Permeability of the plasma membrane for propidium iodide and destruction of cell nuclei in the epidermis of pea leaves: the effect of polyelectrolytes and detergents

Damage to the plasma membrane (PM) of cells in pea leaf epidermis, determined by its permeability to propidium iodide  (PI),  which  binds  to  DNA  of  cell  nuclei, and programmed cell death (PCD) detected by the destruction of cell nuclei was investigated. PM of the epidermal cells in the isolated epidermis was permeable to PI (it stained their nuclei). PM of the guard cells did not allow PI to pass through. KCN, an inducer of PCD, caused the destruction of the both epidermal and guard cell nuclei. KCN-Induced destruction of guard cell nuclei was accompanied by the penetration of PI into the cells. The polycation chitosan at a concentration of 0.1 mg/ ml caused the destruction of the epidermal cell nuclei, but at a concentration of 1 mg/ ml, the permeability of the guard cell PM for PI staining their nuclei was induced. Other polycations (cytochrome c, polylysine, polyethylenimine and protamine) also caused staining of the guard cell nuclei by PI. Polyanions (polyacrylic acid, dextran and heparin) initiated the destruction  of  cell  nuclei,  which  was  accompanied  by the penetration of PI into cells. Detergents Triton X-100 and lauryldimethylamine- N-oxide produced the permeability of the guard cell PM for PI and prevented the destruction of the nuclei that was induced by  KCN.  Treatment  of  the  epidermis with Triton X-100 (for 2 h with its subsequent washing) increased the destruction of the guard cell nuclei that  was  caused  by  KCN.  Polycations  polyethyleneimine and protamine were prevented, while  chitosan,  cytochrome  c,  and  polylysine,  on the contrary, enhanced KCN-induced destruction of the guard cell nuclei. The data obtained shows that the destruction of cell nuclei upon induction of cell death with KCN or polyanions is accompanied  by  damage  to  PM  (producing  its  permeability for PI). Damage to PM caused by detergents or polycations prior to cell treatm       by KCN, can prevent or, on the contrary, intensify the destruction of cell nuclei.

1. van Doorn W.G., Beers E.P., Dangl J.L., et al. Morphological classification of plant cell deaths // Cell Death Differ. 2011. Vol. 18. N 8. P. 1241–1246.

2. Kroemer G., Galluzzi L., Vandenabeele P., et al. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009 // Cell Death Differ. 2009. Vol. 16. N. 1. P. 3–11.

3. Zhang Y., Chen X., Gueydan C., Han J. Plasma membrane changes during programmed cell deaths // Cell Research. 2018. Vol. 28. N 1. P. 9–21.

4. Julien O., Wells J.A. Caspases and their substrates // Cell Death Differ. 2017. Vol. 24. N 8. P. 1380–1389.

5. Chichkova N.V., Tuzhikov A.I., Taliansky M., Vartapetian A.B. Plant phytaspases and animal caspases: structurally unrelated death proteases with a common role and specificity // Physiol. Plant. 2012. Vol. 145. N 1. P. 77–84.

6. Thomas E.L., van der Hoorn R.A.L. Ten prominent host proteases in plant-pathogen interactions // Int. J. Mol. Sci. 2018. Vol. 19. N 2: 639.

7. Samuilov V.D., Lagunova E.M., Kiselevsky D.B., Dzyubinskaya E.V., Makarova Ya.V., Gusev M.V. Participation of chloroplasts in plant apoptosis // Biosci. Rep. 2003. Vol. 23. N 2–3. P. 103–117.

8. Dzyubinskaya E.V., Kiselevsky D.B., Bakeeva L.E., Samuilov V.D. Programmed cell death in plants: effect of protein synthesis inhibitors and structural changes in pea guard cells // Biochemistry (Mosc.). 2006. Vol. 71. N 4. P. 395– 405.

9. Dzyubinskaya E.V., Kiselevsky D.B., Lobysheva N.V., Shestak A.A., Samuilov V.D. Death of stoma guard cells in leaf epidermis under disturbance of energy provision // Biochemistry (Mosc.). 2006. Vol. 71. N 10. P. 1120–1127.

10. Bakeeva L.E., Dzyubinskaya E.V., Samuilov Programmed cell death in plants: ultrastructural changes in pea guard cells // Biochemistry (Mosc.). 2005. Vol. 70. N 9. P. 972– 979.

11. Kiselevsky D.B., Kuznetsova Y.E., Vasil’ev L.A., Lobysheva N.V., Zinovkin R.A., Nesov A.V., Shestak A.A., Samuilov V.D. Effect of Ca2+ on programmed death of guard and epidermal cells of pea leaves // Biochemistry (Mosc.). 2010. Vol. 75. N 5. P. 614–622.

12. Vasil’ev L.A., Dzyubinskaya E.V., Zinovkin R.A., Kiselevsky D.B., Lobysheva N.V., Samuilov V.D. Chitosan-induced programmed cell death in plants // Biochemistry (Mosc.). 2009. Vol. 74. N 9. P. 1035–1043.

13. Liu B., Li J.-F., Ao Y., Qu J., Li Z., Su J., Zhang Y., Liu J., Feng D., Qi K., He Y., Wang J., Wang H.-B. Lysin motif-containing proteins LYP4 and LYP6 play dual roles in peptidoglycan and chitin perception in rice innate immunity // Plant Cell. 2012. Vol. 24. N 8. P. 3406–3419.

14. Hadwiger L.A. Multiple effects of chitosan on plant systems: solid science or hype // Plant Sci. 2013. Vol. 208. P. 42–49.

15. Artemov A.V., Samuilov V.D. Effect of polyelectrolytes on serine proteinase secretion by Bacillus subtilis // FEBS Lett. 1990. Vol. 262. N 1. P. 33–35.

16. Prevette L.E., Mullen D.G., Banaszak Holl Polycation-induced cell membrane permeability does not enhance cellular uptake or expression efficiency of delivered DNA // Mol. Pharm. 2010. Vol. 7. N 3. P. 870–883.

17. Young D.H., Kцhle H., Kauss H. Effect of chitosan on membrane permeability of suspension- cultured Glycine max and Phaseolus vulgaris cells // Plant Physiol. 1982. Vol. 70. N 5. P. 1449– 1454.

18. Kiselevsky D.B., Frolova O.Y., Solovyev A.G., Dorokhov Y.L., Morozov S.Y., Samuilov V.D. Plant cell death caused by fungal, bacterial, and viral elicitors: protective effect of mitochondria-targeted quinones // Biochemistry (Mosc.). 2014. Vol. 79. N 12. P. 1322–1332.

19. Rodi P.M., Bocco Gianello M.D., Corregido M.C., Gennaro A.M. Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane // Biochim. Biophys. Acta. 2014. Vol. 1838. N 3. P. 859–866.

20. Darzynkiewicz Z., Bruno S., Del Bino G., Gorczyca W., Hotz M.A., Lassota P., Traganos F. Features of apoptotic cells measured by flow cytometry // Cytometry. 1992. Vol. 13. N 8. P. 795–808.

21. Luttge U., Higinbotham N. Transport in plants. N.Y.: Springer Verlag, 1979. 468 pp.

22. Iriti M., Sironi M., Gomarasca S., Casazza A.P., Soave C., Faoro F. Cell death-mediated antiviral effect of chitosan in tobacco // Plant Physiol. Biochem. 2006. Vol. 44. N 11–12. P. 893– 900.

23. Zhang X., Li X.H., Ma X., Wang Z.H., Lu S., Guo Y.L. Redox-induced apoptosis of human oocytes in resting follicles in vitro // J. Soc. Gynecol. Investig. 2006. Vol. 13. N 6. P. 451–458.

24. Balhorn R. The protamine family of sperm nuclear proteins // Genome Biol. 2007. Vol. 8. N 9: 227.

25. Vuorimaa-Laukkanen E., Lisitsyna E.S., Ketola T.M., Morin-Pickardat E., Liang H., Hanzlíková M., Yliperttula M. Difference in the core-shell dynamics of polyethyleneimine and poly(l-lysine) DNA polyplexes // Eur. J. Pharm. Sci. 2017. Vol. 103. P. 122–127.