Welcome to Acta Agronomica Sinica，

Acta Agron Sin ›› 2009, Vol. 35 ›› Issue (8): 1445-1450.doi: 10.3724/SP.J.1006.2009.01445

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Expression of a Wheat Endosperm 14-3-3 Protein and Its Interactions with Starch Biosynthetic Enzymes in Amyloplasts

SONG Jian-Min1, DAI Shuang1, LI Hao-Sheng1, LIU Ai-Feng1, CHENG Dun-Gong1, CHU Xiu-Sheng1, Ian J Tetlow2, and Michael J Emes2   

  1. 1Crop Research Institute,Shandong Academy of Agricultural Sciences,Jinan 250100,China;2University of Guelph, Guelph,N2G2W1,Canada
  • Received:2008-12-29 Revised:2009-03-17 Online:2009-08-12 Published:2009-06-10

PDF

1462

Cited

9

Abstract:

Wheat endosperm starch is the major determinant of grain yield and processing quality. The quality and quantity of starch is controlled by a number of starch biosynthetic enzymes. 14-3-3 proteins, involved in many biological processes, are ubiquitous and important regulators in all eukaryotic cells from yeast to mammals and plants. Protein-protein interactions between a wheat endosperm 14-3-3 protein and starch biosynthetic enzymes from amyloplast were investigated in this study. A 14-3-3 gene was cloned from developing wheat endosperm and inserted into plasmid vectors pET29c and pET41c, respectively. The recombinant vectors were transformed into Escherichia coli strain BL21-CodonPlus (DE3)-RP and expressed at very high level. The fusion protein existed mainly as an insoluble inclusion body after extraction by BugBuster Protein Extraction Reagent. The soluble fusion protein was purified by bounding to S-protein agarose, while the inclusion body should be dissolved in 8 mol L-1 urea and refolded firstly. Sucrose synthase activity was shown to be inhibited by exogenous recombinant 14-3-3 protein in a dosage-dependent manner, which suggested the refolded protein was successfully activated and can be used in the following research. The purified recombinant 14-3-3 protein was bound to S-protein agarose as a biochemical bait, and then incubated with wheat amyloplast extract. Proteins interacting specifically with the 14-3-3 protein and remaining on the resin were analyzed by SDS-PAGE and western blotting. These assays showed that starch synthase I (SSI), starch synthase II (SSII), starch branching enzyme IIa (SBEIIa), starch branching enzyme IIb (SBEIIb), and ADP glucose pyrophosphorylase large subunit (SH2) interacted with 14-3-3 protein, whereas SBEI, ADP glucose pyrophosphorylase small subunit (BT2), starch phosphorylase (SP), and D-enzyme (DE) did not bind with the 14-3-3 protein. The results suggest a role for the wheat endosperm 14-3-3 protein in regulation of grain starch biosynthetic enzymes.

Key words: Wheat14-3-3protein, Expression, Starch biosynthetic enzymes, Protein-protein interaction

[1] Aitken A. 14-3-3 proteins: A historic overview. Semin Cancer Biol, 2006, 16: 162-172

[2] Pozuelo-Rubio M, Geraghty K M, Wong B H, Wood N T, Campbell D G, Morrice N, Mackintosh C. 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking. Biochem J, 2004, 379: 395-408

[3] Schoonheim P J, Veiga H, da Costa Pereira D, Friso G, van Wijk K J, de Boer A H. A comprehensive analysis of the 14-3-3 interactome in barley leaves using a complementary proteomics and two-hybrid approach. Plant Physiol, 2007, 143: 670-683

[4] Alexander R D, Morris P C. A proteomic analysis of 14-3-3 binding proteins from developing barley grains. Proteomics, 2006, 6: 1886-1896

[5] Tetlow I J. Understanding storage starch biosynthesis in plants: a means to quality improvement. Can J Bot, 2006, 84: 1167-1185

[6] Sehnke P C, Chung H J, Wu K, Ferl R J. Regulation of starch accumulation by granule-associated plant 14-3-3 proteins. Proc Natl Acad Sci USA, 2001, 98: 765-770

[7] Zuk M, Weber R, Szopa J. 14-3-3 protein down-regulates key enzyme activities of nitrate and carbohydrate metabolism in potato plants. J Agric Food Chem, 2005, 53: 3454-3460

[8] Tetlow I J, Wait R, Lu Z, Akkasaeng R, Bowsher C G, Esposito S, Kosar-Hashemi B, Morell M K, Emes M J. Protein phosphorylation in amyloplasts regulates starch branching enzyme activity and protein-protein interactions. Plant Cell, 2004, 16: 694-708

[9] Zeng Y, Wu Y, Avigne W T, Koch K E. Differential regulation of sugar-sensitive sucrose synthases by hypoxia and anoxia indicate complementary transcriptional and posttranscriptional responses.Plant Physiol, 1998, 116: 1573-1583

[10] Li Z, Rahman S, Kosar-Hashemi B, Mouille G, Appels R, Morell M K. Cloning and characterization of a gene encoding wheat starch synthase I. Theor Appl Genet, 1999, 98: 1208-1216

[11] Rahman S, Regina A, Li Z, Mukai Y, Yamamoto M, Kosar-Hashemi B, Abrahams S, Morell M K. Comparison of starch-branching enzyme genes reveals evolutionary relationships among isoforms. Characterization of a gene for starch-branching enzyme IIa from wheat D genome donor Aegilops tauschii. Plant Physiol, 2001, 125: 1314-1324

[12] Regina A, Kosar-Hashemi B, Li Z, Pedler A, Mukai Y, Yamamoto M, Gale K, Sharp P J, Morell M K, Rahman S. Starch branching enzyme IIb in wheat is expressed at low levels in the endosperm compared to other cereals and encoded at a non-syntenic locus. Planta, 2005, 222: 899-909

[13] Tetlow I J, Beisel K G, Cameron S, Makhmoudova A, Liu F, Bresolin N S, Wait R, Morell M K, Emes M J. Analysis of protein complexes in wheat amyloplasts reveals functional interactions among starch biosynthetic enzymes. Plant Physiol, 2008, 146: 1878-1891

[14] Toroser D, Athwal G S, Huber S C. Site-specific regulatory interaction between spinach leaf sucrose-phosphate synthase and 14-3-3 proteins. FEBS Lett, 1998, 435: 110-114

[15] Moorhead G, Douglas P, Cotelle V, Harthill J, Morrice N, Meek S, Deiting U, Stitt M, Scarabel M, Aitken A, MacKintosh C. Phosphorylation- dependent interactions between enzymes of plant metabolism and 14-3-3 proteins. Plant J, 1999, 18: 1-12

[16] Esposito D, Chatterjee D K. Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotech, 2006, 17: 353-358

[17] Waugh D S. Making the most of affinity tags. Trends Biotechnol, 2005, 23: 316-320

[18] Pnueli L, Gutfinger T, Haraven D, Ben-Naim O. Tomato SP-interacting proteins define a conserved signaling system that regulates shoot architecture and flowering. Plant Cell, 2001, 13: 2687-2702

[19] Miernyk J A, Thelen J J. Biochemical approaches for discovering protein-protein interactions. Plant J, 2008, 53: 597-609

[20] Hennen-Bierwagen T A, Liu F, Marsh R S, Kim S, Gan Q, Tetlow I J, Emes M J, James M G, Myers A M. Starch biosynthetic enzymes from developing maize endosperm associate in multisubunit complexes. Plant Physiol, 2008, 146: 1892-1908

Kosar-Hashemi B, Li Z, Larroque O, Regina A, Yamamori M, Morell M K, Rahman S. Multiple effects of the starch synthase II mutation in developing wheat endosperm. Funct Plant Biol, 2007, 34: 431-438
[1] CHEN Song-Yu, DING Yi-Juan, SUN Jun-Ming, HUANG Deng-Wen, YANG Nan, DAI Yu-Han, WAN Hua-Fang, QIAN Wei. Genome-wide identification of BnCNGC and the gene expression analysis in Brassica napus challenged with Sclerotinia sclerotiorum and PEG-simulated drought [J]. Acta Agronomica Sinica, 2022, 48(6): 1357-1371.
[2] LI Hai-Fen, WEI Hao, WEN Shi-Jie, LU Qing, LIU Hao, LI Shao-Xiong, HONG Yan-Bin, CHEN Xiao-Ping, LIANG Xuan-Qiang. Cloning and expression analysis of voltage dependent anion channel (AhVDAC) gene in the geotropism response of the peanut gynophores [J]. Acta Agronomica Sinica, 2022, 48(6): 1558-1565.
[3] YAO Xiao-Hua, WANG Yue, YAO You-Hua, AN Li-Kun, WANG Yan, WU Kun-Lun. Isolation and expression of a new gene HvMEL1 AGO in Tibetan hulless barley under leaf stripe stress [J]. Acta Agronomica Sinica, 2022, 48(5): 1181-1190.
[4] ZHOU Hui-Wen, QIU Li-Hang, HUANG Xing, LI Qiang, CHEN Rong-Fa, FAN Ye-Geng, LUO Han-Min, YAN Hai-Feng, WENG Meng-Ling, ZHOU Zhong-Feng, WU Jian-Ming. Cloning and functional analysis of ScGA20ox1 gibberellin oxidase gene in sugarcane [J]. Acta Agronomica Sinica, 2022, 48(4): 1017-1026.
[5] JIN Min-Shan, QU Rui-Fang, LI Hong-Ying, HAN Yan-Qing, MA Fang-Fang, HAN Yuan-Huai, XING Guo-Fang. Identification of sugar transporter gene family SiSTPs in foxtail millet and its participation in stress response [J]. Acta Agronomica Sinica, 2022, 48(4): 825-839.
[6] YUAN Da-Shuang, DENG Wan-Yu, WANG Zhen, PENG Qian, ZHANG Xiao-Li, YAO Meng-Nan, MIAO Wen-Jie, ZHU Dong-Ming, LI Jia-Na, LIANG Ying. Cloning and functional analysis of BnMAPK2 gene in Brassica napus [J]. Acta Agronomica Sinica, 2022, 48(4): 840-850.
[7] ZHOU Yue, ZHAO Zhi-Hua, ZHANG Hong-Ning, KONG You-Bin. Cloning and functional analysis of the promoter of purple acid phosphatase gene GmPAP14 in soybean [J]. Acta Agronomica Sinica, 2022, 48(3): 590-596.
[8] HUANG Cheng, LIANG Xiao-Mei, DAI Cheng, WEN Jing, YI Bin, TU Jin-Xing, SHEN Jin-Xiong, FU Ting-Dong, MA Chao-Zhi. Genome wide analysis of BnAPs gene family in Brassica napus [J]. Acta Agronomica Sinica, 2022, 48(3): 597-607.
[9] JIN Rong, JIANG Wei, LIU Ming, ZHAO Peng, ZHANG Qiang-Qiang, LI Tie-Xin, WANG Dan-Feng, FAN Wen-Jing, ZHANG Ai-Jun, TANG Zhong-Hou. Genome-wide characterization and expression analysis of Dof family genes in sweetpotato [J]. Acta Agronomica Sinica, 2022, 48(3): 608-623.
[10] QU Jian-Zhou, FENG Wen-Hao, ZHANG Xing-Hua, XU Shu-Tu, XUE Ji-Quan. Dissecting the genetic architecture of maize kernel size based on genome-wide association study [J]. Acta Agronomica Sinica, 2022, 48(2): 304-319.
[11] CHEN Xin-Yi, SONG Yu-Hang, ZHANG Meng-Han, LI Xiao-Yan, LI Hua, WANG Yue-Xia, QI Xue-Li. Effects of water deficit on physiology and biochemistry of seedlings of different wheat varieties and the alleviation effect of exogenous application of 5-aminolevulinic acid [J]. Acta Agronomica Sinica, 2022, 48(2): 478-487.
[12] YU Hui-Fang, ZHANG Wei-Na, KANG Yi-Chen, FAN Yan-Ling, YANG Xin-Yu, SHI Ming-Fu, ZHANG Ru-Yan, ZHANG Jun-Lian, QIN Shu-Hao. Genome-wide identification and expression patterns in response to signals from Phytophthora infestans of CrRLK1Ls gene family in potato [J]. Acta Agronomica Sinica, 2022, 48(1): 249-258.
[13] YU Guo-Wu, QING Yun, HE Shan, HUANG Yu-Bi. Preparation and application of polyclonal antibody against SSIIb protein from maize [J]. Acta Agronomica Sinica, 2022, 48(1): 259-264.
[14] JIAN Hong-Ju, SHANG Li-Na, JIN Zhong-Hui, DING Yi, LI Yan, WANG Ji-Chun, HU Bai-Geng, Vadim Khassanov, LYU Dian-Qiu. Genome-wide identification and characterization of PIF genes and their response to high temperature stress in potato [J]. Acta Agronomica Sinica, 2022, 48(1): 86-98.
[15] XIE Qin-Qin, ZUO Tong-Hong, HU Deng-Ke, LIU Qian-Ying, ZHANG Yi-Zhong, ZHANG He-Cui, ZENG Wen-Yi, YUAN Chong-Mo, ZHU Li-Quan. Molecular cloning and expression analysis of BoPUB9 in self-incompatibility Brassica oleracea [J]. Acta Agronomica Sinica, 2022, 48(1): 108-120.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
Add: No.12 Zhongguancun South Street, Beijing 100081,China Email:xbzw@chinajournal.net.cn
Supported by Beijing Magtech Co.Ltd