Running Head: Metacaspases and autophagy in microspore embryogenesis

14 p.-7 fig.Microspore embryogenesis is a biotechnological process that allows to rapidly obtain doubled haploid plants for breeding programs. The process is initiated by the application of stress treatment which reprograms microspores to embark on embryonic development. Typically, a part of the microspores undergoes cell death that reduces the efficiency of the process. Metacaspases (MCAs), a phylogenetically broad group of cysteine proteases, and autophagy, the major catabolic process in eukaryotes, are critical regulators of the balance between cell death and survival in various organisms. In this study we analyzed the role of MCAs and autophagy in cell death during stress-induced microspore embryogenesis in Brassica napus. We demonstrate that this cell death is accompanied by transcriptional upregulation of three BnMCA genes (BnMCA-Ia, BnMCA-IIa and BnMCA-IIi), increase in MCA proteolytic activity, and activation of autophagy. Accordingly, inhibition of autophagy and MCA activity, either individually or in combination, suppressed cell death and increased the number of proembryos, indicating that both components play a pro-cell death role and account for decreased efficiency of early embryonic development. Therefore, MCAs and/or autophagy can be used as new biotechnological targets to improve in vitro embryogenesis in Brassica species and doubled-haploid plant production in crop breeding and propagation programmes.The work was supported by the Spanish National Agency of Research (Agencia Estatal de Investigación, AEI) and European Regional Development Fund (ERDF/FEDER) [grant AGL2017- 82447-R to PST], and by the Swedish Foundation for Strategic Research and the research program “Crops for the future” [grants to PVB].Peer reviewe