Microspore embryogenesis is a model system of plant cell reprogramming, totipotency acquisition, stress response and embryogenesis initiation. This in vitro system constitutes an important biotechnological tool for haploid and doubled-haploid plant production, very useful for crop breeding. In this process, microspores (cells that produce pollen grains in planta) are reprogrammed toward embryogenesis by specific stress treatment, but many microspores die after the stress. The occurrence of cell death is a serious limiting problem that greatly reduces microspore embryogenesis yield. In animals, increasing evidence has revealed caspase proteolytic activities as essential executioners of programmed cell death (PCD) processes, however, less is known in plants. Although plant genomes do not contain caspase homologues, caspase-like proteolytic activities have been detected in many plant PCD processes. In the present study, we have analysed caspase 3-like activity and its involvement in stress-induced cell death during initial stages of microspore embryogenesis of Brassica napus. After stress treatment to induce embryogenesis, isolated microspore cultures showed high levels of cell death and caspase 3-like proteolytic activity was induced. Treatments with specific inhibitor of caspase 3-like activity reduced cell death and increased embryogenesis induction efficiency. Our findings indicate the involvement of proteases with caspase 3-like activity in the initiation and/or execution of cell death at early microspore embryogenesis in B. napus, giving new insights into the pathways of stress-induced cell death in plants and opening a new way to improve in vitro embryogenesis efficiency by using chemical modulators of cell death proteases.
1. Maluszynski M, Kasha K, Forster BP, Szarejko I, editors. Doubled haploid production in crop plants: a manual. Springer Science & Business Media; 2013.
2. Ferrie AM, Caswell KL. Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production. Plant Cell, Tissue and Organ Culture (PCTOC). 2011; 104(3): 301-9.
3. Testillano PS. Microspore embryogenesis: targeting the determinant factors of stress-induced cell reprogramming for crop improvement. Journal of Experimental Botany. 2019; 70(11): 2965-78.
4. Germana MA, Lambardi M, editors. In vitro embryogenesis in higher plants. Humana Press; 2016.
5. Bárány I, González-Melendi P, Fadón B, Mitykó J, Risueño MC, Testillano PS. Microspore-derived embryogenesis in pepper (Capsicum annuum L.): subcellular rearrangements through development. Biology of the Cell. 2005; 97(9): 709-22.
6. Prem D, Solís MT, Bárány I, Rodríguez-Sanz H, Risueño MC, Testillano PS. A new microspore embryogenesis system under low temperature which mimics zygotic embryogenesis initials, expresses auxin and efficiently regenerates doubled-haploid plants in Brassica napus. BMC Plant Biology. 2012; 12(1): 127.
7. Shariatpanahi ME, Belogradova K, Hessamvaziri L, Heberle-Bors E, Touraev A. Efficient embryogenesis and regeneration in freshly isolated and cultured wheat (Triticum aestivum L.) microspores without stress pretreatment. Plant Cell Reports. 2006; 25(12): 1294-9.
8. Satpute GK, Long H, Seguí-Simarro JM, Risueño MC, Testillano PS. Cell architecture during gametophytic and embryogenic micro-spore development in Brassica napus L. Acta Physiologiae Plantarum. 2005; 27(4): 665-74.
9. Rodríguez-Serrano M, Bárány I, Prem D, Coronado MJ, Risueño MC, Testillano PS. NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley. Journal of Experimental Botany. 2011; 63(5): 2007-24.
10. Bárány I, Berenguer E, Solís MT, Pérez-Pérez Y, Santamaría ME, Crespo JL, Risueño MC, Díaz I, Testillano PS. Autophagy is activated and involved in cell death with participation of cathepsins during stress-induced microspore embryogenesis in barley. Journal of Experimental Botany. 2018; 69(6): 1387-402.
11. Pérez-Pérez Y, Carneros E, Berenguer E, Solís MT, Bárány I, Pintos B, Gómez-Garay A, Risueño MC, Testillano PS. Pectin de-methylesterification and AGP increase promote cell wall remodeling and are required during somatic embryogenesis of Quercus suber. Frontiers in Plant Science. 2019; 9: 1915.
12. Daneva A, Gao Z, Van Durme M, Nowack MK. Functions and regulation of programmed cell death in plant development. Annual Review of Cell and Developmental Biology. 2016; 32: 441-68.
13. Huysmans M, Lema S, Coll NS, Nowack MK. Dying two deaths — programmed cell death regulation in development and disease. Current Opinion in Plant Biology. 2017; 35: 37-44.
14. Van Doorn WG, Beers EP, Dangl JL, Franklin-Tong VE, Gallois P, Hara-Nishimura I, Jones AM, Kawai-Yamada M, Lam E, Mundy J, Mur LA. Morphological classification of plant cell deaths. Cell Death and Differentiation. 2011; 18(8): 1241.
15. Minina EA, Coll NS, Tuominen H, Bozhkov PV. Metacaspases versus caspases in development and cell fate regulation. Cell Death and Differentiation. 2017; 24(8): 1314.
16. Poręba M, Stróżyk A, Salvesen GS, Drąg M. Caspase substrates and inhibitors. Cold Spring Harbor Perspectives in Biology. 2013; 5(8): a008680.
18. Bozhkov PV, Filonova LH, Suarez MF, Helmersson A, Smertenko AP, Zhivotovsky B, Von Arnold S. VEIDase is a principal caspase-like activity involved in plant programmed cell death and essential for embryonic pattern formation. Cell Death and Differentiation. 2004; 11(2): 175.
19. Gunawardena AH, Greenwood JS, Dengler NG. Programmed cell death remodels lace plant leaf shape during development. The Plant Cell. 2004; 16(1): 60-73.
20. Solís MT, Chakrabarti N, Corredor E, Cortés-Eslava J, Rodríguez-Serrano M, Biggiogera M, Risueño MC, Testillano PS. Epigenetic changes accompany developmental programmed cell death in tapetum cells. Plant and Cell Physiology. 2013; 55(1): 16-29.
21. Van Durme M, Nowack MK. Mechanisms of developmentally controlled cell death in plants. Current Opinion in Plant Biology. 2016; 29: 29-37.
22. Solís MT, Berenguer E, Risueño MC, Testillano PS. BnPME is progressively induced after microspore reprogramming to embryogenesis, correlating with pectin de-esterification and cell differentiation in Brassica napus. BMC Plant Biology. 2016; 16(1): 176.
23. Solís MT, El-Tantawy AA, Cano V, Risueño MC, Testillano PS. 5-azacytidine promotes microspore embryogenesis initiation by decreasing global DNA methylation, but prevents subsequent embryo development in rapeseed and barley. Frontiers in Plant Science. 2015; 6: 472.
24. Berenguer E, Bárány I, Solís MT, Pérez-Pérez Y, Risueño MC, Testillano PS. Inhibition of histone H3K9 methylation by BIX-01294 promotes stress-induced microspore totipotency and enhances embryogenesis initiation. Frontiers in Plant Science. 2017; 8: 1161.
25. Gervais C, Newcomb W, Simmonds DH. Rearrangement of the actin filament and microtubule cytoskeleton during induction of microspore embryogenesis in Brassica napus L. cv. Topas. Proto-plasma. 2000; 213(3-4): 194-202.
26. Rodríguez-Sanz H, Solís MT, López MF, Gómez-Cadenas A, Risueño MC, Testillano PS. Auxin biosynthesis, accumulation, action and transport are involved in stress-induced microspore embryogenesis initiation and progression in Brassica napus. Plant and Cell Physiology. 2015; 56(7): 1401-17.
27. Maraschin SD, Caspers M, Potokina E, Wülfert F, Graner A, Spaink HP, Wang M. cDNA array analysis of stress-induced gene expression in barley androgenesis. Physiologia Plantarum. 2006; 127(4): 535-50.
28. Uren AG, O’Rourke K, Aravind L, Pisabarro MT, Seshagiri S, Koonin EV, Dixit VM. Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma. Molecular Cell. 2000; 6(4): 961-7.
29. Suarez MF, Filonova LH, Smertenko A, Savenkov EI, Clapham DH, von Arnold S, Zhivotovsky B, Bozhkov PV. Metacaspase-dependent programmed cell death is essential for plant embryogenesis. Current Biology. 2004; 14(9): R339-40.
30. Bozhkov PV, Suarez MF, Filonova LH, Daniel G, Zamyatnin AA, Rodriguez-Nieto S, Zhivotovsky B, Smertenko A. Cysteine protease mcII-Pa executes programmed cell death during plant embryogenesis. Proceedings of the National Academy of Sciences. 2005; 102(40): 14463-8.
31. Bollhöner B, Zhang B, Stael S, Denancé N, Overmyer K, Goffner D, Van Breusegem F, Tuominen H. Post mortem function of AtMC9 in xylem vessel elements. New Phytologist. 2013; 200(2): 498-510.
32. Vercammen D, Van De Cotte B, De Jaeger G, Eeckhout D, Casteels P, Vandepoele K, Vandenberghe I, Van Beeumen J, Inzé D, Van Breusegem F. Type II metacaspases Atmc4 and Atmc9 of Arabidopsis thaliana cleave substrates after arginine and lysine. Journal of Biological Chemistry. 2004; 279(44): 45329-36.
33. Tsiatsiani L, Van Breusegem F, Gallois P, Zavialov A, Lam E, Bozhkov PV. Metacaspases. Cell Death and Differentiation. 2011; 18(8): 1279.
34. Danon A, Rotari VI, Gordon A, Mailhac N, Gallois P. Ultraviolet-C overexposure induces programmed cell death in Arabidopsis, which is mediated by caspase-like activities and which can be suppressed by caspase inhibitors, p35 and Defender against Apoptotic Death. Journal of Biological Chemistry. 2004; 279(1): 779-87.
35. Ge Y, Cai YM, Bonneau L, Rotari V, Danon A, McKenzie EA, McLellan H, Mach L, Gallois P. Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in Arabidopsis. Cell Death and Differentiation. 2016; 23(9): 1493.
36. Bonneau L, Ge Y, Drury GE, Gallois P. What happened to plant caspases?. Journal of Experimental Botany. 2008; 59(3): 491-9.
37. Hatsugai N, Iwasaki S, Tamura K, Kondo M, Fuji K, Ogasawara K, Nishimura M, Hara-Nishimura I. A novel membrane fusion-mediated plant immunity against bacterial pathogens. Genes & Development. 2009; 23(21): 2496-506.
38. Balakireva A, Zamyatnin A. Indispensable role of proteases in plant innate immunity. International Journal of Molecular Sciences. 2018; 19(2): 629.
39. Reape TJ, McCabe PF. Apoptotic-like regulation of programmed cell death in plants. Apoptosis. 2010; 15(3): 249-56.