An Integrated Physiological, Cytology and Proteomics Reveals Network of Sugarcane Protoplasts Responses to Enzymolysis

The protoplast experimental system has been becoming a powerful tool for functional genomics and cell fusion breeding. However, the physiology and molecular mechanism during enzymolysis is not completely understood and has become a major obstacle to protoplast regeneration. Our study used physiological, cytology, iTRAQ (Isobaric Tags for Relative and Absolute Quantification) -based proteomic and RT-PCR analyses to compare the young leaves of sugarcane (ROC22) and protoplasts of more than 90% viability. We found that oxidation product MDA content increased in the protoplasts after enzymolysis and several antioxidant enzymes such as POD, CAT, APX, and O2- content significantly decreased. The cytology results showed that after enzymolysis, the cell membranes were perforated to different degrees, the nuclear activity was weakened, the nucleolus structure was not obvious, and the microtubules depolymerized and formed many short rod-like structures in protoplasts. The proteomic results showed that 1,477 differential proteins were down-regulated and 810 were up-regulated after enzymolysis of sugarcane young leaves. The GO terms, KEGG and KOG enrichment analysis revealed that differentially abundant proteins were mainly involved in bioenergetic metabolism, cellular processes, osmotic stress, and redox homeostasis of protoplasts, which would allow protein biosynthesis or / degradation. The RT-PCR analysis revealed the expression of osmotic stress resistance genes such as DREB, WRKY, MAPK4, and NAC were up-regulated. Meanwhile, the expression of key regeneration genes such as CyclinD3, CyclinA, CyclinB, Cdc2, PSK, CESA and GAUT were significantly down-regulated in the protoplasts. Hierarchical clustering, identification of redox proteins and oxidation products showed that these proteins were involved in dynamic networks in response to oxidative stress after enzymolysis. We used a variety of methods to figure out how young sugarcane leaves react to enzymes.