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
收稿日期: 2020-09-29
录用日期: 2020-12-25
网络出版日期: 2020-12-29
基金资助
中央高校基本业务费(KYZ201824);国家自然科学基金No(31871754);国家自然科学基金No(31871793)
Changes of Protein N-glycosylation in the Growth of Arabidopsis thaliana and Effects of Enzymatic Deglycosylation on Root Development
† These authors contributed equally to this paper
Received date: 2020-09-29
Accepted date: 2020-12-25
Online published: 2020-12-29
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BSA溶液为阴性对照, 无菌去离子水为空白对照, 用N-糖酰胺酶(PNGase Rz)溶液处理拟南芥幼苗8小时; 然后继续在MS培养基中培养5天、10天, 测量主根长度并检测N-糖链组成的变化。结果显示, 从拟南芥中解析出12种N-糖链结构, 其中包括4个高甘露糖型和8个复杂型。在拟南芥整个生长周期中, 复杂型N-糖链含量始终高于高甘露糖型, 其中含木糖和岩藻糖的复杂型结构是N-糖链的主要组成, 而Man3XylFucGlcNAc2含量最高。高甘露糖型N-糖链含量由幼苗期的13.87%缓慢上升至抽薹期的19.02%, 盛花期回落至17.98%, 而在长角果成熟期快速下降至最低点2.36%, 衰老期再度小幅回升至5.23%。用高浓度糖酰胺酶液PNGase Rz处理后, 可观察到幼苗主根生长受到显著抑制, 且培养10天后仍然无法恢复正常; 而低浓度酶液处理组与阴性对照组差异不显著, 根长和生长状态基本正常。糖链分析结果显示, 与对照组相比, 高、低浓度酶液处理组的N-糖链组成均发生显著变化, 主要表现为高甘露糖型含量显著低于空白对照组, 同时随生长时间的延长该差异逐渐减小, 最终消失。研究表明, 拟南芥N-糖基化组成随着生长发育发生周期性变化, 且去糖基化酶处理能够瞬时影响拟南芥蛋白质N-糖基化修饰, 进而抑制根的发育。
王婷, 羊欢欢, 赵弘巍, JosefVoglmeir, 刘丽 . 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[J]. 植物学报, 2021 , 56(3) : 262 -274 . DOI: 10.11983/CBB20163
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.
Key words： Arabidopsis thaliana; N-glycan; root development; deglycosylation
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