Integrated flow cytometric and proteomics analyses reveal the regulatory network underlying sugarcane protoplast responses to fusion.

BACKGROUND:Somatic cell fusion is a process that transfers cytoplasmic and nuclear genes to create new germplasm resources. But our limited understanding of the physiological and molecular mechanisms that shape protoplast responses to fusion. METHOD:We employed flow cytometry, cytology, proteomics, and gene expression analysis to examine the sugarcane (Saccharum spp.) protoplast fusion. RESULTS:Flow cytometry analysis revealed the fusion rate of protoplasts was 1.95%, the FSC value and SSC of heterozygous cells was 1.17-1.47 times higher than that of protoplasts. The protoplasts viability decreased and the MDA increased after fusion. During fusion, the cell membranes were perforated to different degrees, nuclear activity was weakened, while microtubules depolymerized and formed several short rod like structures in the protoplasts. The most abundant proteins during fusion were mainly involved in RNA processing and modification, cell cycle control, cell division, chromosome partition, nuclear structure, extracellular structures, and nucleotide transport and metabolism. Moreover, the expression of key regeneration genes, such as WUS, GAUT, CESA, PSK, Aux/IAA, Cdc2, Cyclin D3, Cyclin A, and Cyclin B, was significantly altered following fusion. PURPOSE AND SIGNIFICANCE:Overall, our findings provide a theoretical basis that increases our knowledge of the mechanisms underlying protoplast fusion.