Glyphosate treatment mediates the accumulation of small discrete 5^′ and 3^′-terminal fragments of 18S rRNA in plant cells

Under many kinds of stress, eukaryotic cells rapidly decrease the overall translation level of the majority of mRNAs. However, some molecular mechanisms of protein synthesis inhibition like phosphorylation of eukaryotic elongation factor 2 (eEF2), which are known to be functional in animals and yeast, are not implemented in plants. We suggest that there is an alternative mechanism for the inhibition of protein synthesis in plant cells and possibly, in other eukaryotes, which is based on the discrete fragmentation of 18S rRNA molecules within small ribosomal subunits. We identified four stressinduced small RNAs, which are 5’and 3’-terminal fragments of 18S rRNA. In the present work, we studied the induction of 18S rRNA discrete fragmentation and phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) in germinated wheat embryos in the presence of glyphosate, which imitates the condition of amino acid starvation. Using northern and western blotting, we have shown that stress-induced 18S rRNA fragments started to accumulate in wheat embryos at glyphosate concentrations that did not evoke eIF2α phosphorylation. It was also found that cleavage of 18S rRNA near the 5’-terminus began much earlier than eIF2α phosphorylation, which became noticeable only at higher concentration (500 μM) of glyphosate. This result suggests that discrete fragmentation of 18S rRNA may constitute a regulatory mechanism of mRNA translation in response to stress and may occur in plant cells in parallel with and independently of eIF2α phosphorylation. The identified small 5’and 3’-terminal fragments of 18S rRNA that accumulate during various stresses may serve as stress resistance markers in the breeding of economically important plant crops.

1. Altschuler M., Mascarenhas J.P. Heat shock proteins and effects of heat shock in plants. Plant Mol. Biol. 1982;1(2):103­115. DOI:10.1007/BF00024974.

2. Baird Th.D., Wek R.C. Eukaryotic initiation factor 2 phosphorylation and translational control in metabolism. Adv. Nutr. 2012;3:307­321. DOI:10.3945/an.112.002113.

3. Ballard D.J., Peng H.­Y., Das J.K., Kumar A., Wang L., Ren Y., Xiong X., Ren X., Yang J.­M., Song J. Insights into the pathologic roles and regulation of eukaryotic elongation factor-2 kinase. Front. Mol. Biosci. 2021;8:839. DOI:10.3389/fmolb.2021.727863.

4. Browning K.S., Bailey-Serres J. Mechanism of cytoplasmic mRNA translation. Arabidopsis Book. 2015;13:e0176.

5. Chen Z., Sun Y., Yang X., Wu Z., Guo K., Niu X., Wang Q., Ruan J., Bu W., Gao S. Two featured series of rRNA-derived RNA fragments (rRFs) constitute a novel class of small RNAs. PLoS One. 2017;12: e0176458. DOI:10.1371/journal.pone.0176458.

6. Echevarria­Zomeno S., Yanguez E., Fernandez­Bautista N., Castro­ Sanz A.B., Ferrando A., Castellano M.M. Regulation of translation initiation under biotic and abiotic stresses. Int. J. Molec. Sci. 2013; 14:4670­4683. DOI:10.3390/ijms14034670.

7. Endo Y. Mechanism of action of ricin and related toxins on the inactivation of eukaryotic ribosomes. Cancer Res. Treat. 1988;37:75­89. DOI:10.1007/978-1-4613-1083-9_5.

8. Gallie D.R., Le H., Caldwell C., Tanguay R., Hoang N.X., Brow ning K.S. The phosphorylation state of translation initiation factors is regulated developmentally and following heat shock in wheat. J. Biol. Chem. 1997;272:1046­1053. DOI:10.1074/jbc.272.2.1046.

9. Gallie D.R., Le H., Caldwell C., Browning K.S. Analysis of translation elongation factors from wheat during development and following heat shock. Biochem. Biophys. Res. Comm. 1998;245:295­300. DOI:10.1006/bbrc.1998.8427.

10. Henras A.K., Plisson-Chastang C., O’Donohue M.F., Chakraborty A., Gleizes P.E. An overview of pre-ribosomal RNA processing in eukaryotes. Wiley Interdiscip. Rev. RNA. 2015;6(2):225­242. DOI:10.1002/wrna.1269.

11. Hernandez G., Altmann M., Lasko P. Origins and evolution of the mechanisms regulating translation initiation in eukaryotes. Trends Biochem. Sci. 2010;35(2):63­73. DOI:10.1016/j.tibs.2009.10.009.

12. Immanuel T.M., Greenwood D.R., MacDiarmid R.M. A critical review of translation initiation factor eIF2α kinases in plants – regulating protein synthesis during stress. Funct. Plant Biol. 2012;39(9):717­ 735. DOI:10.1071/FP12116.

13. Kamauchi S., Nakatani H., Nakano C., Urade R. Gene expression in response to endoplasmic reticulum stress in Arabidopsis thaliana. FEBS J. 2005;272(13):3461­3476. DOI:10.1111/j.1742­4658.2005.04770.x.

14. Kast A., Klassen R., Meinhardt F. rRNA fragmentation induced by a yeast killer toxin. Mol. Microbiol. 2014;91(3):606­617. DOI:10.1111/mmi.12481.

15. Knutsen J.H., Rødland G.E., Bøe C.A., Håland T.W., Sunnerhagen P., Grallert B., Boye E. Stress-induced inhibition of translation independently of eIF2α phosphorylation. J. Cell Sci. 2015;128(23): 4420-4427. DOI:10.1242/jcs.176545.

16. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680­685. DOI:10.1038/227680a0.

17. Lageix S., Lanet E., Pouch-Pelissier M.N., Espagnol M.C., Robaglia C., Deragon J.M., Pelissier T. Arabidopsis eIF2α kinase GCN2 is essential for growth in stress conditions and is activated by wounding. BMC Plant Biol. 2008;8:134. DOI:10.1186/1471­2229­8­134.

18. Padgette S.R., Kolacz K.H., Delannay X., Re D.B., LaVallee B.J., Tinius C.N., Rhodes W.K., Otero Y.I., Barry G.F., Eichholtz D.A., Pesch­ ke V.M., Nida D.L., Taylor N.B., Kishore G.M. Development, identification and characterization of a Glyphosate­tolerant soybean line. Crop Sci. 1995;35(5):1451­1461. DOI:10.3389/fpls.2016.01009.

19. Ruberti C., Kim S.J., Stefano G., Brandizzi F. Unfolded protein response in plants: one master, many questions. Curr. Opin. Plant Biol. 2015;27:59­66. DOI:10.1016/j.pbi.2015.05.016.

20. Shaikhin S.M., Smailov S.K., Lee A.V., Kozhanov E.V., Iskakov B.K. Interaction of wheat germ translation initiation factor 2 with GDP and GTP. Biochimie. 1992;74(5):447­454. DOI:10.1016/0300­9084(92)90085-s.

21. Smailov S.K., Lee A.V., Iskakov B.K. Study of phosphorylation of translation elongation factor 2 (EF-2) from wheat germ. FEBS Lett. 1993;321(2­3):219­223. DOI:10.1016/0014­5793(93)80112­8.

22. Zhang Y., Wang Y., Kanyuka K., Parry M.A., Powers S.J., Halford N.G. GCN2-dependent phosphorylation of eukaryotic translation initiation factor-2alpha in Arabidopsis. J. Exp. Bot. 2008;59(11):3131­ 3141. DOI:10.1093/jxb/ern169.

23. Zhanybekova S.S., Polimbetova N.S., Nakisbekov N.O., Iskakov B.K. Detection of a new small RNA, induced by heat shock, in wheat seed ribosomes. Biochemistry (Moscow). 1996;61:862­870.

24. Zhigailov A.V., Alexandrova A.M., Nizkorodova A.S., Stanbekova G.E., Kryldakov R.V., Karpova O.V., Polimbetova N.S., Halford N.G., Iskakov B.K. Evidence that Phosphorylation of the α­subunit of eIF2 does not essentially inhibit mRNA translation in wheat germ cell-free system. Front. Plant Sci. 2020;11:936. DOI:10.3389/fpls.2020.00936.

25. Zhigailov A.V., Polimbetova N.S., Borankul R.I., Iskakov B.K. Investigation of discrete fragmentation of 18S rRNA within 40S ribosomal subparticles of plant cells. Vestnik KazNU. Biological Series. 2013;2:81­87. (in Russian)

26. Zhigailov A.V., Polimbetova N.S., Doshchanov Kh.I., Iskakov B.K. Detection in plant cells of a new 75-nucleotide cytoplasmic RNA corresponding to the 5′­terminal fragment of 18S RNA. Vestnik KazNU. Biological and Medical Series. 2014;1:191­194. (in Russian)

27. Yu Ch.­Y., Cho Y., Sharma O., Kanehara K. What’s unique? The unfolded protein response in plants. J. Exp. Botany. 2021:erab513. DOI:10.1093/jxb/erab513.