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Journal of Integrative Agriculture  2025, Vol. 24 Issue (2): 575-593    DOI: 10.1016/j.jia.2024.07.039
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HpFBH3 transactivates HpCO7 via binding to the E-boxes in the promoter and may accelerate flower formation in pitaya

Xiaowei Cai1, 2, Ling Xiao1, 2, Xiangmei Nie1, 2, Qiandong Hou1, 2, Sulin Wen1, 2, Kun Yang1, 2#, Xiaopeng Wen1, 2#

1 Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Institute of Agro-Bioengineering/College of Life Sciences, Guizhou University, Guiyang 550025, China

2 Guizhou Key Laboratory of Agro-Bioengineering, Guizhou University, Guiyang 550025, China

 Highlights 
A total of eight HpFBHs consisting of three subgroups were identified in pitaya.
HpFBH3 heterodimerizes with HpFBH1 in nucleus and binds to the E-boxes in HpCO7 promoter.
Overexpression of HpFBH3 results in early flowering while the HpFBH8 doesn’t.
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摘要  

火龙果是一种藤本仙人掌科植物,因为其对非生物逆境的高耐受及红紫色的果实而广泛种植于全世界。实践中,诱导成花和促进花芽分化往往可以提高作物的产量。然而,相关基因在火龙果中仍有待进一步挖掘。前人研究发现,FLOWERING BHLHs(FBHs)是促进成花的关键转录因子。本工作中,我们在火龙果中共鉴定到8个FBH转录因子。由于近期发生的全基因组加倍事件,火龙果比甜菜和菠菜编码更多的FBH转录因子。系统进化树显示,火龙果FBHs可以被归为3类,其第II类中,石竹目植物编码的FBH转录因子均含有一个不完整的bHLH结构域,来自基因组的散在重复事件,为非典型FBHKa/Ks值显示,HpFBHs进化受到纯化选择影响。对HpFBHs的启动子和表达进行分析发现,它们在与花相关的组织中被时空特异地激活,且表达受非生物逆境的影响,说明HpFBHs参与了火龙果的成花过程。因此,我们选择HpFBH1HpFBH3两个来自TYPE III的典型FBH与来自TYPE II的非典型FBH进行了功能验证。亚细胞定位、酵母双杂与荧光素酶互补实验结果显示,HpFBH3能够在细胞核内与HpFBH1形成异源二聚体。利用生物信息学手段,HpFBHs被预测通过结合启动子上的E-box参与对下游基因的调控,而这类顺式作用元件广泛存在于HpCOs、HpFTs和HpSOC1s的启动子上。RNA-Seq数据显示,这些成花促进因子的表达与HpFBH3高度协同。酵母单杂及双分子荧光素酶报告实验证实,HpFBH3通过结合HpCO7启动子上的E-box元件反式激活其表达。此外,在拟南芥中异位过表达HpFBH3同样促进了成花过程。综上所述,本研究系统分析了火龙果HpFBH转录因子家族,并将其中的典型FBH尤其是HpFBH3鉴定为成花促进因子,有望成为遗传改良火龙果的目标基因。



Abstract  
Hylocereus polyrhizus, also known as pitaya or dragon fruit, is a climbing cactus grown worldwide because of its excellent performance under drought stress and appealing red-purple fruits.  In practice, accelerating flower formation and inducing more flowers usually result in higher yield.  However, the genes for this purpose have not been well characterized in pitaya.  Previously, FLOWERING BHLHs (FBHs) have been identified as positive regulators of flower formation.  In the present work, a total of eight FBHs were identified in pitaya.  This is a greater number than in beet and spinach, possibly because of the recent whole-genome duplication that occurred in the pitaya genome.  The phylogenetic tree indicated that the FBHs could be divided into three groups.  In TYPE II, the genes of Caryophyllales encode atypical FBHs and are generated by dispersed duplication.  The Ka/Ks ratios indicated that HpFBHs are under purifying selection.  Promoter and expression analysis of HpFBHs revealed that they are spatiotemporally activated in flower-related tissues and responsive to multiple abiotic stresses.  These results indicated that HpFBHs are involved in the flower formation of pitaya.  Therefore, typical HpFBH1/3 from TYPE III and an atypical HpFBH8 from TYPE II were selected for functional verification.  HpFBH3 was found to heterodimerize with HpFBH1 in the nucleus using subcellular localization, yeast two-hybrid and luciferase complementation assays.  With bioinformatic analysis, all HpFBHs were predicted to transactivate downstream genes via binding to the E-boxes, which were frequently detected in the promoters of HpCOs, HpFTs and HpSOC1s.  RNA-Seq datasets showed that these flowering accelerators were expressed in coordination with HpFBH3.  Yeast one-hybrid and dual-luciferase reporter assays further verified that HpFBH3 transactivated HpCO7 by selectively binding to the E-boxes in the promoter.  Moreover, ectopic overexpression of HpFBH3 accelerated flower formation in Arabidopsis.  In summary, this study systematically characterized the typical HpFBHs, especially HpFBH3, as positive regulators of flower formation, which could be target genes for the genetic improvement of pitaya.


Keywords:  pitaya       FLOWERING BHLH        dimerization        transactivation        flower formation  
Received: 06 June 2023   Accepted: 11 August 2023
Fund: 
This work was supported by the National Natural Science Foundation of China (32160681 and 32060663), the National Guidance Foundation for Local Science and Technology Development of China (2023-009), the Guizhou Provincial Basic Research Program (Natural Science) (ZK[2022]YB132) and the Foundation of Postgraduate of Guizhou Province, China (YJSKYJJ[2021]057).
About author:  Xiaowei Cai, E-mail: vera8472@163.com; #Correspondence Kun Yang, E-mail: kyang5@gzu.edu.cn; Xiaopeng Wen, E-mail: xpwensc@hotmail.com

Cite this article: 

Xiaowei Cai, Ling Xiao, Xiangmei Nie, Qiandong Hou, Sulin Wen, Kun Yang, Xiaopeng Wen. 2025. HpFBH3 transactivates HpCO7 via binding to the E-boxes in the promoter and may accelerate flower formation in pitaya. Journal of Integrative Agriculture, 24(2): 575-593.

Ain-Ali Q U, Mushtaq N, Amir R, Gul A, Tahir M, Munir F. 2021. Genome-wide promoter analysis, homology modeling and protein interaction network of Dehydration Responsive Element Binding (DREB) gene family in Solanum tuberosumPLoS ONE16, e0261215.

Alessio V M, Cavaçana N, Dantas L L D B, Lee N, Hotta C T, Imaizumi T, Menossi M. 2018. The FBH family of bHLH transcription factors controls ACC synthase expression in sugarcane. Journal of Experimental Botany69, 2511–2525.

Bailey T L, Johnson J, Grant C E, Noble W S. 2015. The MEME suite. Nucleic Acids Research43, W39–W49.

Boeckmann B, Bairoch A, Apweiler R, Blatter M C, Estreicher A, Gasteiger E, Martin M J, Michoud K, O’Donovan C, Phan I, Pilbout S, Schneider M. 2003. The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003. Nucleic Acids Research31, 365–370.

Carretero-Paulet L, Galstyan A, Roig-Villanova I, Martínez-García J F, Bilbao-Castro J R, Robertson D L. 2010. Genome-wide classification and evolutionary analysis of the bHLH family of transcription factors in Arabidopsis, poplar, rice, moss, and algae. Plant Physiology153, 1398–1412.

Chen C B, Li F P, Xie F F, Chen J X, Hua Q Z, Chen J Y, Wu Z J, Zhang Z K, Zhang R, Zhao J T, Hu G B, Qin Y H. 2022. Pitaya genome and multiomics database (PGMD): A comprehensive and integrative resource of Selenicereus undatusGenes13, 745.

Chen J Y, Xie F F, Cui Y Z, Chen C B, Lu W J, Hu X D, Hua Q Z, Zhao J, Wu Z J, Gao D, Zhang Z K, Jiang W K, Sun Q M, Hu G B, Qin Y H. 2021. A chromosome-scale genome sequence of pitaya (Hylocereus undatus) provides novel insights into the genome evolution and regulation of betalain biosynthesis. Horticulture Research8, 164.

Chen Y C, Rajagopala S V, Stellberger T, Uetz P. 2010. Exhaustive benchmarking of the yeast two-hybrid system. Nature Methods7, 667–668.

Chu Y C, Chang J C. 2020. Regulation of floral bud development and emergence by ambient temperature under a long-day photoperiod in white-fleshed pitaya (Hylocereus undatus). Scientia Horticulturae271, 109479.

Falcon S, Gentleman R. 2007. Using GOstats to test gene lists for GO term association. Bioinformatics23, 257–258.

Feller A, Machemer K, Braun E L, Grotewold E. 2011. Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. The Plant Journal66, 94–116.

Gasteiger E, Hoogland C, Gattiker A, Duvaud S E, Wilkins M R, Appel R D, Bairoch A. 2005. Protein identification and analysis tools on the ExPASy server. In: Walker J M, eds., The Proteomics Protocols Handbook. Humana press, Totowa, NJ. pp. 571–607.

Goodstein D M, Shu S, Howson R, Neupane R, Hayes R D, Fazo J, Mitros T, Dirks W, Hellsten U, Putnam N, Rokhsar D S. 2012. Phytozome: a comparative platform for green plant genomics. Nucleic Acids Research40, D1178–D1186.

Ito S, Song Y H, Josephson-Day A R, Miller R J, Breton G, Olmstead R G, Imaizumi T. 2012. FLOWERING BHLH transcriptional activators control expression of the photoperiodic flowering regulator CONSTANS in ArabidopsisProceedings of the National Academy of Sciences of the United States of America109, 3582–3587.

Jiang Y L, Liao Y Y, Lin T S, Lee C L, Yen C R, Yang W J. 2012. The photoperiod-regulated bud formation of red pitaya (Hylocereus sp.). Hortscience47, 1063–1067.

Katoh K, Rozewicki J, Yamada K D. 2019. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics20, 1160–1166.

Kim D, Langmead B, Salzberg S L. 2015. HISAT: a fast spliced aligner with low memory requirements. Nature Methods12, 357–360.

Krueger F. 2015. Trim Galore!: A wrapper around Cutadapt and FastQC to consistently apply adapter and quality trimming to FastQ files, with extra functionality for RRBS data. [2023-6-3]. https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/

Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones S J, Marra M A. 2009. Circos: An information aesthetic for comparative genomics. Genome Research19, 1639–1645.

Le Bellec F, Vaillant F, Imbert E. 2006. Pitahaya (Hylocereus spp.): A new fruit crop, a market with a future. Fruits61, 237–250.

Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouzé P, Rombauts, S. 2002. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Research30, 325–327.

Letunic I, Bork P. 2021. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Research49, W293–W296.

Li X Y. 2011. Infiltration of Nicotiana benthamiana protocol for transient expression via AgrobacteriumBio-protocol1, e95.

Liao Y, Smyth G K, Shi W. 2014. featureCounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics30, 923–930.

Lin L, Fan J L, Li P P, Liu D X, Ren S C, Lin K Y, Fang Y J, Lin C, Wang Y P, Wu J. 2022. The Sclerotinia sclerotiorum-inducible promoter pBnGH17D7 in Brassica napus: isolation, characterization, and application in host-induced gene silencing. Journal of Experimental Botany73, 6663–6677.

Lu S N, Wang J Y, Chitsaz F, Derbyshire M K, Geer R C, Gonzales N R, Gwadz M, Hurwitz D, Marchler G H, Song J S, Thanki N, Yamashita R A, Yang M Z, Zhang D C, Zhang C J, Lanczycki C J, Marchler-Bauer A. 2020. CDD/SPARCLE: the conserved domain database in 2020. Nucleic Acids Research48, D265–D268.

Minh B Q, Schmidt H A, Chernomor O, Schrempf D, Woodhams M D, Von Haeseler A, Lanfear R. 2020. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Molecular Biology and Evolution37, 1530–1534.

Mistry J, Finn R D, Eddy S R, Bateman A, Punta M. 2013. Challenges in homology search: HMMER3 and convergent evolution of coiled-coil regions. Nucleic Acids Research41, e121.

Morgan M, Falcon S, Gentleman R. 2018. GSEABase: Gene set enrichment data structures and methods. R package version 1.64.0. [2023-6-3]. https://bioconductor.org/packages/release/bioc/html/GSEABase.html

Nair S K, Burley S K. 2000. Recognizing DNA in the library. Nature404, 715–717.

Okada M, Lanzatella C, Saha M C, Bouton J, Wu R, Tobias C M. 2010. Complete switchgrass genetic maps reveal subgenome collinearity, preferential pairing and multilocus interactions. Genetics185, 745–760.

Popescu S C, Popescu G V, Bachan S, Zhang Z M, Gerstein M, Snyder M, Dinesh-Kumar, S P. 2009. MAPK target networks in Arabidopsis thaliana revealed using functional protein microarrays. Genes & Development23, 80–92.

Sanagi M, Aoyama S, Kubo A, Lu Y, Sato Y, Ito S, Abe M, Mitsuda N, Ohme-Takagi M, Kiba T, Nakagami H, Rolland F, Yamaguchi J, Imaizumi T, Sato T. 2021. Low nitrogen conditions accelerate flowering by modulating the phosphorylation state of FLOWERING BHLH 4 in ArabidopsisProceedings of the National Academy of Sciences of the United States of America118, e2022942118.

Schmittgen T D, Livak K J. 2008. Analyzing real-time PCR data by the comparative CT method. Nature Protocols3, 1101–1108.

Takahashi Y, Ebisu Y, Kinoshita T, Doi M, Okuma E, Murata Y, Shimazaki K I. 2013. bHLH transcription factors that facilitate K+ uptake during stomatal opening are repressed by abscisic acid through phosphorylation. Science Signaling6, ra48.

Takahashi Y, Kinoshita T, Matsumoto M, Shimazaki K I. 2016. Inhibition of the Arabidopsis bHLH transcription factor by monomerization through abscisic acidinduced phosphorylation. The Plant Journal87, 559–567.

Thumuluri V, Almagro Armenteros J J, Johansen A R, Nielsen H, Winther O. 2022. DeepLoc 2.0: multi-label subcellular localization prediction using protein language models. Nucleic Acids Research50, W228–W234.

Tian H N, Guo H Y, Dai X M, Cheng Y X, Zheng K J, Wang X P, Wang S. 2015. An ABA down-regulated bHLH transcription repressor gene, bHLH129 regulates root elongation and ABA response when overexpressed in ArabidopsisScientific Reports5, 17587.

Toledo-Ortiz G, Huq E, Quail P H. 2003. The Arabidopsis basic/helix-loop-helix transcription factor family. The Plant Cell15, 1749–1770.

Törönen P, Medlar A, Holm L. 2018. PANNZER2: A rapid functional annotation web server. Nucleic Acids Research46, W84–W88.

Wang L P, Yu G, Macho A P, Lozano-Durán R. 2021. Split-luciferase complementation imaging assay to study protein–protein interactions in Nicotiana benthamianaBio-protocol11, e4237.

Wang Y P, Tang H B, DeBarry J D, Tan X, Li J P, Wang X Y, Lee T, Jin H Z, Marler B, Guo H, Kissinger J C, Paterson A H. 2012. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Research40, e49.

Wu L C, Hsu H W, Chen Y C, Chiu C C, Lin Y I, Ho J A A. 2006. Antioxidant and antiproliferative activities of red pitaya. Food Chemistry95, 319–327.

Yin J, Chang X X, Kasuga T, Bui M, Reid M S, Jiang C Z. 2015. A basic helix-loop-helix transcription factor, PhFBH4, regulates flower senescence by modulating ethylene biosynthesis pathway in petunia. Horticulture Research2, 15059.

Yoo S K, Chung K S, Kim J, Lee J H, Hong S M, Yoo S J, Yoo S Y, Lee J S, Ahn J H. 2005. CONSTANS activates SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 through FLOWERING LOCUS T to promote flowering in ArabidopsisPlant Physiology139, 770–778.

Zhang S C, Ma J Q, Zou H Y, Zhang L, Li S H, Wang Y P. 2020. The combination of blue and red LED light improves growth and phenolic acid contents in Salvia miltiorrhiza Bunge. Industrial Crops and Products158, 112959.

Zhang X R, Henriques R, Lin S S, Niu Q W, Chua N H. 2006. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature Protocols1, 641–646.

Zhang Z. 2022. KaKs_Calculator 3.0: Calculating selective pressure on coding and non-coding sequences. Genomics, Proteomics and Bioinformatics20, 536–540.

Zhang Z, Xiao J F, Wu J Y, Zhang H Y, Liu G M, Wang X M, Dai L. 2012. ParaAT: A parallel tool for constructing multiple protein-coding DNA alignments. Biochemical and Biophysical Research Communications419, 779–781.

Zhao Y, Zhang G H, Tang Q Y, Song W L, Gao Q Q, Xiang G S, Li X, Liu G Z, Fan W, Li X N, Yang S C, Zhai C X. 2022. EbMYBP1, a R2R3-MYB transcription factor, promotes flavonoid biosynthesis in Erigeron breviscapusFrontiers in Plant Science13, 946827.

Zinzen R P, Girardot C, Gagneur J, Braun M, Furlong E E. 2009. Combinatorial binding predicts spatio-temporal cis-regulatory activity. Nature462, 65–70.

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