Welcome to Acta Agronomica Sinica，

Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (9): 2390-2399.doi: 10.3724/SP.J.1006.2022.11070

• RESEARCH NOTES • Previous Articles     Next Articles

Preparation of highly specific wheat ATG8 antibody and its application in the detection of autophagy

LI Yong-Bo1(), CUI De-Zhou1, HUANG Chen1, SUI Xin-Xia1, FAN Qing-Qi1,*(), CHU Xiu-Sheng1,2,*   

  1. 1. Crop Research Institute, Shandong Academy of Agricultural Sciences / Key Laboratory of Wheat Biology and Genetic improvement in North Huang-Huai River Valley, Ministry of Agriculture and Rural Affairs / National Engineering Laboratory for Wheat and Maize, Jinan 250100, Shandong, China
    2. School of Life Science, Shandong Normal University, Jinan 250000, Shandong, China
  • Received:2021-08-06 Accepted:2021-11-30 Online:2022-09-12 Published:2022-07-15
  • Contact: FAN Qing-Qi,CHU Xiu-Sheng E-mail:lyb920327@sina.com;xschu2007@sina.cn
  • Supported by:
    Agricultural Variety Improvement Project of Shandong Province(2019LZGC01702);National Natural Science Foundation of China (Youth Project)(32001542);Shandong Natural Science Foundation (Youth Project)(ZR2020QC114);“Key Crop Breeding Technology”(CXGC2021A09)

RichHTML402

9

PDF

147

Abstract:

Autophagy is an evolutionarily highly conserved lysosomal/vacuolar degradation pathway, which is of great significance for the body to adapt to various biotic or abiotic stresses and maintain its normal growth and development. Autophagy-related protein 8 (ATG8) is the gold standard for detecting autophagy. At present, the lack of specific wheat ATG8 antibody has led to slow progress in the research of wheat autophagy. In this study, an 18-amino acid polypeptide of wheat ATG8 protein sequence was artificially synthesized as an antigen to immunize rabbits, and a highly specific wheat ATG8 polyclonal antibody was successfully obtained. The antibody can not only recognize the ATG8 protein bands in roots, leaves, and seeds of wheat, but also can detect autophagy structures in roots and leaves, and autophagy structures in wheat grains were detected for the first time. Besides eight typical types of ATG8a-h antibody found in wheat, a typical new ATG8 protein was detected by wheat ATG8 antibody developed in this study, These results provide the best methodological basis for further study on the regulation mechanism of plant autophagy and excavation of excellent genes.

Key words: wheat, autophagy, polyclonal antibody, autophagy-related protein 8

Fig. 1

Titer analysis of ATG8 antiserum"

Fig. 2

Specific detection of ATG8 antibody The concentration of SDS gel is 12.5%."

Table 1

Identification of ATG8 protein by mass spectrometry"

起始
Start		 序列
Sequence		 结束
End		 蛋白鉴定
Protein ID		 名称
Name
4 SSFKLEHPLER 14 U3Q019 Autophagy-related protein 8
4 TCFKTEHPLER 14 Q7XY24 Autophagy-related protein 8
24 EKYSDRIPVIVEK 36 U3Q019 Autophagy-related protein 8
24 EKYADRIPVIVEK 36 Q7XY24 Autophagy-related protein 8
37 ADKSDVPEIDKKK 49 Q7XY24 Autophagy-related protein 8
37 AGKSDIPDIDK 47 U3Q019 Autophagy-related protein 8
49 KYLVPADLTVGQFVYVVR 66 U3Q019 Autophagy-related protein 8
69 IKLSAEKAIFIFVK 82 U3Q019 Autophagy-related protein 8
83 NTLPPTAALMSAIYEENKDEDGFLYMTYSGENTFG 117 U3Q019 Autophagy-related protein 8

Fig. 3

Western blot detection of ATG8 protein α-Tubulin is used as an internal control. The concentration of SDS gel is 15%; 3-MA: 3-MA+PEG-8000; the concentration is 10 mmol L-1; the treatment time is 48 h; the total protein load is 1 mg ; the error bar is the mean ± SD of three experimental data. Different letters represent significant differences."

Fig. 4

Detection of autophagy structures in wheat root cells The white arrow indicates autophagy structures, and the scale bar: 500 μm. The same parts of the roots of different treatments were selected, placed under a 10×10 microscope, and the number of autophagic structures in each field of view was statistically analyzed by ANOVA software. Different letters represent significant differences. The error is the mean ± SD of three experimental data."

Fig. 5

Statistical analysis of autophagy structures in wheat root cells The same parts of the roots of different treatments were selected, placed under a 10×10 microscope, and the number of autophagic structures in each field of view was statistically analyzed by ANOVA software. Different letters represent significant differences. The error is the mean ± SD of three experimental data."

Fig. 6

Detection of autophagy structures in wheat leaf cells The white arrow indicates autophagy structures, and the scale bar: 50 μm. The same parts of the leaves of different treatments were selected, and placed under a 10×20 microscope, and the number of autophagic structures in each field of view was statistically analyzed by ANOVA software. Different letters represent significant differences. The error is the mean ± SD of three experimental data."

Fig. 7

Statistical analysis of autophagy structures in wheat leaf cells The same parts of the leaves of different treatments were selected, and placed under a 10×20 microscope, and the number of autophagic structures in each field of view was statistically analyzed by ANOVA software. Different letters represent significant differences. The error is the mean ± SD of three experimental data."

Fig. 8

Detection of autophagy structures in wheat grains The short white arrow indicates autophagy structures in endosperm; the short yellow arrow indicates autophagy structures in pericarp; the long white arrows indicate the different structures of the grain, and the scale bar: 200 μm. Al: aleurone layer; En: endosperm; Pe: pericarp."

Fig. 9

Different types of wheat ATG8 protein expression, purification, and antibody recognition The concentration of SDS gel is 15%."

Fig. 10

Phylogenetic tree analysis of homology The red rectangular area is the new ATG8 protein, ATG8j; ATG8a (AGW81789.1), ATG8b (AGW81790.1), ATG8c (AGW81791.1), ATG8d (AGW81792.1), ATG8e (AGW81793.1), ATG8f (AGW81794.1), ATG8g (AGW81795.1), ATG8h (AGW81796.1), ATG8j (A0A3B6PL31)."

[1] Ghosh A, Jana M, Modi K, Gonzalez F J, Sims K B, Berry-Kravis E, Pahan K. Activation of peroxisome proliferator-activated receptor alpha induces lysosomal biogenesis in brain cells: implications for lysosomal storage disorders. J Biol Chem, 2015, 290: 10309-10324.
[2] Feng Y, He D, Yao Z, Klionsky D J. The machinery of macroautophagy. Cell Res, 2014, 24: 24-41.
doi: 10.1038/cr.2013.168
[3] Yin Z, Pascual C, Klionsky D J. Autophagy: machinery and regulation. Microb Cell, 2016, 3: 588-596.
doi: 10.15698/mic2016.12.546
[4] Nakatogawa H, Ichimura Y, Ohsumi Y. Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion. Cell, 2007, 130: 165-178.
pmid: 17632063
[5] Ichimura Y, Kirisako T, Takao T, Satomi Y, Shimonishi Y, Ishihara N, Mizushima N, Tanida I, Kominami E, Ohsumi M, Noda T, Ohsumi Y. A ubiquitin-like system mediates protein lipidation. Nature, 2000, 408: 488-492.
doi: 10.1038/35044114
[6] Yoshimoto K, Hanaoka H, Sato S, Kato T, Tabata S, Noda T, Ohsumi Y. Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy. Plant Cell, 2004, 16: 2967-2983.
pmid: 15494556
[7] Bassham D C. Methods for analysis of autophagy in plants. Methods, 2015, 75: 181-188.
doi: 10.1016/j.ymeth.2014.09.003 pmid: 25239736
[8] Thompson A R, Doelling J H, Suttangkakul A, Vierstra R D. Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways. Plant Physiol, 2005, 138: 2097-2110.
pmid: 16040659
[9] Chung T, Phillips A R, Vierstra R D. ATG8 lipidation and ATG8-mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A AND ATG12B loci. Plant J, 2010, 62: 483-493.
doi: 10.1111/j.1365-313X.2010.04166.x
[10] Kuzuoglu-Ozturk D, Cebeci Yalcinkaya O, Akpinar B A, Mitou G, Korkmaz G, Gozuacik D, Budak H. Autophagy-related gene, TdAtg8, in wild emmer wheat plays a role in drought and osmotic stress response. Planta, 2012, 236: 1081-1092.
doi: 10.1007/s00425-012-1657-3 pmid: 22569921
[11] Merkulova E A, Guiboileau A, Naya L, Masclaux-Daubresse C, Yoshimoto K. Assessment and optimization of autophagy monitoring methods in Arabidopsis roots indicate direct fusion of autophagosomes with vacuoles. Plant Cell Physiol, 2014, 55: 715-726.
doi: 10.1093/pcp/pcu041
[12] Lin W, Zhuang X. Using microscopy tools to visualize autophagosomal structures in plant cells. Methods Mol Biol, 2017, 1662: 257-266.
[13] Pu Y, Bassham D C. Detection of autophagy in plants by fluorescence microscopy. Methods Mol Biol, 2016, 1450: 161-172.
[14] Fan T, Yang W, Zeng X, Xu X, Xu Y, Fan X, Luo M, Tian C, Xia K, Zhang M. A rice autophagy gene OsATG8b is involved in nitrogen remobilization and control of grain quality. Front Plant Sci, 2020, 11: 588.
doi: 10.3389/fpls.2020.00588 pmid: 32582228
[15] 吴洪波, 刘刚, 张路路, 王冬梅. 小麦ATG8的原核表达及其抗血清制备. 华北农学报, 2013, 28(1): 101-105.
Wu H B, Liu G, Zhang L L, Wang D M. Cloning and prokaryotic expression of ATG8 (a molecular marker of autophagy) gene from wheat and preparation of antiserum. Acta Agric Boreali-Sin, 2013, 28(1): 101-105. (in Chinese with English abstract)
[16] Rappsilber J, Mann M, Ishihama Y. Protocol for micro- purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc, 2007, 2: 1896-1906.
doi: 10.1038/nprot.2007.261 pmid: 17703201
[17] Li Y B, Cui D Z, Sui X X, Huang C, Huang C Y, Fan Q Q, Chu X S. Autophagic survival precedes programmed cell death in wheat seedlings exposed to drought stress. Int J Mol Sci, 2019, 20: 5777.
doi: 10.3390/ijms20225777
[18] Martinet W, Roth L, De Meyer G R Y. Standard Immunohistochemical assays to assess autophagy in mammalian tissue. Cells, 2017, 6: 17.
doi: 10.3390/cells6030017
[19] Ktistakis N T. Monitoring the localization of MAP1LC3B by indirect immunofluorescence. Cold Spring Harb Protoc, 2015, 2015: 751-755.
doi: 10.1101/pdb.prot086264 pmid: 26240409
[20] Zhang Q, Wang D, Zhang H, Wang M, Li P, Fang X, Cai X. Detection of autophagy processes during the development of nonarticulated laticifers in Euphorbia kansui Liou. Planta, 2018, 247: 845-861.
doi: 10.1007/s00425-017-2835-0 pmid: 29260395
[21] Bao Y, Mugume Y, Bassham D C. Biochemical methods to monitor autophagic responses in plants. Methods Enzymol, 2017, 588: 497-513.
[22] 林静. 腐败梭菌毒素单抗、多抗的制备及DAS_ELISA方法的初步建立. 山东农业大学硕士毕业论文, 山东泰安, 2019.
Lin J. Preparation of Monoclonal Antibody and Multiple Antibody against Clostridium septicums Alpha Toxin and Preliminary Establishment of DAS-ELISA. MS Thesis of Shandong Agricultural University, Tai’an, Shandong, China, 2019. (in Chinese with English abstract)
[23] 王传武, 赵德明. 用实验动物制备多克隆抗体: I. 免疫方案的优化. 实验动物科学与管理, 2002, 19(4): 40-44.
Wang C W, Zhao D M. Preparation of polyclonal antibody from experimental animals: I. Optimization of immololunization scheme. Lab Anim Sci Administr, 2002, 19(4): 40-44. (in Chinese with English abstract)
[24] 刘希, 李齐, 郁飞. 拟南芥自噬蛋白ATG8e的原核表达及多克隆抗体制备. 江苏农业科学, 2018, 46(18): 47-51.
Liu X, Li Q, Yu F. Prokaryotic expression and polyclonal antibody preparation of Arabidopsis autophagy protein ATG8e. Jiangsu Agric Sci, 2018, 46(18): 47-51. (in Chinese with English abstract)
[25] Hernandez-Garcia M S, Miranda-Ozuna J F T, Salazar-Villatoro L, Vazquez-Calzada C, Avila-Gonzalez L, Gonzalez-Robles A, Ortega-Lopez J, Arroyo R. Biogenesis of autophagosome in trichomonas vaginalis during macroautophagy induced by rapamycin-treatment and iron or glucose starvation conditions. J Eukaryot Microbiol, 2019, 66: 654-669.
doi: 10.1111/jeu.12712
[26] Bu F, Yang M, Guo X, Huang W, Chen L. Multiple functions of atg8 family proteins in plant autophagy. Front Cell Dev Biol, 2020, 8: 466.
doi: 10.3389/fcell.2020.00466
[27] 胡蕊洁, 杨向云, 贾磊, 李玉如, 项月, 岳洁瑜, 王华忠. 病毒介导的GFP-ATG8在小麦上的表达和在自噬活性监测上的应用. 作物学报, 2021, 47: 2371-2378.
doi: 10.3724/SP.J.1006.2021.01094
Hu X J, Yang X Y, Jia L, Li Y R, Xiang Y, Yue J Y, Wang H J. Virus-mediated expression of GFP-ATG8 for autophagy monitoring in wheat. Acta Agron Sin, 2021, 47: 2371-2378. (in Chinese with English abstract)
[28] Pottier M, Dumont J, Masclaux-Daubresse C, Thomine S. Autophagy is essential for optimal translocation of iron to seeds in Arabidopsis. J Exp Bot, 2019, 70: 859-869.
[29] Zhen X, Li X, Yu J, Xu F. OsATG8c-mediated increased autophagy regulates the yield and nitrogen use efficiency in rice. Int J Mol Sci, 2019, 20: 4956.
doi: 10.3390/ijms20194956
[30] Sera Y, Hanamata S, Sakamoto S, Ono S, Kaneko K, Mitsui Y, Koyano T, Fujita N, Sasou A, Masumura T, Saji H, Nonomura K I, Mitsuda N, Mitsui T, Kurusu T, Kuchitsu K. Essential roles of autophagy in metabolic regulation in endosperm development during rice seed maturation. Sci Rep, 2019, 9: 18544.
[31] Hanaoka H, Noda T, Shirano Y, Kato T, Hayashi H, Shibata D, Tabata S, Ohsumi Y. Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene. Plant Physiol, 2002, 129: 1181-1193.
pmid: 12114572
[32] Chung T, Suttangkakul A, Vierstra R D. The ATG autophagic conjugation system in maize: ATG transcripts and abundance of the ATG8-lipid adduct are regulated by development and nutrient availability. Plant Physiol, 2009, 149: 220-234.
doi: 10.1104/pp.108.126714
[33] Maqbool A, Hughes R K, Dagdas Y F, Tregidgo N, Zess E, Belhaj K, Round A, Bozkurt T O, Kamoun S, Banfield M J. Structural basis of host autophagy-related protein 8 (ATG8) binding by the Irish potato famine pathogen effector protein PexRD54. J Biol Chem, 2016, 291: 20270-20282.
[34] Xia K, Liu T, Ou Y J, Wang R, Fan T, Zhang M. Genome-wide identification, classification, and expression analysis of autophagy-associated gene homologues in rice (Oryza sativa L.). DNA Res, 2011, 18: 363-377.
doi: 10.1093/dnares/dsr024
[35] Zess E K, Jensen C, Cruz-Mireles N, De la Concepcion J C, Sklenar J, Stephani M, Imre R, Roitinger E, Hughes R, Belhaj K, Mechtler K, Menke F L H, Bozkurt T, Banfield M J, Kamoun S, Maqbool A, Dagdas Y F. N-terminal beta-strand underpins biochemical specialization of an ATG8 isoform. PLoS Biol, 2019, 17: e3000373.
[36] Pei D, Zhang W, Sun H, Wei X, Yue J, Wang H. Identification of autophagy-related genes ATG4 and ATG8 from wheat (Triticum aestivum L.) and profiling of their expression patterns responding to biotic and abiotic stresses. Plant Cell Rep, 2014, 33: 1697-1710.
doi: 10.1007/s00299-014-1648-x
[37] Seo E, Woo J, Park E, Bertolani S J, Siegel J B, Choi D, Dinesh-Kumar S P. Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4. Autophagy, 2016, 12: 2054-2068.
doi: 10.1080/15548627.2016.1217373
[1] ZHANG Yi-Duo, LI Guo-Qiang, KONG Zhong-Xin, WANG Yu-Quan, LI Xiao-Li, RU Zhen-Gang, JIA Hai-Yan, MA Zheng-Qiang. Breeding of FHB-resistant wheat line Bainong 4299 by gene pyramiding [J]. Acta Agronomica Sinica, 2022, 48(9): 2221-2227.
[2] TAN Zhao-Guo, YUAN Shao-Hua, LI Yan-Mei, BAI Jian-Fang, YUE Jie-Ru, LIU Zi-Han, ZHANG Tian-Bao, ZHAO Fu-Yong, ZHAO Chang-Ping, XU Ben-Bo, ZHANG Sheng-Quan, PANG Bin-Shuang, ZHNAG Li-Ping. Cloning of TaPIP1 gene and its potential function in anther dehiscence in wheat [J]. Acta Agronomica Sinica, 2022, 48(9): 2242-2254.
[3] FENG Zi-Heng, LI Xiao, DUAN Jian-Zhao, GAO Fei, HE Li, YANG Tian-Chong, RONG Ya-Si, SONG Li, YIN Fei, FENG Wei. Hyperspectral remote sensing monitoring of wheat powdery mildew based on feature band selection and machine learning [J]. Acta Agronomica Sinica, 2022, 48(9): 2300-2314.
[4] CAO Ji-Ling, ZENG Qing, ZHU Jian-Guo. Responses of photosynthetic characteristics and gene expression in different wheat cultivars to elevated ozone concentration at grain filling stage [J]. Acta Agronomica Sinica, 2022, 48(9): 2339-2350.
[5] WANG Yun-Qi, GAO Fu-Li, LI Ao, GUO Tong-Ji, QI Liu-Ran, ZENG Huan-Yu, ZHAO Jian-Yun, WANG Xiao-Ge, GAO Guo-Ying, YANG Jia-Peng, BAI Jin-Ze, MA Ya-Huan, LIANG Yue-Xin, ZHANG Rui. Variation of ear temperature after anthesis and its relationship with yield in wheat [J]. Acta Agronomica Sinica, 2022, 48(9): 2400-2408.
[6] DU Qi-Di, GUO Hui-Jun, XIONG Hong-Chun, XIE Yong-Dun, ZHAO Lin-Shu, GU Jia-Yu, ZHAO Shi-Rong, DING Yu-Ping, SONG Xi-Yun, LIU Lu-Xiang. Gene mapping of apical spikelet degeneration mutant asd1 in wheat [J]. Acta Agronomica Sinica, 2022, 48(8): 1905-1913.
[7] FENG Ya-Juan, LI Ting-Xuan, PU Yong, ZHANG Xi-Zhou. Characteristics of cadmium accumulation and distribution in different organs of wheat with different cadmium-accumulating type [J]. Acta Agronomica Sinica, 2022, 48(7): 1761-1770.
[8] LIU A-Kang, MA Rui-Qi, WANG De-Mei, WANG Yan-Jie, YANG Yu-Shuang, ZHAO Guang-Cai, CHANG Xu-Hong. Effects of filming and supplemental nitrogen fertilizer application on plant growth and population quality of late sowing winter wheat before winter [J]. Acta Agronomica Sinica, 2022, 48(7): 1771-1786.
[9] WANG Juan, LIU Yi, YAO Dan-Yu, ZOU Jing-Wei, XIAO Shi-He, SUN Guo-Zhong. Identification on sensitivity of wheat to low temperature at reproductive stages [J]. Acta Agronomica Sinica, 2022, 48(7): 1721-1729.
[10] ZHANG Shao-Hua, DUAN Jian-Zhao, HE Li, JING Yu-Hang, Urs Christoph Schulthess, Azam Lashkari, GUO Tian-Cai, WANG Yong-Hua, FENG Wei. Wheat yield estimation from UAV platform based on multi-modal remote sensing data fusion [J]. Acta Agronomica Sinica, 2022, 48(7): 1746-1760.
[11] HU Wen-Jing, LI Dong-Sheng, YI Xin, ZHANG Chun-Mei, ZHANG Yong. Molecular mapping and validation of quantitative trait loci for spike-related traits and plant height in wheat [J]. Acta Agronomica Sinica, 2022, 48(6): 1346-1356.
[12] GUO Xing-Yu, LIU Peng-Zhao, WANG Rui, WANG Xiao-Li, LI Jun. Response of winter wheat yield, nitrogen use efficiency and soil nitrogen balance to rainfall types and nitrogen application rate in dryland [J]. Acta Agronomica Sinica, 2022, 48(5): 1262-1272.
[13] LEI Xin-Hui, WAN Chen-Xi, TAO Jin-Cai, LENG Jia-Jun, WU Yi-Xin, WANG Jia-Le, WANG Peng-Ke, YANG Qing-Hua, FENG Bai-Li, GAO Jin-Feng. Effects of soaking seeds with MT and EBR on germination and seedling growth in buckwheat under salt stress [J]. Acta Agronomica Sinica, 2022, 48(5): 1210-1221.
[14] FU Mei-Yu, XIONG Hong-Chun, ZHOU Chun-Yun, GUO Hui-Jun, XIE Yong-Dun, ZHAO Lin-Shu, GU Jia-Yu, ZHAO Shi-Rong, DING Yu-Ping, XU Yan-Hao, LIU Lu-Xiang. Genetic analysis of wheat dwarf mutant je0098 and molecular mapping of dwarfing gene [J]. Acta Agronomica Sinica, 2022, 48(3): 580-589.
[15] FENG Jian-Chao, XU Bei-Ming, JIANG Xue-Li, HU Hai-Zhou, MA Ying, WANG Chen-Yang, WANG Yong-Hua, MA Dong-Yun. Distribution of phenolic compounds and antioxidant activities in layered grinding wheat flour and the regulation effect of nitrogen fertilizer application [J]. Acta Agronomica Sinica, 2022, 48(3): 704-715.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
Add: No. 80 Xueyuan South Rd, Beijing 100081, P. R. China E-mail: zwxb301@caas.cn
Supported by Beijing Magtech Co.Ltd