) Effect of ionic strength on the pH optimum, specificity and mechanism of action of acid DNase from mature eggs of the sea urchin Strongylocentrotus intermedius. Biochemistry, 65, pp. 1121-1129 [in Russian].
19. Bradford, M.M. (1976). A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, pp. 248-254. https://doi.org/10.1006/abio.1976.9999
20. Ernst, O. & Zor, T. (2010). Linearization of the bradford protein assay. J. Vis. Exp., 38, p. 1918. https://doi.org/10.3791/1918
21. Sugiyama, M., Ito, J., Aoyagi, S. & Fukuda, H. (2000). Endonucleases. Plant Mol. Biol., 44, No. 3, pp. 387-397. https://doi.org/10.1023/A:1026504911786
22. Bai, M., Liang, M., Huai, B., Gao, H., Tong, P., Shen, R., He, H. & Wu, H. (2020). Ca2+-dependent nuclease is involved in DNA degradation during the formation of the secretory cavity by programmed cell death in fruit of Citrus grandis 'Tomentosa'. J. Exp. Bot., 71, No. 16, pp. 4812-4827. https://doi.org/10.1093/jxb/eraa199
23. Jones, M.L., Bai, S., Lin, Y. & Chapin, L.J. (2021). The senescence-associated endonuclease, PhENDO1 is upregulated by ethylene and phosphorus deficiency in petunia. Horticulturae, 7, p. 46. https://doi.org/10.3390/horticulturae7030046
24. Locato, V. & De Gara, L. (Eds.). (2018). Programmed cell death in plants: An Overview. Methods in Molecular Biology, 1743. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7668-3_1
25. De Pinto, M., Locato, V. & de Gara, L. (2012). Redox regulation in plant programmed cell death. Plant Cell Environ., 35, pp. 234-244. https://doi.org/10.1111/j.1365-3040.2011.02387.x
26. Radchenko, M.P., Sychuk, A.M. & Morderer, Ye.Yu. (2013). Increasing of selective phytotoxicity and prooxidant-antioxidant equilibrium under the graminicide fenoxaprop-P-ethyl use with the herbicide synergist and antagonist. Fiziol. rast. genet., 45, No. 4, pp. 306-312 [in Ukrainian]. http://dspace.nbuv.gov.ua/handle/12345 6789/159324
27. Radchenko, M.P., Sychuk, A.M. & Morderer, Ye.Yu. (2013). Reducing antagonism in mixtures of herbicides by a specific inhibitor of superoxide dismutase activity. Scientific notes of the Taurida VI Vernadsky National University. Series: Biology, Chemistry, 26, No. 65, pp. 161-168 [in Ukrainian]. http://sn-biolchem.cfuv.ru/wp-content/uploads/ 2016/11/016_radc.pdf
28. Radchenko, M.P., Sychuk, A.M. & Morderer, Ye.Yu. (2014). Decrease of the herbicide fenoxaprop phytotoxicity in drought conditions: the role of the antioxidant enzymatic system. J. Plant Prot. Research., 54, No. 4, pp. 390-394. https://doi.org/10.2478/jppr-2014-0058
29. Aleksandrushkina, N.I., Seredina, A.V. & Vanyushin, B.F. (2009). Endonuclease activities in the coleoptile and the first leaf of developing etiolated wheat seedlings. Russ. J. Plant Physiol., 56, pp. 154-163. https://doi.org/10.1134/S1021443709020022
30. Radchenko, M.P., Sychuk, A.M. & Morderer, Ye.Yu. (2016). The activity of NADPH oxidase in corn seedlings root meristems under the herbicide acetyl-CoA-carboxylase inhibitor action. Fiziol. rast. genet., 48, No. 6, pp. 544-547 [in Ukrainian]. https://doi.org/10.15407/frg2016.06.544
31. Kraehmer, H., Laber, B., Rosinger, C. & Shulz, A.(2014). Herbicides as weed control agents: state of the art: I. Weed control research and safener technology: the path to modern agriculture. Plant Physiol., 166, pp. 1119-1131. https://doi.org/10.1104/pp.114.241901
32. Sanchez-Pons, N. & Vicient, C.M. (2013). Identification of a type I Ca2+/Mg2+-dependent endonuclease induced in maize cells exposed to camptothecin. BMC Plant Biol., 13, p. 186. https://doi.org/10.1186/1471-2229-13-186
33. Islam, F., Xie, Y. & Farooq, M.A. (2016). Salinity reduces 2,4-D efficacy in Echinochloa crusgalli by affecting redox balance, nutrient acquisition and hormonal regulation. Protoplasma, 255, pp. 785-802. https://doi.org/10.1007/s00709-017-1159-z
34. Radchenko, M., Ponomareva, I., Pozynych, I. & Morderer, Ye. (2021). Stress and use of herbicides in field crops. Agricult. Sci. Pract., 8, No. 3, pp. 50-70. https://doi.org/10.15407/agrisp8.03.050
35. Morderer, Ye.Yu., Nizkov, Y.I., Radchenko, M.P., Rodzevich, O.P. & Sychuk, A.M. (2014). Weed control in crops by herbicides using. Kyiv: Logos [in Ukrainian].
36. Langaro, A.C., Agostinetto, D. & Ruchel, Q. (2017). Oxidative stress caused by the use of preemergent herbicides in rice crops. Revista Ciencia Agronomica, 48, pp. 358-364. https://doi.org/10.5935/1806-6690.20170041
37. Wang, J., Lv, M., Islam, F., Gill, R.A, Yang, C., Ali, B. & Zhou W. (2016). Salicylic acid mediates antioxidant defense system an Yan G., Zhoud ABA pathway related gene expression in Oryza sativa against quinclorac toxicity. Ecotoxicol Environ. Saf., 133, pp. 146-156. https://doi.org/10.1016/j.ecoenv.2016.07.002
38. Serrano, I., Romero-Puertas, M.C., Sandalio, L.M. & Olmedilla, A. (2015). The role of reactive oxygen species and nitric oxide in programmed cell death associated with self-incompatibility. J. Exp. Bot., 66, No. 10, pp. 2869-2876. https://doi.org/10.1093/jxb/erv083
39. Sychuk, A.M., Radchenko, M.P. & Morderer, Ye.Yu. (2013). The increase of phytotoxic action of graminicide fenoxaprop-P-ethyl by NO donor sodium nitroprusside. Science and Education a New Dimension: Natural and Technical Sciences, 2, No. 15, pp 21-22.
40. Maroli, A.S., Nandula, V.K., Dayan, F.E., Duke, S.O., Gerard, P. & Tharayil, N. (2015). Metabolic profiling and enzyme analyses indicate a potential role of antioxidant systems in complementing glyphosate resistance in an amaranthus palmeri Biotype. J. Agric. Food Chem., 63, No. 41, pp. 9199-9209. https://doi.org/10.1021/acs.jafc.5b04223
41. Harre, N.T., Nie, H., Jiang, Y. & Young, B.G. (2018). Differential antioxidant enzyme activity in rapid-response glyphosate-resistant Ambrosia trifida. Pest. Manag. Sci., 74, No. 9, pp. 2125-2132. https://doi.org/10.1002/ps.4909
42. Piasecki, C., Yang, Y., Benemann, D.P., Kremer, F.S., Galli, V., Millwood, R.J., Cechin, J., Agostinetto, D., Maia, L.C., Vargas, L.C. & Stewart, N.Jr. (2019). Transcriptomic analysis identifies new non-target site glyphosate-resistance genes in Conyza bonariensis. Plants, 8, No. 6, 157. https://doi.org/10.3390/plants8060157
43. Benedetti, L., Rangani, G., Viana, V.E. , Carvalho-Moore, P., Merotto, A.Jr., Camargo, E.R., de Avila, L.A. & Roma-Burgos, N. (2020). Rapid reduction of herbicide susceptibility in junglerice by recurrent selection with sublethal dose of herbicides and heat stress. Agronomy, 10, p. 1761. https://doi.org/10.3390/agronomy10111761
44. Fipke, M.V. Feijo, R., Garcia, N.S., Heck, T., Viana, V.E., Dayan, F.E., Agostinetto, D., Lamego, P.F., Souza, G.M. & Camargo, E.R. (2022). Transgenerational effect of drought stress and sub-lethal doses of quizalofop-p-ethyl: decreasing sensitivity to herbicide and biochemical adjustment in eragrostis plana. Agriculture, 12, p. 396. https://doi.org/10.3390/agriculture12030396
45. Mohammad, V.H., Osborne, C.P. & Freckleton, R.P. (2022). Drought exposure leads to rapid acquisition and inheritance of herbicide resistance in the weed Alopecurus myosuroides. Ecology and Evolution, 12, No. 2. https://doi.org/10.1002/ece3.8563
46. Vanderauwera, S., Suzuki, N., Miller, G., van de Cotte, B., Morsa, S., Ravanat, J.L., Hegie, A., Triantaphylides, C., Shulaev, V., Van Montagu, M.C., Van Breusegem, F. & Mittler, R. (2011). Extranuclear protection of chromosomal DNA from oxidative stress. Proc. Natl. Acad. Sci. USA, 108, No. 4, pp. 1711-1716. https://doi.org/10.1073/pnas.1018359108