The Cloning and Expression Analysis of FtWRKY6 Gene in Roots of Fagopyrum tataricum Treated by Low Phosphorus and Hormones

doi:10.7668/hbnxb.20193338

Abstract

Abstract:

WRKY transcription factors act important regulators in plant response to low phosphorus.Buckwheat performs well in under-fertilized soils with higher phosphorus use efficiency.Taking tartary buckwheat as experimental materials,this study aims to explore the possible regulatory roles of WRKY genes in phosphorus starvation response of buckwheat.The entire coding sequence(CDS)of FtWRKY6 gene was cloned from RNA samples generated from roots treated by low phosphorus using reverse transcription PCR.The obtained CDS of FtWRKY6 was 1 572 bp in length,encoded a polypeptide of 524 amino acid residues which consists of two conserved WRKY domain each with a zinc finger motif of CCHH,and belonged to the WRKY group Ⅰ.FtWRKY6 shared the highest identity(55.5%)at the amino acid level with Camellia sinensis CsWRKY24.Transient expression assay in protoplasts showed that FtWRKY6 protein was localized in nucleus.Yeast one-hybrid assay revealed that FtWRKY6 had transcription-activating activity.Real-time fluorescence quantitative PCR analysis showed that the expression of FtWRKY6 in roots was significantly induced by low phosphorus and three related hormones such as indole acetic acid(IAA),gibberellin(GA)and cytokinin(CTK).Taken together,FtWRKY6 possesses basic structural and biochemical characteristics as a putative transcription factor,and may be involved in low phosphorus response in roots possibly by crosstalk of IAA,GA and CTK signaling pathways.

Key words: Tartary buckwheat, Low phosphorus, WRKY gene, Gene expression, Transcription-activating activity, Subcellular localization

TIAN Jianhong, PENG Xixu, WU Qingtao, WEN Biyao, DENG Chuchu, TANG Xinke, WANG Haihua. The Cloning and Expression Analysis of FtWRKY6 Gene in Roots of Fagopyrum tataricum Treated by Low Phosphorus and Hormones[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(1): 32-39. doi: 10.7668/hbnxb.20193338.

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Figures/Tables 8

Tab.1 Primers used in this study

基因(登录号) Gene (Accession number)	正向引物序列(5'—3') Forward primer sequence	反向引物序列(5'—3') Reverse primer sequence	用途 Use
FtWRKY6	ATGAATTTCACCTTCACATCCGA	GTAGAGCAATGACTCGAAGAACAAGT	cDNA克隆
(FtPinG0001577500.01)	CGCCTGCAGATGAATTTCACCTTCACATCCGA^*	GGAATTCCATATGGTAGAGCAATGACTCGAAGAACAAGT^**	构建转录激活载体
	caccATGGAGATCAATAATTATAGCC	AAAAGATCCCTTCTGCTCATAG
	TGATGGACCTCCTGATGATGG	AGACTTGGACCGACGAACC	构建亚细胞定位载体
FtHiston 3	GAAATTCGCAAGTACCAGAAGAG	CCAACAAGGTATGCCTCAGC	qPCR内参基因
(HM628903.1)

Fig.1 Electrophoresis detection for PCR products of FtWRKY6 coding sequence Marker.DNA Molecular size standard;1,2.PCR amplification products.

Fig.2 Structures of FtWRKY6 gene and its deduced protein A.Structure of FtWRKY6 gene:Orange boxes,black lines and gray boxes indicate exons,introns,and 5' untranslated regions;this gene has no predicted 3' untranslated region.B.The structure of FtWRKY6 protein:^*.Dense phosphorylation sites in the C-terminal acidic region of FtWRKY6。

Tab.2 Prediction of cis-elements in the promoter of FtWRKY6

顺式作用元件 cis-acting element	一致序列 Consensus sequence	功能 Function	数量 Number
GT1CONSENSUS	GRWAAW	光响应元件	4
POLLEN1LELAT52	AGAAA	花粉特异性表达元件	8
CACTFTPPCA1	YACT	叶肉特异性表达元件	19
DOFCOREZM	AAAG	茎特异性表达元件	15
RAV1AAT	CAACA	根特异性表达元件	8
GARE	TCTGTTG	GA响应元件	4
NTBBF1ARROLB	ACTTTA	IAA响应元件	2
ARR1AT	NGATT	CTK响应元件	14
Helix-loop-helix	CANNTG		4
ABRELATERD1	ACGTG	低磷响应元件	1
W-box	TGACC/T		7

Fig.3 Phylogenetic tree of FtWRKY6 and its homologous proteins from different species The numbers marked on the branches are Bootstrap values,of which only≥50% were shown.FtWRKY6 in this study is highlighted by shading.Protein accession numbers were retrieved from NCBI(http://ncbi.nlm.nih.gov).

Fig.4 Subcellular localization of FtWRKY6

Fig.5 Analysis of transcription-activating activity of FtWRKY6

Fig.6 Analysis for differential expression of FtWRKY6 after the treatment of low phosphorus(LP),IAA,GA₃ and CTK Data are presented as the mean±standard deviation of three biological replicates,different letters indicate significant difference at P<0.05(n=6;Duncan multiple-range test).

References 31

[1]	Rubio V, Linhares F, Solano R, Martín A C, Iglesias J, Leyva A, Paz-Ares J. A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae[J]. Genes & Development, 2001, 15(16): 2122-2133.doi:10.1101/gad.204401. doi: 10.1101/gad.204401 URL
[2]	Pérez-Torres C A, LÓpez-Bucio J, Herrera Estrella L. Low phosphate signaling induces changes in cell cycle gene expression by increasing auxin sensitivity in the Arabidopsis root system[J]. Plant Signaling & Behavior, 2009, 4(8): 781-783.doi:10.4161/psb.4.8.9230. doi: 10.4161/psb.4.8.9230
[3]	Zhong Y J, Wang Y G, Guo J F, Zhu X L, Shi J, He Q J, Liu Y, Wu Y R, Zhang L, Lü Q D, Mao C Z. Rice SPX6 negatively regulates the phosphate starvation response through suppression of the transcription factor PHR2[J]. The New Phytologist, 2018, 219(1): 135-148.doi:10.1111/nph.15155. doi: 10.1111/nph.15155 URL
[4]	Wang Y, Wang F, Lu H, Liu Y, Mao C Z. Phosphate uptake and transport in plants: An elaborate regulatory system[J]. Plant and Cell Physiology, 2021, 62(4): 564-572.doi:10.1093/pcp/pcab011. doi: 10.1093/pcp/pcab011 pmid: 33508131
[5]	Shukla D, Waigel S, Rouchka E C, Sandhu G, Trivedi P K, Sahi S V. Genome-wide expression analysis reveals contrasting regulation of phosphate starvation response(PSR)in root and shoot of Arabidopsis and its association with biotic stress[J]. Environmental and Experimental Botany, 2021, 188: 104483.doi:10.1016/j.envexpbot.2021.104483. doi: 10.1016/j.envexpbot.2021.104483 URL
[6]	Devaiah B N, Nagarajan V K, Raghothama K G. Phosphate homeostasis and root development in Arabidopsis are synchronized by the zinc finger transcription factor ZAT6[J]. Plant Physiology, 2007, 145(1): 147-159.doi:10.1104/pp.107.101691. doi: 10.1104/pp.107.101691 URL
[7]	Devaiah B N, Madhuvanthi R, Karthikeyan A S, Raghothama K G. Phosphate starvation responses and gibberellic acid biosynthesis are regulated by the MYB62 transcription factor in Arabidopsis[J]. Molecular Plant, 2009, 2(1): 43-58.doi:10.1093/mp/ssn081. doi: 10.1093/mp/ssn081 URL
[8]	Yi K K, Wu Z C, Zhou J, Du L M, Guo L B, Wu Y R, Wu P. OsPTF1,a novel transcription factor involved in tolerance to phosphate starvation in rice[J]. Plant Physiology, 2005, 138(4): 2087-2096.doi:10.1104/pp.105.063115. doi: 10.1104/pp.105.063115 URL
[9]	Gross M. Fears over phosphorus supplies[J]. Current Biology, 2010, 20(9): R386-R387.doi:10.1016/j.cub.2010.04.031. doi: 10.1016/j.cub.2010.04.031
[10]	Eulgem T. Regulation of the Arabidopsis defense transcriptome[J]. Trends in Plant Science, 2005, 10(2): 71-78.doi:10.1016/j.tplants.2004.12.006. doi: 10.1016/j.tplants.2004.12.006 URL
[11]	Chen F, Hu Y, Vannozzi A, Wu K C, Cai H Y, Qin Y, Mullis A, Lin Z G, Zhang L S. The WRKY transcription factor family in model plants and crops[J]. Critical Reviews in Plant Sciences, 2017, 36(5/6): 311-335.doi:10.1080/07352689.2018.1441103. doi: 10.1080/07352689.2018.1441103 URL
[12]	Devaiah B N, Karthikeyan A S, Raghothama K G. WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis[J]. Plant Physiology, 2007, 143(4): 1789-1801.doi:10.1104/pp.106.093971. doi: 10.1104/pp.106.093971 URL
[13]	Chen Y F, Li L Q, Xu Q, Kong Y H, Wang H, Wu W H. The WRKY6 transcription factor modulates PHOSPHATE1 expression in response to low pi stress in Arabidopsis[J]. The Plant Cell, 2009, 21(11): 3554-3566.doi:10.1105/tpc.108.064980. doi: 10.1105/tpc.108.064980 URL
[14]	Dai X Y, Wang Y Y, Zhang W H. OsWRKY74,a WRKY transcription factor,modulates tolerance to phosphate starvation in rice[J]. Journal of Experimental Botany, 2016, 67(3): 947-960.doi:10.1093/jxb/erv515. doi: 10.1093/jxb/erv515 URL
[15]	Li C, Liu X Y, Ruan H, Zhang J Y, Xie F B, Gai J Y, Yang S P. GmWRKY45 enhances tolerance to phosphate starvation and salt stress,and changes fertility in transgenic Arabidopsis[J]. Frontiers in Plant Science, 2020, 10: 1714.doi:10.3389/fpls.2019.01714. doi: 10.3389/fpls.2019.01714 URL
[16]	Zhang X R, Wang B M, Zhao Y J, Zhang J R, Li Z X. Auxin and GA signaling play important roles in the maize response to phosphate deficiency[J]. Plant Science, 2019, 283: 177-188.doi:10.1016/j.plantsci.2019.02.011. doi: S0168-9452(18)31198-1 pmid: 31128687
[17]	Yan H M, Wang Y L, Chen B, Wang W J, Sun H Z, Sun H W, Li J Z, Zhao Q Z. OsCKX2 regulates phosphate deficiency tolerance by modulating cytokinin in rice[J]. Plant Science, 2022, 319: 111257.doi:10.1016/j.plantsci.2022.111257. doi: 10.1016/j.plantsci.2022.111257 URL
[18]	林汝法. 中国荞麦[M]. 北京: 中国农业出版社,1994.
	Lin R F. Chinese buckwheat[M]. Beijing: China Agriculture Press,1994.
[19]	Amann C, Amberger A. Phosphorus efficiency of buckwheat(Fagopyrum esculentum)[J]. Zeitschrift Für Pflanzenernährung Und Bodenkunde, 1989, 152(2): 181-189.doi:10.1002/jpln.19891520208. doi: 10.1002/jpln.19891520208 URL
[20]	He X, Li J J, Chen Y, Yang J Q, Chen X Y. Genome-wide analysis of the WRKY gene family and its response to abiotic stress in buckwheat(Fagopyrum tataricum)[J]. Open Life Sciences, 2019, 14(1):80-96.doi:10.1515/biol-2019-0010. doi: 10.1515/biol-2019-0010 URL
[21]	邬清韬, 王海华, 申权, 田建红, 唐新科. 苦荞麦叶斑病原诱导的FtWRKY39基因克隆、表达与生化特性分析[J]. 植物生理学报, 2021, 57(7): 1480-1490.doi:10.13592/j.cnki.ppj.2021.0050. doi: 10.13592/j.cnki.ppj.2021.0050
	Wu Q T, Wang H H, Shen Q, Tian J H, Tang X K. Cloning of leaf spot pathogen-induced WRKY39 gene in Fagopyrum tataricum and analysis of its expression and biochemical characteristics[J]. Plant Physiology Journal, 2021, 57(7): 1480-1490.
[22]	杨春婷, 张永清, 马星星, 陈伟, 董璐, 张楚, 路之娟. 苦荞耐低磷基因型筛选及评价指标的鉴定[J]. 应用生态学报, 2018, 29(9): 2997-3007.doi:10.13287/j.1001-9332.201809.021. doi: 10.13287/j.1001-9332.201809.021
	Yang C T, Zhang Y Q, Ma X X, Chen W, Dong L, Zhang C, Lu Z J. Screening genotypes and identifying indicators of different Fagopyrum tataricum varieties with low phosphorus tolerance[J]. Chinese Journal of Applied Ecology, 2018, 29(9): 2997-3007.
[23]	Liu J, Ha D, Xie Z M, Wang C M, Wang H W, Zhang W K, Zhang J S, Chen S Y. Ectopic expression of soybean GmKNT1 in Arabidopsis results in altered leaf morphology and flower identity[J]. Journal of Genetics and Genomics, 2008, 35(7): 441-449.doi:10.1016/S1673-8527(08)60061-2. doi: 10.1016/S1673-8527(08)60061-2 URL
[24]	Schwechheimer C, Bevan M. The regulation of transcription factor activity in plants[J]. Trends in Plant Science, 1998, 3(10): 378-383.doi:10.1016/S1360-1385(98)01302-8. doi: 10.1016/S1360-1385(98)01302-8 URL
[25]	黄晨晨, 宋晓, 黄绍敏, 张珂珂, 岳克. 不同磷效率小麦根系形态、磷素转运及产量的差异分析[J]. 华北农学报, 2021, 36(2): 169-175. doi: 10.7668/hbnxb.20191728. doi: 10.7668/hbnxb.20191728
	Huang C C, Song X, Huang S M, Zhang K K, Yue K. Analysis of differences in root morphology, phosphorus transport and yield of wheat with different phosphorus efficiency[J]. Acta Agriculturae Boreali-Sinica, 2021, 36(2): 169-175. doi: 10.7668/hbnxb.20191728
[26]	文亦芾, 单贵莲, 姜飞, 白喻, 刘金凤, 韩博. 柱花草WRKY转录因子在低磷胁迫下的克隆与分析[J]. 西北植物学报, 2019, 39(2): 226-233.doi:10.7606/j.issn.1000-4025.2019.02.0226. doi: 10.7606/j.issn.1000-4025.2019.02.0226
	Wen Y F, Shan G L, Jiang F, Bai Y, Liu J F, Han B. Cloning and analysis of WRKY transcription factors of Stylosanthes guianensis under low phosphorus stress[J]. Acta Botanica Boreali-Occidentalia Sinica, 2019, 39(2): 226-233.
[27]	Hamburger D, Rezzonico E, MacDonald-Comber Petétot J, Somerville C, Poirier Y. Identification and characterization of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem[J]. The Plant Cell, 2002, 14(4): 889-902.doi:10.1105/tpc.000745. doi: 10.1105/tpc.000745 URL
[28]	Wang H, Xu Q, Kong Y H, Chen Y, Duan J Y, Wu W H, Chen Y F. Arabidopsis WRKY45 transcription factor activates PHOSPHATE TRANSPORTER1;1 expression in response to phosphate starvation[J]. Plant Physiology, 2014, 164(4): 2020-2029.doi:10.1104/pp.113.235077. doi: 10.1104/pp.113.235077 pmid: 24586044
[29]	Rouached H, Arpat A B, Poirier Y. Regulation of phosphate starvation responses in plants: Signaling players and cross-talks[J]. Molecular Plant, 2010, 3(2): 288-299.doi:10.1093/mp/ssp120. doi: 10.1093/mp/ssp120 pmid: 20142416
[30]	Giri J, Bhosale R, Huang G Q, Pandey B K, Parker H, Zappala S, Yang J, Dievart A, Bureau C, Ljung K, Price A, Rose T, Larrieu A, Mairhofer S, Sturrock C J, White P, Dupuy L, Hawkesford M, Perin C, Liang W Q, Peret B, Hodgman C T, Lynch J, Wissuwa M, Zhang D B, Pridmore T, Mooney S J, Guiderdoni E, Swarup R, Bennett M J. Rice auxin influx carrier OsAUX1 facilitates root hair elongation in response to low external phosphate[J]. Nature Communications, 2018, 9(1): 1408.doi:10.1038/s41467-018-03850-4. doi: 10.1038/s41467-018-03850-4 pmid: 29650967
[31]	Liu X Q, Yang Y M, Wang R Y, Cui R F, Xu H Q, Sun C Y, Wang J S, Zhang H Y, Chen H T, Zhang D. GmWRKY46,a WRKY transcription factor,negatively regulates phosphorus tolerance primarily through modifying root morphology in soybean[J]. Plant Science, 2022, 315: 111148.doi:10.1016/j.plantsci.2021.111148. doi: 10.1016/j.plantsci.2021.111148 URL

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