Welcome to Acta Agronomica Sinica，

Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (7): 1332-1341.doi: 10.3724/SP.J.1006.2021.02049

• TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY • Previous Articles     Next Articles

Preliminary study of the peptide aptamer R18 promotes grain filling of rice inferior spikelets

ZHANG Zhi-Xing1,2, CHEN Hua1, MIN Xiu-Mei1, XU Hai-Long1, SONG Guo1, LIN Wen-Xiong1,2,*()   

  1. 1College of Life Sciences, Fujian Agricultural and Forestry University, Fuzhou 350002, Fujiang, China
    2Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujiang, China
  • Received:2020-07-18 Accepted:2020-12-01 Online:2021-07-12 Published:2021-01-09
  • Contact: LIN Wen-Xiong E-mail:wenxiong181@163.com
  • Supported by:
    This work was supported by the National Natural Science Foundation of China(31871542);the National Key Research and Development Program of China(2018YFD0301105)

RichHTML385

10

PDF

156

Abstract:

14-3-3 protein plays an important role in plant growth and development. The high expression abundant of 14-3-3 isoform (GF14f) was an important reason for the poor grain-filling of rice inferior spikelets. In the present study, firstly we used molecular docking to study the most possible binding sites between GF14f protein and peptide aptamer R18. Furthermore, in vitro competitive experiment showed that the R18 could block GF14f binding to its interaction proteins, in turn, lead to a weakened interaction of GF14f with the SuS2, SS, and AGPS. Meanwhile, in vitro enzymatic assays showed that exogenous added R18 significantly increased the activity of sucrose synthase (SuSase), adenosine diphosphate-glucose pyrophosphorylase (AGPase), and starch synthase (StSase). Coimmunoprecipitation (Co-IP) experiments revealed that, in addition to GF14f, R18 could interacted with the other 14-3-3 isoforms, such as GF14b, GF14c, GF14d, and GF14e, which were further confirmed by Bimolecular Fluorescent Complimentary (BiFC) assay. These results also showed that the exogenous application (60 mg L -1) of R18 at grain-filling stage could significantly increase the activity of StSase, SuSase, and AGPase, resulting in the improvement of 1000-grain weight and seed-setting rate of rice inferior spikelets. In the present study, R18 had been investigated to competitively interfere with the interaction of rice 14-3-3 protein with multiple client proteins, weak the binding force, which increased the activity of sucrose conversion and starch synthesis related enzyme, resulting in the improvement of grain filling of rice inferior spikelets.

Key words: rice, inferior spikelets, grain filling, 14-3-3 protein, peptide aptamer

Table 1

Primers used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 用途
Usage
pet32a_His_R18-F GCTGATATCGGATCCGAATTCCCCCACTGCGTGCCCAGG 载体构建
Vector construct
pet32a_His_R18-R GTGGTGGTGGTGGTGCTCGAGGGGGGGCAGGCACATGTT
YFPN-GF14b-F TATACTAGTATGTCGGCACAGGCGGAGC 双分子荧光互补实验载体
Vector for BiFC
YFPN-GF14b-R ATATACTAGTCTGCCCCTCGCTGGAGTCG
YFPN-GF14f-F TTTTGCCACTAGTATGTCGCCTGCTGAGGCATCGCGT 双分子荧光互补实验载体
Vector for BiFC
YFPN-GF14f-R TTTTCCACTAGTGTGGCCCTCTCCTTCAGGCTTCGCT
YFPN- R18-F CTAGAATGCCCCACTGCGTGCCCAGGGACCTGAGCTGGCTGGACCTGGAGGCCAACATGTGCCTGCCCCCCT 双分子荧光互补实验载体
Vector for BiFC
YFPN- R18-R CTAGAGGGGGGCAGGCACATGTTGGCCTCCAGGTCCAGCCAGCTCAGGTCCCTGGGCACGCAGTGGGGCATT

Fig. 1

Molecular docking between GF14f protein and R18 polypeptide"

Fig. 2

Peptide aptamer R18 competes with client proteins for binding to rice GF14f proteins A: the in vitro interaction between GST-GF14f and His-SUS2 is gradually weakened with an increase in the concentration of His-R18; B: the in vitro interaction between GST-GF14f and His-AGPS is weakened at 60 mg L-1 of His-R18; C: the in vitro interaction between GST-GF14f and His-SS is weakened at 60 mg L-1 of His-R18."

Fig. 3

Effects of in vitro with the exogenous R18 on the activities of sucrose breakdown and starch synthesis-related enzyme A: the enzyme activity of SuSase is gradually increased with an increase in the concentration of R18. B: the enzyme activity of AGPase is significantly increase at R18 concentrations of 60 mg L-1. C: the enzyme activity of StSase is significantly increase at R18 concentrations of 60 mg L-1."

Fig. 4

Western blot verification of R18 in vivo DAF: days after flowering; CBB: coomassie brilliant blue."

Table 2

List of interaction isoforms between R18 and rice 14-3-3 proteins"

蛋白ID
Protein IDs		 蛋白名称
Protein name		 独特肽数量
Unique peptides
LOC_Os04g38870.5 GF14b 2
LOC_Os08g33370.2 GF14c 4
LOC_Os11g34450.1 GF14d 3
LOC_Os02g36974.4 GF14e 2
LOC_Os03g50290.3 GF14f 5

Fig. 5

Interaction between R18 with 14-3-3 family members GF14b and GF14f in pavement cells of Nicotiana benthamiana A: the interaction between R18 with GF14b; B: the interaction between R18 with GF14f."

Fig. 6

Effects of the exogenous application R18 on the activities of sucrose breakdown and starch synthesis-related enzyme at grain-filling stage A: the enzyme activity of AGPase is significantly increase with the 60 mg L-1 R18 solution; B: the enzyme activity of SuSase is significantly increase with the 60 mg L-1 R18 solution; C: the enzyme activity of StSase is significantly increase with the 60 mg L-1 R18 solution. Different lowercase letters indicate significant difference at P < 0.05 among the treatments."

Fig. 7

Effects of the exogenous application R18 on 1000-grain weight and seed setting rate of rice inferior spikelets A: the change in the 1000-grain weight of rice inferior spikelets; B: the change in the seed setting rate of rice inferior spikelets. Different lowercase letters indicate significant difference at P < 0.05 among the treatments."

[1] 顾世梁, 朱庆森, 杨建昌, 彭少兵. 不同水稻材料籽粒灌浆特性的分析. 作物学报, 2001, 27:7-14.
Gu S L, Zhu Q S, Yang J C, Peng S B. Analysis on grain filling characteristics for different rice types. Acta Agron Sin, 2001,27:7-14 (in Chinese with English abstract).
[2] Yang J C, Zhang J H. Grain-filling problem in ‘super’ rice. J Exp Bot, 2010,61:1-4.
[3] Zhu G H, Ye N H, Yang J C, Peng X X, Zhang J H. Regulation of expression of starch synthesis genes by ethylene and ABA in relation to the development of rice inferior and superior spikelets. J Exp Bot, 2011,11:3907-3916.
[4] Sekhar S, Gharat S A, Panda B B, Mohaptra T, Das K, Kariali E, Mohapatra P K, Shaw B P. Identification and characterization of differentially expressed genes in inferior and superior spikelets of rice cultivars with contrasting panicle-compactness and grain-filling properties. PLoS One, 2015,10:e0145749.
[5] Sun H, Peng T, Zhao Y, Du Y, Zhang J, Li J, Xin Z, Zhao Q. Dynamic analysis of gene expression in rice superior and inferior grains by RNA-Seq. PLoS One, 2015, 10:e0137168.
[6] You C, Zhu H, Xu B, Huang W, Wang S, Ding Y, Liu Z, Li G, Chen L, Ding C, Tang S. Effect of removing superior spikelets on grain filling of inferior spikelets in rice. Front Plant Sci, 2016,7:1161.
[7] Zhang Z X, Zhao H, Huang F L, Long J F, Song G, Lin W X. The 14-3-3 protein GF14f negatively affects grain filling of inferior spikelets of rice (Oryza sativa L.). Plant J, 2019,99:344-358.
doi: 10.1111/tpj.14329 pmid: 30912217
[8] Sluchanko N N. Association of multiple phosphorylated proteins with the 14-3-3 regulatory hubs: problems and perspectives. J Mol Biol, 2018, 430:20-26.
[9] Denison F C, Paul A L, Zupanska A K, Ferl R J. In 14-3-3 Proteins in Plant Physiology, Seminars in Cell and Developmental Biology, Elsevier, 2011. pp 720-727.
[10] de Boer A H, van Kleeff P J, Gao J. Plant 14-3-3 proteins as spiders in a web of phosphorylation. Protoplasma, 2013, 250:425-440.
[11] Zhou H, Lin H, Chen S, Becker K, Yang Y, Zhao J, Kudla J, Schumaker K S, Guo Y. Inhibition of the Arabidopsis salt overly sensitive pathway by 14-3-3 proteins. Plant Cell, 2014, 26:1166-1182.
[12] Colombo M, Mizzotti C, Masiero S, Kater M M, Pesaresi P. Peptide aptamers: the versatile role of specific protein function inhibitors in plant biotechnology. J Integr Plant Biol, 2015, 57:892-901.
[13] Hao Z, Gong P, He C, Lin J. Peptide aptamers to inhibit protein function in plants. Trends Plant Sci, 2018, 23:281-284.
[14] Wang B C, Yang H Z, Liu Y C, Jelinek T, Zhang L, Ruoslahti E, Fu H. Isolation of high-affinity peptide antagonists of 14-3-3 proteins by phage display. Biochemistry, 1999,38:12499-12504.
doi: 10.1021/bi991353h pmid: 10493820
[15] Ishimaru T, Matsuda T, Ohsugi R, Yamagishi T. Morphological development of rice caryopses located at the different positions in a panicle from early to middle stage of grain filling. Funct Plant Biol, 2003,30:1139-1149.
pmid: 32689096
[16] Zhang Z X, Zhang Y P, Zhao H, Huang F L, Zhang Z F, Lin W X. The important functionality of 14-3-3 isoforms in rice roots revealed by affinity chromatography. J Proteom, 2017,158:20-30.
[17] Morrison D K . The 14-3-3 proteins: integrators of diverse signaling cues that impact cell fate and cancer development. Trends Cell Biol, 2009,19:16-23.
doi: 10.1016/j.tcb.2008.10.003 pmid: 19027299
[18] Molzan M, Kasper S, Röglin L, Skwarczynska M, Sassa T, Inoue T, Breitenbuecher F, Ohkanda J, Kato N, Schuler M. Stabilization of physical RAF/14-3-3 interaction by cotylenin A as treatment strategy for RAS mutant cancers. ACS Chem Biol, 2013,8:1869-1875.
[19] Cornell B, Toyo-Oka K. 14-3-3 proteins in brain development: neurogenesis, neuronal migration and neuromorphogenesis. Front Mol Neurosci, 2017,10:318.
[20] Masters S C, Fu H. 14-3-3 proteins mediate an essential anti-apoptotic signal. J Biol Chem, 2001,276:45193-45200.
pmid: 11577088
[21] 王志琴, 叶玉秀, 杨建昌, 袁莉民, 王学明, 朱庆森. 水稻灌浆期籽粒中蔗糖合成酶活性的变化与调节. 作物学报, 2004,30:634-643.
Wang Z Q, Ye Y X, Yang J C, Yuang L M, Wang X M, Zhu Q S. Changes and regulations of sucrose synthase activity in rice grains during grain filling. Acta Agron Sin, 2004,30:634-643 (in Chinese with English abstract).
[22] Ishimaru T, Hirose T, Matsuda T, Goto A, Takahashi K, Sasaki H, Terao T, Ishii R, Ohsugi R, Yamagishi T. Expression patterns of genes encoding carbohydrate-metabolizing enzymes and their relationship to grain filling in rice (Oryza sativa L.): comparison of caryopses located at different positions in a panicle. Plant Cell Physiol, 2005,46:620-624.
[23] Zhang Z X, Zhao H, Tang J, Li Z, Li Z, Chen D M, Lin W X. A proteomic study on molecular mechanism of poor grain-filling of rice (Oryza sativa L.) inferior spikelets. PLoS One, 2014,9:e89140.
[24] Zhang L, Chen J, Fu H. Suppression of apoptosis signal-regulating kinase 1-induced cell death by 14-3-3 proteins. Proc Natl Acad Sci USA, 1999,96:8511-8515.
doi: 10.1073/pnas.96.15.8511 pmid: 10411906
[25] Gong P, Quan H, He C. Targeting MAGO proteins with a peptide aptamer reinforces their essential roles in multiple rice developmental pathways. Plant J, 2014,80, 905-914.
doi: 10.1111/tpj.12672 pmid: 25230811
[26] 李俊周, 李磊, 孙传范, 赵全志, 杜彦修, 张静, 彭庭. 水氮互作对水稻籽粒充实及产量的影响. 中国农业大学学报, 2011,16:42-47.
Li J Z, Li L, Sun C F, Zhao Q Z, Du Y X, Zhang J, Peng T. Effects of water-nitrogen interaction on rice grain plumpness and yield. J China Agric Univ, 2011,16:42-47 (in Chinese with English abstract).
[27] 张志兴, 陈军, 李忠, 李兆伟, 黄锦文, 陈婷, 方长旬, 陈鸿飞, 林文雄. 水稻籽粒灌浆过程中蛋白质表达特性及其对氮肥运筹的响应. 生态学报, 2012,32, 3209-3224.
Zhang Z X, Chen J, Li Z, Li Z W, Huang J W, Chen T, Fang C X, Chen H F, Lin W X. Protein expression characteristics and their response to nitrogen application during grain-filling stage of rice (Oryza sativa L.). Acta Ecol Sin, 2012,32:3209-3224 (in Chinese with English abstract).
[28] Zhang H, Li H W, Yuan L M, Wang Z Q, Yang J C, Zhang J H. Post-anthesis alternate wetting and moderate soil drying enhances activities of key enzymes in sucrose-to-starch conversion in inferior spikelets of rice. J Exp Bot, 2012,63:215-227.
doi: 10.1093/jxb/err263 pmid: 21926094
[29] Zhang Z X, Tang J, Du T W, Zhao H, Li Z, Li Z, Lin W X. Mechanism of developmental stagnancy of rice inferior spikelets at early grain-filling stage as revealed by proteomic analysis. Plant Mol Biol Rep, 2015,33:1844-1863.
[1] TIAN Tian, CHEN Li-Juan, HE Hua-Qin. Identification of rice blast resistance candidate genes based on integrating Meta-QTL and RNA-seq analysis [J]. Acta Agronomica Sinica, 2022, 48(6): 1372-1388.
[2] ZHENG Chong-Ke, ZHOU Guan-Hua, NIU Shu-Lin, HE Ya-Nan, SUN wei, XIE Xian-Zhi. Phenotypic characterization and gene mapping of an early senescence leaf H5(esl-H5) mutant in rice (Oryza sativa L.) [J]. Acta Agronomica Sinica, 2022, 48(6): 1389-1400.
[3] ZHOU Wen-Qi, QIANG Xiao-Xia, WANG Sen, JIANG Jing-Wen, WEI Wan-Rong. Mechanism of drought and salt tolerance of OsLPL2/PIR gene in rice [J]. Acta Agronomica Sinica, 2022, 48(6): 1401-1415.
[4] ZHENG Xiao-Long, ZHOU Jing-Qing, BAI Yang, SHAO Ya-Fang, ZHANG Lin-Ping, HU Pei-Song, WEI Xiang-Jin. Difference and molecular mechanism of soluble sugar metabolism and quality of different rice panicle in japonica rice [J]. Acta Agronomica Sinica, 2022, 48(6): 1425-1436.
[5] YAN Jia-Qian, GU Yi-Biao, XUE Zhang-Yi, ZHOU Tian-Yang, GE Qian-Qian, ZHANG Hao, LIU Li-Jun, WANG Zhi-Qin, GU Jun-Fei, YANG Jian-Chang, ZHOU Zhen-Ling, XU Da-Yong. Different responses of rice cultivars to salt stress and the underlying mechanisms [J]. Acta Agronomica Sinica, 2022, 48(6): 1463-1475.
[6] CHEN Jing, REN Bai-Zhao, ZHAO Bin, LIU Peng, ZHANG Ji-Wang. Regulation of leaf-spraying glycine betaine on yield formation and antioxidation of summer maize sowed in different dates [J]. Acta Agronomica Sinica, 2022, 48(6): 1502-1515.
[7] XU Tian-Jun, ZHANG Yong, ZHAO Jiu-Ran, WANG Rong-Huan, LYU Tian-Fang, LIU Yue-E, CAI Wan-Tao, LIU Hong-Wei, CHEN Chuan-Yong, WANG Yuan-Dong. Canopy structure, photosynthesis, grain filling, and dehydration characteristics of maize varieties suitable for grain mechanical harvesting [J]. Acta Agronomica Sinica, 2022, 48(6): 1526-1536.
[8] YANG Jian-Chang, LI Chao-Qing, JIANG Yi. Contents and compositions of amino acids in rice grains and their regulation: a review [J]. Acta Agronomica Sinica, 2022, 48(5): 1037-1050.
[9] DENG Zhao, JIANG Nan, FU Chen-Jian, YAN Tian-Zhe, FU Xing-Xue, HU Xiao-Chun, QIN Peng, LIU Shan-Shan, WANG Kai, YANG Yuan-Zhu. Analysis of blast resistance genes in Longliangyou and Jingliangyou hybrid rice varieties [J]. Acta Agronomica Sinica, 2022, 48(5): 1071-1080.
[10] YANG De-Wei, WANG Xun, ZHENG Xing-Xing, XIANG Xin-Quan, CUI Hai-Tao, LI Sheng-Ping, TANG Ding-Zhong. Functional studies of rice blast resistance related gene OsSAMS1 [J]. Acta Agronomica Sinica, 2022, 48(5): 1119-1128.
[11] ZHU Zheng, WANG Tian-Xing-Zi, CHEN Yue, LIU Yu-Qing, YAN Gao-Wei, XU Shan, MA Jin-Jiao, DOU Shi-Juan, LI Li-Yun, LIU Guo-Zhen. Rice transcription factor WRKY68 plays a positive role in Xa21-mediated resistance to Xanthomonas oryzae pv. oryzae [J]. Acta Agronomica Sinica, 2022, 48(5): 1129-1140.
[12] WANG Xiao-Lei, LI Wei-Xing, OU-YANG Lin-Juan, XU Jie, CHEN Xiao-Rong, BIAN Jian-Min, HU Li-Fang, PENG Xiao-Song, HE Xiao-Peng, FU Jun-Ru, ZHOU Da-Hu, HE Hao-Hua, SUN Xiao-Tang, ZHU Chang-Lan. QTL mapping for plant architecture in rice based on chromosome segment substitution lines [J]. Acta Agronomica Sinica, 2022, 48(5): 1141-1151.
[13] WANG Ze, ZHOU Qin-Yang, LIU Cong, MU Yue, GUO Wei, DING Yan-Feng, NINOMIYA Seishi. Estimation and evaluation of paddy rice canopy characteristics based on images from UAV and ground camera [J]. Acta Agronomica Sinica, 2022, 48(5): 1248-1261.
[14] KE Jian, CHEN Ting-Ting, WU Zhou, ZHU Tie-Zhong, SUN Jie, HE Hai-Bing, YOU Cui-Cui, ZHU De-Quan, WU Li-Quan. Suitable varieties and high-yielding population characteristics of late season rice in the northern margin area of double-cropping rice along the Yangtze River [J]. Acta Agronomica Sinica, 2022, 48(4): 1005-1016.
[15] CHEN Yue, SUN Ming-Zhe, JIA Bo-Wei, LENG Yue, SUN Xiao-Li. Research progress regarding the function and mechanism of rice AP2/ERF transcription factor in stress response [J]. Acta Agronomica Sinica, 2022, 48(4): 781-790.
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