Unresolved ER stress restricts in vitro plant cell totipotency.

Many plant cells can be induced to regenerate in vitro. We show that successful regeneration during microspore-derived embryo culture relies in part on the ability of embryogenic cells to resolve tissue culture-induced ER stress. During Brassica napus microspore embryogenesis, the immature male gametophyte is induced by a heat stress treatment to develop into a haploid embryo. Different multicellular embryogenic structures develop in response to heat stress, each with a different potential to complete embryo development. The underlying factors that determine the ability of these initially embryogenic structures to successfully complete embryo development are not known. We show that all embryogenic structures exhibit elements of endoplasmic reticulum (ER) stress, like ER expansion and protein-filled ER cisternae, but that the ER stress response is amplified in embryogenic structures with a low potential to complete embryo development. ER stress was amplified even further by treating heat-stressed cultures with trichostatin A, a histone deacetylase inhibitor epidrug that promotes embryogenic cell formation. Pharmacological treatment of microspore-derived embryo cultures with small molecule modulators of ER stress provided further evidence for the role of ER stress in microspore embryo development. Our results suggest that (1) the inability of certain embryogenic structures to resolve their ER stress responses restricts their ability to complete embryo development, and (2) histone deacetylation enhances microspore embryogenesis in B. napus, in part through its activity as an abiotic stress inducer.