An integrated physiology, cytology, and proteomics analysis reveals a network of sugarcane protoplast responses to enzymolysis.
An integrated physiology, cytology, and proteomics analysis reveals a network of sugarcane protoplast responses to enzymolysis.
复制标题
综合生理学、细胞学和蛋白质组学分析揭示了甘蔗原生质体对酶解反应的网络
DOI：
10.3389/fpls.2022.1066073
复制
发表时间：
2022
影响因子：
5.6
通讯作者：
中科院分区：
文献类型：
--
作者：
原生质体实验系统是功能基因组学和细胞融合育种的有效工具。然而，原生质体对外源激素反应的生理和分子机制尚不清楚，这已成为原生质体再生的主要障碍。本研究采用生理学、细胞学、蛋白质组学和基因表达分析等方法，对甘蔗幼叶和酶解原生质体进行比较。酶消化后，我们获得了活力> 90%的原生质体。同时，原生质体氧化产物丙二醛含量增加，过氧化物酶（POD）、过氧化氢酶（CAT）、酸性过氧化物酶（APX）和O2-等抗氧化酶活性显著降低。细胞学分析结果表明，原生质体细胞膜不同程度穿孔，核活性减弱，核仁结构不明显，微管解聚，形成短棒状结构。本研究利用蛋白质组学方法对酶解过程中原生质体的蛋白质进行了鉴定。GO、KEGG和KOG富集分析表明，丰富的蛋白质主要参与原生质体的生物能代谢、细胞过程、渗透胁迫和氧化还原稳态，从而允许蛋白质的生物合成或降解。RT-qPCR分析表明，DREB、WRKY、MAPK 4和NAC等渗透胁迫抗性基因在原生质体中表达上调，而CyclinD 3、CyclinA、CyclinB、Cdc 2、PSK、CESA和GAUT等关键再生基因在原生质体中表达显著下调。系统聚类和氧化还原蛋白和氧化产物的鉴定表明，这些蛋白参与了动态网络，在响应氧化应激后的氧化。我们的研究结果可以促进一个标准系统的发展，以产生再生原生质体的分子标记和抗体检测的细菌。
The protoplast experimental system eis an effective tool for functional genomics and cell fusion breeding. However, the physiological and molecular mechanisms of protoplast response to enzymolysis are not clear, which has become a major obstacle to protoplast regeneration. Here, we used physiological, cytological, proteomics and gene expression analysis to compare the young leaves of sugarcane and enzymolized protoplasts. After enzymatic digestion, we obtained protoplasts with viability of > 90%. Meanwhile, the content of malondialdehyde, an oxidation product, increased in the protoplasts following enzymolysis, and the activity of antioxidant enzymes, such as peroxidase (POD), catalase (CAT), acid peroxidase (APX), and O2-, significantly decreased. Cytologic analysis results showed that, post 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 several short rod-like structures in protoplasts. In this study, a proteomics approaches was used to identify proteins of protoplasts in response to the enzymatic digestion process. GO, KEGG, and KOG enrichment analyses revealed that the abundant proteins were mainly involved in bioenergetic metabolism, cellular processes, osmotic stress, and redox homeostasis of protoplasts, which allow for protein biosynthesis or degradation. RT-qPCR analysis revealed that the expression of osmotic stress resistance genes, such as DREB, WRKY, MAPK4, and NAC, was upregulated, while that of key regeneration genes, such as CyclinD3, CyclinA, CyclinB, Cdc2, PSK, CESA, and GAUT, was significantly downregulated in the protoplasts. Hierarchical clustering and identification of redox proteins and oxidation products showed that these proteins were involved in dynamic networks in response to oxidative stress after enzymolysis. Our findings can facilitate the development of a standard system to produce regenerated protoplasts using molecular markers and antibody detection of enzymolysis.
登录
查看更多内容
影响因子：
4.9
作者：
Chen, Songbiao;Tao, Lizen;Wang, Guo-Liang
通讯作者：
Wang, Guo-Liang
影响因子：
4.4
作者：
Holmes, Peta;Farquharson, Ryan;Rolfe, Barry G.
通讯作者：
Rolfe, Barry G.
影响因子：
5
作者：
Khatri, Kusum;Rathore, Mangal S.
通讯作者：
Rathore, Mangal S.
DOI：
10.1007/978-1-62703-631-3_31
发表时间：
2014-01-01
期刊：
PLANT PROTEOMICS: METHODS AND PROTOCOLS, 2ND EDITION
影响因子：
--
作者：
Estavillo, Gonzalo M.;Verhertbruggen, Yves;Ito, Jun
通讯作者：
Ito, Jun
影响因子：
2.8
作者：
Meena, Rajendra Prasad;Ghosh, Gourab;Padaria, Jasdeep Chatrath
通讯作者：
Padaria, Jasdeep Chatrath