蛋白质N-糖基化在拟南芥生长周期中的变化规律及去糖基化对根发育的影响

蛋白质N-糖基化修饰在植物生长发育中发挥重要作用。为探究蛋白质N-糖基化在拟南芥(Arabidopsis thaliana)整个生长周期中的变化规律以及去N-糖基化对拟南芥生根发育的影响，通过N-糖链酶解和HPLC与MALDI-TOF-MS分析解析了不同生长时期的拟南芥Col-0植株的N-糖链组成(结构和含量)变化。以...

Changes of Protein N-glycosylation in the Growth of Arabidopsis thaliana and Effects of Enzymatic Deglycosylation on Root Development

N-glycosylation of proteins plays an important role in plant growth and development. This study explored the changes in protein N-glycosylation in Arabidopsis thaliana at different growth stages and its role in the root development. N-glycans of Arabidopsis Col-0 plants at different growth stages were enzymatically released with N-glycanase and analyzed by HPLC and MALDI-TOF-MS. In addition, Arabidopsis seedlings were treated with N-glycanase, PNGase Rz, for 8 hours before further cultivation in MS medium for five and ten days. The group treated with BSA solution was used as the negative control and the group treated with sterile deionized water was used as blank control. The changes in the primary root length and N-glycosylation composition of the seedlings were measured after treatment. A total number of 12 N-glycan structures were deduced from Arabidopsis, including 4 high-mannose types and 8 complex types. Throughout the entire period, the content of complex type N-glycan was always higher than that of high-mannose; the complex structures modified with xylose and fucose were the dominant component, among which Man3XylFucGlcNAc2 is the highest. The changes of high-mannose N-glycan were as follows: the content steadily increased from 13.87% (seedling stage) to 19.02% (bolting stage), slightly decreased to 17.98% (flowering stage), and dramatically dropped to 2.36% (longhorn ripening stage), then returned 5.23% (aging stage). After treatment with PNGase Rz at high concentration, a significant inhibition of the primary root’s growth was observed, which could not be recovered after cultivation in MS medium for ten days. However, no statistical differences of root length and growth state were found in the treatment group of low concentration (0.05 mg·mL ^-1), compared with the negative group. N-glycan analysis of seedlings revealed that compared with the control, both treatment groups showed significant changes in the composition of N-glycans. Especially, the total content of high-mannose N-glycans was dramatically lower than that of the blank control group. Meanwhile, these glycoform differences shrank with prolonged time of cultivation and finally disappeared. In conclusion, Arabidopsis thaliana has unique pattern of N-glycosylation during the whole growing period; and glycanase treatment could intermediately alter the N-glycosylation pattern and subsequently inhibit the development of root.