冷冻电镜技术在分子植物学研究中的应用

doi:10.13560/j.cnki.biotech.bull.1985.2021-1562

摘要/Abstract

摘要：

植物体的各项生理活动依赖于分子水平上多种植物蛋白质/蛋白质复合体的相互作用和动态变化,了解这些蛋白质/蛋白质复合体的结构和功能对于研究相关植物生理活动的分子机理至关重要。得益于最近的技术进步——包括直接电子探测器的发展和先进的图像处理算法,冷冻电镜技术已经逐步发展成为研究蛋白质/蛋白质复合体的重要技术手段,这也为深入理解植物生理活动分子机理提供了结构生物学研究利器。目前,冷冻电镜技术在分子植物学研究领域的应用仍处于早期,对于一些重要蛋白质复合体的结构功能研究还不够深入。本文在对冷冻电镜技术发展历史进行简要回顾的基础上,对近年来人们利用冷冻电镜技术在植物光合作用、胁迫响应等方面进行的分子机理研究进行了梳理,旨在为加强分子植物学和冷冻电镜技术两个研究领域的合作提供有益参考。

关键词: 冷冻电镜, 分子植物学, 光合作用, 胁迫响应

Abstract:

Various physiological activities of plants depend on the interaction and dynamic changes of many plant proteins/protein complexes at the molecular level. Understanding their structure and function is very important to study the molecular mechanism of plant physiological activities. With the recent technology advances,including the development of direct electron detectors and sophisticated image processing algorithms,cryo-electron microscopy（cryo-EM）has gradually become an important technique for studying proteins/protein complexes,which provides an efficient research tool to study the molecular mechanism of plant physiological activities. At present,the application of cryo-EM in molecular botany is still in the early stage,and researches on the structure and function of important plant protein complexes are not sufficient. Starting from a brief introduction of the history of cryo-EM development,this review summarizes the recent applications of cryo-EM in studying photosynthesis and plant stress response,aiming to promote the cooperation in the study field of molecular botany and cryo-EM.

Key words: cryo-EM, molecular botany, photosynthesis, stress response

殷国良, 孙文浩, 庞效云, 孙飞. 冷冻电镜技术在分子植物学研究中的应用[J]. 生物技术通报, 2022, 38(1): 15-32.

YIN Guo-liang, SUN Wen-hao, PANG Xiao-yun, SUN Fei. Application of cryo-Electron Microscopy in Molecular Botany Research[J]. Biotechnology Bulletin, 2022, 38(1): 15-32.

图/表 11

图1 光合作用相关蛋白结构解析采用的手段

Fig. 1 Methods used for structural analysis of photosyn-thesis-related proteins

图2 光系统I的冷冻电镜结构 A：豌豆PSI-FD复合体;B：玉米PSI-LHCI-LHCII复合体;C：杜氏盐藻PSI复合体;D：红藻PSI复合体;E：莱茵衣藻PSI复合体;F：纤细角毛藻PSI-FCP复合体;G：小立碗藓PSI复合体;H：嗜热蓝藻PSI-isiA复合体

Fig. 2 cryo-EM structure of photosystem I A：PSI-FD complex of Pisum sativum;B：PSI-LHCI-LHCII complex of Zea mays;C：PSI complex of Dunaliella salina;D：PSI complex of Cyanidioschyzon merolae;E：PSI complex of Chlamydomonas reinhardtii;F：PSI-FCP complex of Chaeto-ceros gracilis;G：PSI complex of Physcomitrium patens;H：PSI-isiA complex of Thermosynechococcus vulcanus

图3 光系统II的冷冻电镜结构 A：菠菜PSII-LHCII复合体;B：豌豆C2S2型PSII-LHCII复合体;C：拟南芥PSII复合体;D：纤细角毛藻C2S2型PSII-FCPII复合体;E：莱茵衣藻C2S2型PSII-LHCII复合体

Fig.3 Cryo-EM structure of photosystem II A：PSII-LHCII complex of Spinacia oleracea. B：PSII-LHCII complex of Pisum sativum. C：PSII complex of Arabidopsis thaliana. D：C2S2 type PSII-FCPII complex of Chaetoceros gracilis. E：C2S2 type PSII-LHCII complex of Chlamydomonas reinhardtii

图4 菠菜细胞色素b6f复合体冷冻电镜结构

Fig. 4 cryo-EM structure of spinach cytochrome b6f complex

图5 藻胆体的冷冻电镜结构[80]

Fig. 5 cryo-EM structure of phycobilisome[80]

图6 冷冻电镜技术在植物光合作用研究领域的应用

Fig. 6 Application of cryo-EM in the field of plant photos-ynthesis research

图7 植物NLR蛋白ZAR1由静息状态进入激活状态并寡聚化形成抗病小体的构象变化

Fig. 7 Conformational changes of plant NLR protein ZAR1 from inactive state to active state and then oligomerized to form resistsome

图8 RPP1-ATR1与ROQ1-XopQ分别寡聚化组装形成抗病小体[103]

Fig. 8 Resistosomes oligomerized respectively from RPP1-ATR1 and ROQ1-XopQ

图9 OSCA1蛋白的冷冻电镜结构及其激活构象 AtOSCA1.1（A）、AtOSCA3.1（B）的冷冻电镜结构比对;（C）AtOSCA1.1构象变化以允许Ca2+进入

Fig. 9 cryo-EM structure and activated conformation of OSCA1 Cryo-EM structure of AtOSCA1.1（A）,AtOSCA3.1（B）;（C）Conformational changes of AtOSCA1.1 to allow Ca2 + entry

图10 AtOSCA1.2单体在多个方向的构象

Fig. 10 Conformations of AtOSCA1.2 monomer in mult-iple directions

表1 冷冻电镜技术解析获得的植物相关重要生物大分子结构

Table 1 Structure of important plant-related biological macromolecules solved by cryo-EM

	Name	Organism（s）	Resolution	Year	PDB	Reference
Photosynthesis	PSII-LHCII supercomplex	Spinacia oleracea	3.20 Å	2016	3JCU	[71]
	M-LHCII and CP24 complexes	Pisum sativum	3.50 Å	2017	5XNO	[72]
	C2S2M2N2-type PSII-LHCII	Pisum sativum	2.70 Å	2017	5XNL	[72]
	Phycobilisome	Griffithsia pacifica	3.50 Å	2017	5Y6P	[78]
	PSI-LHCR	Cyanidioschyzon merolae strain 10D	3.82 Å	2018	5ZGH	[63]
	Photosystem I supercomplex with light-harvesting complexes I and II	Zea mays,Zea mays subsp. mays	3.30 Å	2018	5ZJI	[68]
	PSII-FCP supercomplex	Chaetoceros gracilis	3.02 Å	2019	6JLU	[74]
	C2S2M2L2-type PSII-LHCII supercomplex	Chlamydomonas reinhardtii	3.40 Å	2019	6KAD	[75]
	C2S2-type PSII-LHCII supercomplex	Chlamydomonas reinhardtii	2.70 Å	2019	6KAC	[75]
	Photosystem I	Chlamydomonas reinhardtii	3.30 Å	2019	6IJO	[65]
	Cytochrome b6f complex	Spinacia oleracea	3.58 Å	2019	6RQF	[76]
	Phycobilisome	Porphyridium purpureum	2.80 Å	2019	6KGX	[79]
	NDH	Thermosynechococcus vestitus BP-1	3.10 Å	2019	6NBY	[77]
	Photosystem I	Dunaliella salina	3.20 Å	2020	6RHZ	[66]
	PSI-FCPI supercomplex	Chaetoceros gracilis	2.40 Å	2020	6L4U	[67]
	Photosystem I complex	Synechocystis sp. PCC 6803 substr. Kazusa	3.10 Å	2020	6UZV	[62]
	Fd-NDH-1L complex	Thermosynechococcus elongatus BP-1	3.20 Å	2020	6L7O	[70]
	NDH-1LdelV complex	Thermosynechococcus elongatus BP-1	3.60 Å	2020	6L7P	[70]
	PSI-NDH supercomplex	Hordeum vulgare subsp. spontaneum	4.50 Å	2021	7F9O	[69]
	Chloroplast NDH complex	Hordeum vulgare subsp. spontaneum	3.70 Å	2021	7EU3	[69]
	PSI-LHCI-Lhca5 supercomplex	Hordeum vulgare subsp. spontaneum	3.40 Å	2021	7EW6	[69]
	PSI-LHCI-Lhca6 supercomplex	Hordeum vulgare subsp. spontaneum	3.88 Å	2021	7EWK	[69]
Immunity	NLR complex	Arabidopsis thaliana	3.70 Å	2019	6J5W	[99]
	NLR RPP1 LRR-ID domain in complex with ATR1	Hyaloperonospora arabidopsidis Emoy2,Arabidopsis thaliana	3.16 Å	2020	7CRB	[102]
	Activated Roq1 resistosome	Nicotiana benthamiana,Xanthomonas euvesicatoria	3.80 Å	2020	7JLU	[103]
Transportation	atOSCA3.1 channel	Arabidopsis thaliana	4.80 Å	2018	5Z1F	[112]
	atOSCA1.1 channel	Arabidopsis thaliana	3.52 Å	2018	6JPF	[112]
	Activated ion channel OSCA1.2	Arabidopsis thaliana	3.10 Å	2018	6MGV	[113]
	Cation channel	Arabidopsis thaliana	3.68 Å	2018	6IJZ	[115]
	Ion channel OSCA1.2	Oryza sativa	4.90 Å	2019	6OCE	[116]
	MSL1	Arabidopsis thaliana	3.06 Å	2020	6VXM	[118]

表1 冷冻电镜技术解析获得的植物相关重要生物大分子结构

Table 1 Structure of important plant-related biological macromolecules solved by cryo-EM

	Name	Organism（s）	Resolution	Year	PDB	Reference
Photosynthesis	PSII-LHCII supercomplex	Spinacia oleracea	3.20 Å	2016	3JCU	[71]
	M-LHCII and CP24 complexes	Pisum sativum	3.50 Å	2017	5XNO	[72]
	C2S2M2N2-type PSII-LHCII	Pisum sativum	2.70 Å	2017	5XNL	[72]
	Phycobilisome	Griffithsia pacifica	3.50 Å	2017	5Y6P	[78]
	PSI-LHCR	Cyanidioschyzon merolae strain 10D	3.82 Å	2018	5ZGH	[63]
	Photosystem I supercomplex with light-harvesting complexes I and II	Zea mays,Zea mays subsp. mays	3.30 Å	2018	5ZJI	[68]
	PSII-FCP supercomplex	Chaetoceros gracilis	3.02 Å	2019	6JLU	[74]
	C2S2M2L2-type PSII-LHCII supercomplex	Chlamydomonas reinhardtii	3.40 Å	2019	6KAD	[75]
	C2S2-type PSII-LHCII supercomplex	Chlamydomonas reinhardtii	2.70 Å	2019	6KAC	[75]
	Photosystem I	Chlamydomonas reinhardtii	3.30 Å	2019	6IJO	[65]
	Cytochrome b6f complex	Spinacia oleracea	3.58 Å	2019	6RQF	[76]
	Phycobilisome	Porphyridium purpureum	2.80 Å	2019	6KGX	[79]
	NDH	Thermosynechococcus vestitus BP-1	3.10 Å	2019	6NBY	[77]
	Photosystem I	Dunaliella salina	3.20 Å	2020	6RHZ	[66]
	PSI-FCPI supercomplex	Chaetoceros gracilis	2.40 Å	2020	6L4U	[67]
	Photosystem I complex	Synechocystis sp. PCC 6803 substr. Kazusa	3.10 Å	2020	6UZV	[62]
	Fd-NDH-1L complex	Thermosynechococcus elongatus BP-1	3.20 Å	2020	6L7O	[70]
	NDH-1LdelV complex	Thermosynechococcus elongatus BP-1	3.60 Å	2020	6L7P	[70]
	PSI-NDH supercomplex	Hordeum vulgare subsp. spontaneum	4.50 Å	2021	7F9O	[69]
	Chloroplast NDH complex	Hordeum vulgare subsp. spontaneum	3.70 Å	2021	7EU3	[69]
	PSI-LHCI-Lhca5 supercomplex	Hordeum vulgare subsp. spontaneum	3.40 Å	2021	7EW6	[69]
	PSI-LHCI-Lhca6 supercomplex	Hordeum vulgare subsp. spontaneum	3.88 Å	2021	7EWK	[69]
Immunity	NLR complex	Arabidopsis thaliana	3.70 Å	2019	6J5W	[99]
	NLR RPP1 LRR-ID domain in complex with ATR1	Hyaloperonospora arabidopsidis Emoy2,Arabidopsis thaliana	3.16 Å	2020	7CRB	[102]
	Activated Roq1 resistosome	Nicotiana benthamiana,Xanthomonas euvesicatoria	3.80 Å	2020	7JLU	[103]
Transportation	atOSCA3.1 channel	Arabidopsis thaliana	4.80 Å	2018	5Z1F	[112]
	atOSCA1.1 channel	Arabidopsis thaliana	3.52 Å	2018	6JPF	[112]
	Activated ion channel OSCA1.2	Arabidopsis thaliana	3.10 Å	2018	6MGV	[113]
	Cation channel	Arabidopsis thaliana	3.68 Å	2018	6IJZ	[115]
	Ion channel OSCA1.2	Oryza sativa	4.90 Å	2019	6OCE	[116]
	MSL1	Arabidopsis thaliana	3.06 Å	2020	6VXM	[118]

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