Mitochondrial activity changes during somatic embryo development in Abies alba Mill

Somatic embryo development in conifers includes the development of different stages of proliferation, named pro-embryogenic cell masses (PEMs), and subsequent maturation of somatic embryos. Besides to be recognized as a useful method for clonal propagation, somatic embryogenesis can also be used for the study of plant embryogenesis and developmental programmed cell death (PCD). In the present work, different morphological structures of proliferating PEMs and early maturing embryos of Abies alba, respectively, were observed under histological preparations and were recognized to be similar to those previously described by others in the somatic embryo model of Picea abies. Cell death and two parameters of PCD, DNA laddering and cytochrome c release, resulted to decrease passing from the proliferating stage to the maturing embryogenic one. Furthermore, some bioenergetic parameters and mitochondrial activities were analysed during the manifestation of the two events of PCD, linked to the A. alba somatic embryogenesis. The major cellular energetic metabolites (ATP, NAD(P)H and glucose-6-phosphate) increased during maturation, while the major plant mitochondrial activities showed different patterns during the two developmental stages. In particular, mitochondria, isolated from maturing somatic embryos, with respect to proliferating PEMs, showed an increased activity and expression of the alternative oxidase, together with other energy-dissipating mitochondrial activities, such as external NADH dehydrogenase and free fatty-acid mediated uncoupling. Conversely, the mitochondrial cyclosporin A-sensitive K+ ATP channel activity was higher during proliferation step and associated to the first event of PCD. In conclusion, these results show a correlation between mitochondrial activities and the manifestation of PCD during somatic embryo differentiation of A. alba, and suggest the involvement of K+ ATP channel in the cytochrome c release during proliferation. Vice versa, it was shown that the alternative pathways could be activated and act as anti-apoptotic factors during maturation of somatic embryos.