PcERF5通过激活并与PcMYB互作促进红肉梨花青苷合成

doi:10.1016/j.jia.2023.07.007

摘要/Abstract

摘要：

红皮梨因鲜艳的果皮色泽受消费者喜爱，对其果皮花青苷生物合成分子调控机制已有较多的研究；而红肉梨仅存在于少数西洋梨中，目前对红肉梨中花青苷合成的分子调控机制知之甚少。本研究基于红肉与白肉西洋梨的转录组比较分析，鉴定到一个在果肉中表达趋势与花青苷含量变化显著相关的乙烯响应因子PcERF5。进一步通过瞬时转化梨果皮和稳定转化苹果愈伤组织，验证了候选基因PcERF5调控花青苷生物合成的功能。研究表明，PcERF5可通过不同途径调控花青苷的生物合成。一方面，PcERF5激活了花青苷生物合成途径中结构基因(PcDFR、PcANS和PcUFGT)以及PcMYB10和PcMYB114关键转录因子的表达。另一方面，PcERF5与PcMYB10互作形成ERF5-MYB10蛋白复合体，增强了PcERF5对其靶基因的转录激活。因此，本研究揭示了PcERF5作为转录激活子促进西洋梨果肉中花青苷合成的功能，研究结果不仅为花青苷生物合成的调控机制提供了新的见解，也为红肉梨的分子育种提供了理论指导

Abstract:

As there is a strong interest in red-skinned pears, the molecular mechanism of anthocyanin regulation in red-skinned pears has been widely investigated; however, little is known about the molecular mechanism of anthocyanin regulation in red-fleshed pears due to limited availability of such germplasm, primarily found in European pears (Pyrus communis). In this study, based on transcriptomic analysis in red-fleshed and white-fleshed pears, we identified an ethylene response factor (ERF) from P. communis, PcERF5, of which expression level in fruit flesh was significantly correlated with anthocyanin content. We then verified the function of PcERF5 in regulating anthocyanin accumulation by genetic transformation in both pear skin and apple calli. PcERF5 regulated anthocyanin biosynthesis by different regulatory pathways. On the one hand, PcERF5 can activate the transcription of flavonoid biosynthetic genes (PcDFR, PcANS and PcUFGT) and two key transcription factors encoding genes PcMYB10 and PcMYB114. On the other hand, PcERF5 interacted with PcMYB10 to form the ERF5-MYB10 protein complex that enhanced the transcriptional activation of PcERF5 on its target genes. Our results suggested that PcERF5 functioned as a transcriptional activator in regulating anthocyanin biosynthesis, which provides new insights into the regulatory mechanism of anthocyanin biosynthesis. This new knowledge will provide guidance for molecular breeding of red-fleshed pear.

Key words: Pyrus communis , red-fleshed , anthocyanin biosynthesis , PcERF5 , PcMYB10/PcMYB114

CHANG Yao-jun, CHEN Guo-song, YANG Guang-yan, SUN Cong-rui, WEI Wei-lin, Schuyler S. KORBAN, WU Jun. PcERF5通过激活并与PcMYB互作促进红肉梨花青苷合成[J]. Journal of Integrative Agriculture, 2023, 22(9): 2687-2704.

CHANG Yao-jun, CHEN Guo-song, YANG Guang-yan, SUN Cong-rui, WEI Wei-lin, Schuyler S. KORBAN, WU Jun. The PcERF5 promotes anthocyanin biosynthesis in red-fleshed pear (Pyrus communis) through both activating and interacting with PcMYB transcription factors[J]. Journal of Integrative Agriculture, 2023, 22(9): 2687-2704.

参考文献

Allan A C, Hellens R P, Laing W A. 2008. MYB transcription factors that colour our fruit. Trends in Plant Science, 13, 99–102.

An J P, An X H, Yao J F, Wang X N, You C X, Wang X F, Hao Y J. 2018. BTB protein MdBT2 inhibits anthocyanin and proanthocyanidin biosynthesis by triggering MdMYB9 degradation in apple. Tree Physiology, 38, 1578–1587.

An J P, Zhang X W, Bi S Q, You C X, Wang X F, Hao Y J. 2020. The ERF transcription factor MdERF38 promotes drought stress-induced anthocyanin biosynthesis in apple. The Plant Journal, 101, 573–589.

Bai S L, Sun Y W, Qian M J, Yang F X, Ni J B, Tao R Y, Li L, Shu Q, Zhang D, Teng Y W. 2017. Transcriptome analysis of bagging-treated red Chinese sand pear peels reveals light-responsive pathway functions in anthocyanin accumulation. Scientific Reports, 7, 63.

Bai S L, Tao R Y, Tang Y X, Yin L, Ma Y J, Ni J B, Yan X H, Yang Q S, Wu Z Y, Zeng Y L, Teng Y W. 2019a. BBX16, a B-box protein, positively regulates light-induced anthocyanin accumulation by activating MYB10 in red pear. Plant Biotechnology Journal, 17, 1985–1997.

Bai S L, Tao R Y, Yin L, Ni J B, Yang Q S, Yan X H, Yang F, Guo X P, Li H X, Teng Y W. 2019b. Two B-box proteins, PpBBX18 and PpBBX21, antagonistically regulate anthocyanin biosynthesis via competitive association with Pyrus pyrifolia ELONGATED HYPOCOTYL 5 in the peel of pear fruit. The Plant Journal, 100, 1208–1223.

Chagné D, Carilisle C M, Blond C, Volz R K, Whitworth C J, Oraguzie N C, Crowhurst R N, Allan A C, Espley R V, Hellens R P, Gardiner S E. 2007. Mapping a candidate gene (MdMYB10) for red flesh and foliage colour in apple. BMC Genomics, 8, 212.

Chagné D, Lin-Wang K, Espley R V, Volz R K, How N M, Rouse S, Brendolise C, Carlisle C M, Kumar S, Silva N D, Micheletti D, McGhie T, Crowhurst R N, Storey R D, Velasco R, Hellens R P, Gardiner S E, Allan A C. 2013. An ancient duplication of apple MYB transcription factors is responsible for novel red fruit-flesh phenotypes. Plant Physiology, 161, 225–239.

Chakravarthy S, Tuori R P, D’Ascenzo M D, Fobert P R, Després C, Martin G B. 2003. The tomato transcription factor Pti4 regulates defense-related gene expression via GCC box and non-GCC box cis elements. The Plant Cell, 15, 3033–3050.

Dare A P, Schaffer R J, Lin-Wang K, Allan A C, Hellens R P. 2008. Identification of a cis-regulatory element by transient analysis of co-ordinately regulated genes. Plant Methods, 4, 17.

Dondini L, Pierantoni L, Ancarani V, Angelo M D, Cho K H, Shin I S, Musacchi S, Kang S J, Sansavini S. 2008. The inheritance of the red colour character in European pear (Pyrus communis) and its map position in the mutated cultivar ‘Max Red Bartlett’. Plant Breeding, 127, 524–526.

Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L. 2010. MYB transcription factors in Arabidopsis. Trends in Plant Science, 15, 573–581.

Espley R V, Brendolise C, Chagné D, Kutty-Amma S, Green S, Volz R, Putterill J, Schouten H J, Gardiner S E, Hellens R P, Allan A C. 2009. Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples. The Plant Cell, 21, 168–183.

Espley R V, Hellens R P, Putterill J, Stevenson D E, Kutty-Amma S, Allan A C. 2007. Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. The Plant Journal, 49, 414–427.

Feller A, Machemer K, Braun E L, Grotewold E. 2011. Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. The Plant Journal, 66, 94–116.

Feng S Q, Sun S S, Chen X L, Wu S J, Wang D Y, Chen X S. 2015. PyMYB10 and PyMYB10.1 interact with bHLH to enhance anthocyanin accumulation in pears. PLoS ONE, 10, e0142112.

Feng S Q, Wang Y L, Yang S, Xu Y T, Chen X S. 2010. Anthocyanin biosynthesis in pears is regulated by a R2R3-MYB transcription factor PyMYB10. Planta, 232, 245–255.

Gonzalez A, Zhao M, Leavitt J M, Lloyd A M. 2008. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. The Plant Journal, 53, 814–827.

Gou J Y, Felippes F F, Liu C J, Weigel D, Wang J W. 2011. Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor. The Plant Cell, 23, 1512–1522.

Grotewold E. 2006. The genetics and biochemistry of floral pigments. Annual Review of Plant Biology, 57, 761–780.

Hellens R P, Allan A C, Friel E N, Bolitho K, Grafton K, Templeton M D, Karunairetnam S, Gleave A P, Laing W A. 2005. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods, 1, 13.

Holton T A, Cornish E C. 1995. Gentics and biochemistry of anthocyanin biosynthesis. The Plant Cell, 7, 1071–1083.

Huang C H, Yu B, Teng Y W, Su J, Shu Q, Cheng Z Q, Zeng L Q. 2009. Effects of fruit bagging on coloring and related physiology, and qualities of red Chinese sand pears during fruit maturation. Scientia Horticulturae, 121, 149–158.

Kumar K R, Kirti P B. 2010. A mitogen-activated protein kinase, AhMPK6 from peanut localizes to the nucleus and also induces defense responses upon transient expression in tobacco. Plant Physiology and Biochemistry, 48, 481–486.

Li C, Wu J, Hu K D, Wei S W, Sun H Y, Hu L Y, Han Z, Yao G F, Zhang H. 2020. PyWRKY26 and PybHLH3 cotargeted the PyMYB114 promoter to regulate anthocyanin biosynthesis and transport in red-skinned pears. Horticulture Research, 7, 37.

Liu H N, Su J, Zhu Y F, Yao G F, Allan A C, Ampomah-Dwamena C, Shu Q, Lin-Wang K, Zhang S L, Wu J. 2019. The involvement of PybZIPa in light-induced anthocyanin accumulation via the activation of PyUFGT through binding to tandem G-boxes in its promoter. Horticulture Research, 6, 134.

Liu Y F, Zhou B, Qi Y W, Chen X, Liu C H, Liu Z D, Ren X L. 2017. Expression differences of pigment structural genes and transcription factors explain flesh coloration in three contrasting kiwifruit cultivars. Frontiers in Plant Science, 8, 1507.

Luo X N, Luo S, Fu Y Q, Kong C, Wang K, Sun D Y, Li M C, Yan Z G, Shi Q Q, Zhang Y L. 2022. Genome-wide identification and comparative profiling of microRNAs reveal flavonoid biosynthesis in two contrasting flower color cultivars of tree peony. Frontiers in Plant Science, 12, 797799.

Maier A, Schrader A, Kokkelink L, Falke C, Welter B, Iniesto E, Rubio V, Uhrig J F, Hülskamp M, Hoecker U. 2013. Light and the E3 ubiquitin ligase COP1/SPA control the protein stability of the MYB transcription factors PAP1 and PAP2 involved in anthocyanin accumulation in Arabidopsis. The Plant Journal, 74, 638–651.

Matus J T, Aquea F, Arce-Johnson P. 2008. Analysis of the grape MYB R2R3 subfamily reveals expanded wine quality related clades and conserved gene structure organization across Vitis and Arabidopsis genomes. BMC Plant Biology, 8, 83.

Morishita T, Kojima Y, Maruta T, Nishizawa-Yokoi A, Yabuta Y, Shigeoka S. 2009. Arabidopsis NAC transcription factor, ANAC078, regulates flavonoid biosynthesis under high-light. Plant and Cell Physiology, 50, 2210–2222.

Nesi N, Debeaujon I, Jond C, Stewart A J, Jenkins G I, Caboche M, Lepiniec L. 2002. The TRANSPARENT TESTA16 locus encodes the ARABIDOPSIS BSISTER MADS domain protein and is required for proper development and pigmentation of the seed coat. The Plant Cell, 14, 2463–2479.

Ni J B, Bai S L, Zhao Y, Qian M J, Tao R Y, Yin L, Gao L, Teng Y W. 2019. Ethylene response factors Pp4ERF24 and Pp12ERF96 regulate blue light-induced anthocyanin biosynthesis in ‘Red Zaosu’ pear fruits by interacting with MYB114. Plant Molecular Biology, 99, 67–78.

Ni J B, Premathilake A T, Gao Y H, Yu W J, Tao R Y, Teng Y W, Bai S L. 2021. Ethylene-activated PpERF105 induces the expression of the repressor-type R2R3-MYB gene PpMYB140 to inhibit anthocyanin biosynthesis in red pear fruit. The Plant Journal, 105, 167–181.

Ou C Q, Zhang X L, Wang F, Zhang L Y, Zhang Y J, Fang M, Wang J H, Wang J X, Jiang S L, Zhang Z H. 2020. A 14 nucleotide deletion mutation in the coding region of the PpBBX24 gene is associated with the red skin of “Zaosu Red” pear (Pyrus pyrifolia White Pear Group): a deletion in the PpBBX24 gene is associated with the red skin of pear. Horticulture Research, 3, 39.

Pierantoni L, Dondini L, Franceschi P D, Musacchi S, Winke B S J, Sansavini S. 2010. Mapping of an anthocyanin-regulating MYB transcription factor and its expression in red and green pear Pyrus communis. Plant Physiology and Biochemistry, 48, 1020–1026.

Qi T C, Song S S, Ren Q C, Wu D W, Huang H, Chen Y, Fan M, Peng W, Ren C M, Xie D X. 2011. The jasmonate-ZIM-domain proteins interact with the WD-repeat/bHLH/MYB complexes to regulate jasmonate-mediated anthocyanin accumulation and trichome initiation in Arabidopsis thaliana. The Plant Cell, 23, 1795–1814.

Rubtsov G. 1944. Geographical distribution of the genus Pyrus and trends and factors in its evolution. The American Naturalist, 78, 358–366.

Salvatierra A, Pimentel P, Moya-León M A, Herrera R. 2013. Increased accumulation of anthocyanins in Fragaria chiloensis fruits by transient suppression of FcMYB1 gene. Phytochemistry, 90, 25–36.

Sasaki K, Mitsuhara I, Seo S, Ito H, Matsui H, Ohashi Y. 2007. Two novel AP2/ERF domain proteins interact with cis-element VWRE for wound-induced expression of the tobacco tpoxN1 gene. The Plant Journal, 50, 1079–1092.

Shin D H, Choi M, Kim K, Bang G, Cho M, Choi S B, Choi G, Park Y I. 2013. HY5 regulates anthocyanin biosynthesis by inducing the transcriptional activation of the MYB75/PAP1 transcription factor in Arabidopsis. FEBS Letters, 587, 1543–1547.

Shin J, Park E, Choi G. 2007. PIF3 regulates anthocyanin biosynthesis in an HY5-dependent manner with both factors directly binding anthocyanin biosynthetic gene promoters in Arabidopsis. The Plant Journal, 49, 981–994.

Steyn W J, Wand S J E, Holcroft D M, Jacobs G. 2005. Red colour development and loss in pears. Acta Horticulturae, 671, 79–85.

Wang N, Liu W J, Zhang T L, Jiang S H, Xu H F, Wang Y C, Zhang Z Y, Wang C Z, Chen X S. 2018. Transcriptomic analysis of red-fleshed apples reveals the novel role of MdWRKY11 in flavonoid and anthocyanin biosynthesis. Journal of Agricultural and Food Chemistry, 66, 7076–7086.

Wang X P, Niu Y L, Zheng Y. 2017. Multiple functions of MYB transcription factors in abiotic stress responses. International Journal of Molecular Sciences, 22, 6125.

Winkel-Shirley B. 2001. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiology, 126, 485–493.

Wu J, Wang Z W, Shi Z B, Zhang S, Ming R, Zhu S L, Khan M A, Tao S T, Korban S S, Wang H, Chen N J, Nishio T, Xu X, Cong L, Qi K J, Huang X S, Wang Y T, Zhao X, Wu J Y, Deng C, et al. 2013. The genome of the pear (Pyrus bretschneideri Rehd.). Genome Research, 23, 396–408.

Wu L J, Zhang Z J, Zhang H W, Wang X C, Huang R F. 2008. Transcriptional modulation of ethylene response factor protein JERF3 in the oxidative stress response enhances tolerance of tobacco seedlings to salt, drought, and freezing. Plant Physiology, 148, 1953–1963.

Wu T, Liu H T, Zhao G P, Song J X, Wang X L, Yang C Q, Zhai R, Wang Z G, Ma F W, Xu L F. 2020. Jasmonate and ethylene-regulated ethylene response factor 22 promotes lanolin-induced anthocyanin biosynthesis in ‘Zaosu’ pear (Pyrus bretschneideri Rehd.) fruit. Biomolecules, 10, 278.

Yao G F, Ming M L, Allan A C, Gu C, Li L T, Wu X, Wang R Z, Chang Y J, Qi K J, Zhang S L, Wu J. 2017. Map-based cloning of the pear gene MYB114 identifies an interaction with other transcription factors to coordinately regulate fruit anthocyanin biosynthesis. The Plant Journal, 92, 437–451.

Yu B, Zhang D, Huang C H, Qian M J, Zheng X Y, Teng Y W. 2012. Isolation of anthocyanin biosynthetic genes in red Chinese sand pear (Pyrus pyrifolia Nakai) and their expression as affected by organ, tissue, cultivar, bagging and fruit size. Scientia Horticulturae, 136, 29–37.

Zhang J, Xu H F, Wang N, Jiang S H, Fang H C, Zhang Z Y, Yang G X, Wang Y C, Su M Y, Xu L, Chen X S. 2018. The ethylene response factor MdERF1B regulates anthocyanin and proanthocyanidin biosynthesis in apple. Plant Molecular Biology, 98, 205–218.

Zhang S Y, Chen Y X, Zhao L L, Li C Q, Yu J Y, Li T T, Wang W Y, Zhang S N, Su H Y, Wang L. 2020. A novel NAC transcription factor, MdNAC42, regulates anthocyanin accumulation in red-fleshed apple by interacting with MdMYB10. Tree Physiology, 40, 413–423.

Zhang X D, Allan A C, Yi Q, Chen L M, Li K Z, Su J. 2011. Differential gene expression analysis of Yunnan Red Pear, Pyrus pyrifolia, during fruit skin coloration. Plant Molecular Biology Reports, 29, 305–314.

Zhou H, Lin-Wang K, Wang H L, Gu C, Dare A P, Espley R V, He H P, Allan A C, Han Y P. 2015. Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors. The Plant Journal, 82, 105–121.

Zhou Y, Zhou H, Lin-Wang K, Vimolmangkang S, Espley R V, Wang L, Allan A C, Han Y P. 2014. Transcriptome analysis and transient transformation suggest an ancient duplicated MYB transcription factor as a candidate gene for leaf red colration in peach. BMC Plant Biology, 14, 388.