Combined Transcriptome and Proteome Analysis Identifies Pathways and Markers Associated with the Establishment of Rapeseed Microspore-Derived

Microspore-derivedembryo(MDE)culturesareusedasamodelsystemtostudyplantcelltotipotencyandasaninvitrosystemto study embryo development. We characterized and comparedthe transcriptome and proteome of rapeseed (Brassica napus) MDEs from the few-celled stage to the globular/heart stage using two MDE culture systems: conventional cultures in which MDEs initially develop as unorganized clusters that usually lack a suspensor, and a novel suspensor-bearing embryo culture system in which the embryo proper originates from the distal cell of a suspensor-like structure and undergoes the same ordered cell divisions as the zygotic embryo. Improved histodifferentiationofsuspensor-bearingMDEssuggests anew rolefor the suspensor indrivingembryocellidentityandpatterning.AnMDEculturecDNAarrayandtwo-dimensionalgelelectrophoresisandprotein sequencing were used to compile global and specific expression profiles for the two types of MDE cultures. Analysis of the identities of 220 candidate embryo markers, as well as the identities of 32 sequenced embryo up-regulated protein spots, indicate general roles for protein synthesis, glycolysis, and ascorbate metabolism in the establishment of MDE development. A collection of135robustmarkersforthetransitiontoMDEdevelopmentwasidentified,anumberofwhichmaybecoregulatedatthegeneand protein expression level. Comparison of the expression profiles of preglobular-stage conventional MDEs and suspensor-bearing MDEs identified genes whose differential expression may reflect improved histodifferentiation of suspensor-bearing embryos. This collection of early embryo-expressed genes and proteins serves as a starting point for future marker development and gene function studies aimed at understanding the molecular regulation of cell totipotency and early embryo development in plants. Microspore embryogenesis describes the process in which the immature male gametophyte is induced to