‘秋姬李’PsMYB18基因克隆与功能分析
|
摘要
【目的】克隆在采后‘秋姬李’果皮积累花色苷过程中高表达的MYB抑制因子PsMYB18基因,研究其序列特征、表达特点与功能。【方法】以‘秋姬李’为试材,采用qRT-PCR分析不同温度和光照处理条件下‘秋姬李’果皮中PsMYB18基因的转录水平,采用RT-PCR克隆PsMYB18基因,并通过烟草叶片瞬时表达试验分析PsMYB18的功能。【结果】q RT-PCR分析表明20℃和光照处理可促进‘秋姬李’果皮PsMYB18基因表达。PsMYB18基因的开放阅读框(ORF)为702 bp,编码233个氨基酸的蛋白。进化树分析表明PsMYB18与其他植物的花色苷合成抑制因子亲缘关系较近。序列比对结果表明其具有保守的R2R3结构域和抑制基序C1和C2。烟草瞬时表达试验表明,PsMYB18可抑制正调控因子PsMYB10.1和PsbHLH3的花色苷合成诱导功能。【结论】‘秋姬李’PsMYB18为花色苷合成抑制因子。
【Objective】Temperature and light are important environmental factors that affect the biosynthesis and accumulation of anthocyanin in the fruit peel. Our previous study indicated that 20 ℃ temperature and light treatment(150 mol·m~(-2)·s~(-1)) could induce anthocyanin accumulation in the peel of postharvest'Akihime'plums. Comparative transcriptomic analysis results demonstrated that the expression of a R2 R3-MYB gene(designated as PsMYB18) was significantly upregulated in the fruit peel treated at20 ℃+ light. The objective of the study was to isolate and elucidate the function of PsMYB18 gene during anthocyanin accumulation in the peel of'Akihime'plum.【Methods】The total RNA of the plum fruit peels was extracted using EZNA Plant RNA Kit(Omega Bio-tek, USA) according to manufacturer's instructions. Real-time quantitative PCR(qRT-PCR) was employed to determine the expression of PsMYB18 in the peel of'Akihime'plum fruits treated at 20 ℃ + light, 20 ℃+ dark, 30 ℃+ light and30 ℃+ dark. First-strand cDNA was synthesized from 500 ng of total RNA using the PrimeScript RT reagent kit with gDNA Eraser(Takara, China). The qRT-PCR was performed using the Eppendorf Realplex4 real-time PCR system(Hamburg, Germany) in a total volume of 20 μL in each well containing 10μL of 2×SYBR~? Premix Ex Taq? II(Tli RNaseH Plus, TaKaRa), 1 μL of cDNA(in 1:10 dilution), and0.4 μL 10 μmol primers. The qPCR conditions were 30 s at 95 ℃, followed by 40 cycles of 5 s at 95 ℃,15 s at 60 ℃, and 30 s at 72 ℃, followed by 60 ℃ to 95 ℃ melting curve detection. Actin gene was used as the reference. For gene cloning, total RNA was extracted from the fruit peel treated with 20 ℃and light. The first-strand cDNA was synthesized using First Strand cDNA Synthesis Kit(Thermo Scientific, USA). PsMYB18 gene was isolated from the skin of'Akihime'plums by reverse transcription polymerase chain reaction(RT-PCR). Phylogenetic tree was constructed using the MEGA 5.02 software to investigate the evolution relationship between PsMYB18 and MYB proteins from other plant species.Multiple sequence alignments of PsMYB18 protein with MYB repressors from peach, apple, grape, poplar, Medicago truncatula, Fragaria × ananassa and Antirrhinum majus were performed using DNAMAN7.02. p CAMBIA1302 vector, which was linearized by restriction enzyme NcoI and BstEII was used to construct overexpression vectors. The coding regions of PsMYB18, PsMYB10.1 and PsbHLH3 were isolated using I-5? 2×High-Fidelity Master Mix(MCLAB, San Francisco, CA) and then cloned into the pCAMBIA1302 vector using One Step Seamless Cloning kit(Aidlab, China). All constructs were introduced into Agrobacterium tumefaciens GV3101. Tobacco(Nicotiana tabacum L.) leaf transient expression assay was performed to verify the function of PsMYB18 in anthocyanin biosynthesis. The Agrobacterium strain GV3101 containing overexpression vectors were grown at 28 ℃ in LB medium containing with antibiotics. Then Agrobacterium cells were harvested and resuspended in the infiltration buffer(10 mmol·L~(-1) MgCl2, 10 mmol·L~(-1) MES, pH 5.6, 100 μmol·L~(-1) acetosyringone) to a final OD600 of 0.8. The bacteria were incubated at room temperature for 3 h before infiltration. Digital photographs of anthocyanin development in tobacco leaves were taken at 7 days after infiltration.【Results】The qRTPCR analysis revealed that the expression of PsMYB18 in the fruit peel treated at 20 ℃ and light was significantly enhanced after 3 d of treatment and remained at a high level thereafter. However, no obvious expression upregulation of PsMYB18 was detected in the fruit peel treated at 20 ℃ + dark, 30 ℃ +light and 30 ℃ + dark. These results were consistent with RNA-seq data. The cDNA sequence of PsMYB18 was isolated from the peel of'Akihime'fruits treated at 20 ℃ and light. PsMYB18 contained open reading frame(ORF) of 702 bp in length, which was predicted to encode a protein of 539 amino acid residues. The results of phylogenetic analysis revealed that PsMYB18 and repressors of anthocyanin and proanthocyanidin biosynthesis, including FaMYB1, MtMYB2, PtMYB182, VvMYBC2-L3, VvMYBC2-L1 and PpMYB18, from other plants belonging to the same class. It had the closest relationship with PpMYB18 from peach. Multiple alignment of the deduced amino acid sequences of PsMYB18 and MYB repressors showed that PsMYB18 shared similarity of 96.57% with the PpMYB18 protein and contained the conserved R2 and R3 domains. It also had C1 and C2 repression motifs in the C-terminus. These results implicated that PsMYB18 could be a repressor of anthocyanin biosynthesis.To investigate the role of PsMYB18 in anthocyanin biosynthesis, the ORF of PsMYB18 was amplified and ligated into the pCAMBIA1302 vector. Transient color assays in tobacco leaves showed that coinfiltration of PsMYB18 with PsbHLH3 could not induce anthocyanin accumulation. Furthermore, coinfiltration of PsMYB10.1 with PsbHLH3 resulted in red pigmentation after 7 days, while no pigmentation was observed when PsMYB10.1 and PsbHLH3 were co-infiltrated with PsMYB18.【Conclusion】In this study, PsMYB18 was isolated and its expression was enhanced by the treatment at 20 ℃ and light. Phylogenetic and sequence analysis suggested that Pp MYB18 was likely a repressor of flavonoid biosynthesis. Transient color assay results showed that PsMYB18 was a negative regulator of anthocyanin biosynthesis.
【Objective】Temperature and light are important environmental factors that affect the biosynthesis and accumulation of anthocyanin in the fruit peel. Our previous study indicated that 20 ℃ temperature and light treatment(150 mol·m~(-2)·s~(-1)) could induce anthocyanin accumulation in the peel of postharvest'Akihime'plums. Comparative transcriptomic analysis results demonstrated that the expression of a R2 R3-MYB gene(designated as PsMYB18) was significantly upregulated in the fruit peel treated at20 ℃+ light. The objective of the study was to isolate and elucidate the function of PsMYB18 gene during anthocyanin accumulation in the peel of'Akihime'plum.【Methods】The total RNA of the plum fruit peels was extracted using EZNA Plant RNA Kit(Omega Bio-tek, USA) according to manufacturer's instructions. Real-time quantitative PCR(qRT-PCR) was employed to determine the expression of PsMYB18 in the peel of'Akihime'plum fruits treated at 20 ℃ + light, 20 ℃+ dark, 30 ℃+ light and30 ℃+ dark. First-strand cDNA was synthesized from 500 ng of total RNA using the PrimeScript RT reagent kit with gDNA Eraser(Takara, China). The qRT-PCR was performed using the Eppendorf Realplex4 real-time PCR system(Hamburg, Germany) in a total volume of 20 μL in each well containing 10μL of 2×SYBR~? Premix Ex Taq? II(Tli RNaseH Plus, TaKaRa), 1 μL of cDNA(in 1:10 dilution), and0.4 μL 10 μmol primers. The qPCR conditions were 30 s at 95 ℃, followed by 40 cycles of 5 s at 95 ℃,15 s at 60 ℃, and 30 s at 72 ℃, followed by 60 ℃ to 95 ℃ melting curve detection. Actin gene was used as the reference. For gene cloning, total RNA was extracted from the fruit peel treated with 20 ℃and light. The first-strand cDNA was synthesized using First Strand cDNA Synthesis Kit(Thermo Scientific, USA). PsMYB18 gene was isolated from the skin of'Akihime'plums by reverse transcription polymerase chain reaction(RT-PCR). Phylogenetic tree was constructed using the MEGA 5.02 software to investigate the evolution relationship between PsMYB18 and MYB proteins from other plant species.Multiple sequence alignments of PsMYB18 protein with MYB repressors from peach, apple, grape, poplar, Medicago truncatula, Fragaria × ananassa and Antirrhinum majus were performed using DNAMAN7.02. p CAMBIA1302 vector, which was linearized by restriction enzyme NcoI and BstEII was used to construct overexpression vectors. The coding regions of PsMYB18, PsMYB10.1 and PsbHLH3 were isolated using I-5? 2×High-Fidelity Master Mix(MCLAB, San Francisco, CA) and then cloned into the pCAMBIA1302 vector using One Step Seamless Cloning kit(Aidlab, China). All constructs were introduced into Agrobacterium tumefaciens GV3101. Tobacco(Nicotiana tabacum L.) leaf transient expression assay was performed to verify the function of PsMYB18 in anthocyanin biosynthesis. The Agrobacterium strain GV3101 containing overexpression vectors were grown at 28 ℃ in LB medium containing with antibiotics. Then Agrobacterium cells were harvested and resuspended in the infiltration buffer(10 mmol·L~(-1) MgCl2, 10 mmol·L~(-1) MES, pH 5.6, 100 μmol·L~(-1) acetosyringone) to a final OD600 of 0.8. The bacteria were incubated at room temperature for 3 h before infiltration. Digital photographs of anthocyanin development in tobacco leaves were taken at 7 days after infiltration.【Results】The qRTPCR analysis revealed that the expression of PsMYB18 in the fruit peel treated at 20 ℃ and light was significantly enhanced after 3 d of treatment and remained at a high level thereafter. However, no obvious expression upregulation of PsMYB18 was detected in the fruit peel treated at 20 ℃ + dark, 30 ℃ +light and 30 ℃ + dark. These results were consistent with RNA-seq data. The cDNA sequence of PsMYB18 was isolated from the peel of'Akihime'fruits treated at 20 ℃ and light. PsMYB18 contained open reading frame(ORF) of 702 bp in length, which was predicted to encode a protein of 539 amino acid residues. The results of phylogenetic analysis revealed that PsMYB18 and repressors of anthocyanin and proanthocyanidin biosynthesis, including FaMYB1, MtMYB2, PtMYB182, VvMYBC2-L3, VvMYBC2-L1 and PpMYB18, from other plants belonging to the same class. It had the closest relationship with PpMYB18 from peach. Multiple alignment of the deduced amino acid sequences of PsMYB18 and MYB repressors showed that PsMYB18 shared similarity of 96.57% with the PpMYB18 protein and contained the conserved R2 and R3 domains. It also had C1 and C2 repression motifs in the C-terminus. These results implicated that PsMYB18 could be a repressor of anthocyanin biosynthesis.To investigate the role of PsMYB18 in anthocyanin biosynthesis, the ORF of PsMYB18 was amplified and ligated into the pCAMBIA1302 vector. Transient color assays in tobacco leaves showed that coinfiltration of PsMYB18 with PsbHLH3 could not induce anthocyanin accumulation. Furthermore, coinfiltration of PsMYB10.1 with PsbHLH3 resulted in red pigmentation after 7 days, while no pigmentation was observed when PsMYB10.1 and PsbHLH3 were co-infiltrated with PsMYB18.【Conclusion】In this study, PsMYB18 was isolated and its expression was enhanced by the treatment at 20 ℃ and light. Phylogenetic and sequence analysis suggested that Pp MYB18 was likely a repressor of flavonoid biosynthesis. Transient color assay results showed that PsMYB18 was a negative regulator of anthocyanin biosynthesis.
引文
[1]刘晓芬,李方,殷学仁,徐昌杰,陈昆松.花青苷生物合成转录调控研究进展[J].园艺学报,2013,40(11):2295-2306.LIU Xiaofen,LI Fang,YIN Xueren,XU Changjie,CHEN Kunsong. Recent advances in the transcriptional regulation of anthocyanin biosynthesis[J]. Acta Horticulturae Sinica,2013,40(11):2295-2306.
[2] CHENG Y,LIU L,YUAN C,GUAN J. Molecular characterization of ethylene-regulated anthocyanin biosynthesis in plums during fruit ripening[J]. Plant Molecular Biology Reporter,2016,34(4):777-785.
[3] BAI S,SUN Y,QIAN M,YANG F,NI J,TAO R,LI L,SHU Q,ZHANG D,TENG Y. Transcriptome analysis of bagging-treated red Chinese sand pear peels reveals light-responsive pathway functions in anthocyanin accumulation[J]. Scientific Reports,2017,7(1):63.
[4] TAO R,BAI S,NI J,YANG Q,ZHAO Y,TENG Y. The blue light signal transduction pathway is involved in anthocyanin accumulation in‘Red Zaosu’pear[J]. Planta,2018,248(1):37-48.
[5] WANG N,QU C,JIANG S,CHEN Z,XU H,FANG H,SU M,ZHANG J,WANG Y,LIU W,ZHANG Z,LU N,CHEN X. The proanthocyanidin-specific transcription factor MdMYBPA1 initiates anthocyanin synthesis under low-temperature conditions in red-fleshed apples[J]. The Plant Journal,2018,96(1):39-55.
[6] PAN H,WANG R,LI L,WANG J,CAO J,JIANG W. Manipulation of ripening progress of different plum cultivars during shelf life by post-storage treatments with ethylene and 1-methylcyclopropene[J]. Scientia Horticulturae,2016,198:176-182.
[7] ZHAO J,DIXON R A. The‘ins’and‘outs’of flavonoid transport[J]. Trends in Plant Science,2010,15(2):72-80.
[8] ALLAN A C,HELLENS R P,LAING W A. MYB transcription factors that colour our fruit[J]. Trends in Plant Science,2008,13(3):99-102.
[9] ESPLEY R V,HELLENS R P,PUTTERILL J,STEVENSON D E,KUTTY-AMMA S,ALLAN A C. Red colouration in apple fruit is due to the activity of the MYB transcription factor,MdMYB10[J].The Plant Journal,2007,49(3):414-427.
[10] ZHANG Y,FANG Z,YE X,PAN S. Identification of candidate genes involved in anthocyanin accumulation in the peel of jaboticaba(Myrciaria cauliflora)fruits by transcriptomic analysis[J]. Gene,2018,676:202-213.
[11] WANG N,LIU W,ZHANG T,JIANG S,XU H,WANG Y,ZHANG Z,WANG C,CHEN X. Transcriptomic analysis of redfleshed apples reveals the novel role of MdWRKY11 in flavonoid and anthocyanin biosynthesis[J]. Journal of Agricultural and Food Chemistry,2018,66(27):7076-7086.
[12] LIU C C,CHI C,JIN L J,ZHU J,YU J Q,ZHOU Y H. The bZip transcription factor HY5 mediates CRY1a-induced anthocyanin biosynthesis in tomato[J]. Plant,Cell and Environment,2018,41(8):1762-1775.
[13] AN J P,YAO J F,XU R R,YOU C X,WANG X F,HAO Y J.Apple bZIP transcription factor MdbZIP44 regulates ABA-promoted anthocyanin accumulation[J]. Plant,Cell and Environment,2018,41(11):2678-2692.
[14] ZHOU H,WANG L K,WANG H,GU C,DARE A P,ESPLEY R V,HE H,ALLAN A C,HAN Y. Molecular genetics of bloodfleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors[J]. The Plant Journal,2015,82(1):105-121.
[15] PREZ-DAZ J,PREZ-DAZ J,MADRID-ESPINOZA J,GONZ LEZ-VILLANUEVA E,MORENO Y,RUIZ-LARA S. New member of the R2R3-MYB transcription factors family in grapevine suppresses the anthocyanin accumulation in the flowers of transgenic tobacco[J]. Plant Molecular Biology,2015,90(1):63-76.
[16] HUANG Y F,VIALET S,GUIRAUD JL,TORREGROSA L,BERTRAND Y,CHEYNIER V,THIS P,TERRIER N. A negative MYB regulator of proanthocyanidin accumulation,identified through expression quantitative locus mapping in the grape berry[J]. New Phytologist,2014,201(3):795-809.
[17] ZHOU H,LIN-WANG K,WANG F,ESPLEY R V,REN F,ZHAO J,OGUTU C,HE H,JIANG Q,ALLAN A C,HAN Y.Activator-type R2R3-MYB genes induce a repressor-type R2R3-MYB gene to balance anthocyanin and proanthocyanidin accumulation[J]. New Phytologist,2019,221(4):1919-1934.
[18] KOES R,VERWEIJ W,QUATTROCCHIO F. Flavonoids:a colorful model for the regulation and evolution of biochemical pathways[J]. Trends in Plant Science,2005,10(5):236-242.
[19] ZHANG X Y,ZHANG S L,LUO J,YE Z W,GAO Q H,DU J H,WANG X Q. Developmental changes in anthocyanin biosynthesis in plum skins and the effects of ethephon[J]. The Journal of Horticultural Science and Biotechnology,2009,84(5):495-498.
[20] JUN J H,LIU C,XIAO X,DIXON R A. The transcriptional repressor MYB2 regulates both spatial and temporal patterns of proanthocyandin and anthocyanin pigmentation in Medicago truncatula[J]. The Plant Cell,2015,27(10):2860-2879.
[21] TOMINAGA R,IWATA M,OKADA K,WADA T. Functional analysis of the epidermal-specific MYB genes CAPRICE and WEREWOLF in Arabidopsis[J]. The Plant Cell,2007,19(7):2264-2277.
[22] WANG S,HUBBARD L,CHANG Y,GUO J,SCHIEFELBEIN J,CHEN J G. Comprehensive analysis of single-repeat R3 MYB proteins in epidermal cell patterning and their transcriptional regulation in Arabidopsis[J]. BMC Plant Biology,2008,8(1):81.
[23] ZHU H F,FITZSIMMONS K,KHANDELWAL A,KRANZ R G. CPC,a single-repeat R3 MYB,is a negative regulator of anthocyanin biosynthesis in Arabidopsis[J]. Molecular Plant,2009,2(4):790-802.
[24] MATSUI K,UMEMURA Y,OHME-TAKAGI M. AtMYBL2,a protein with a single MYB domain,acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis[J]. The Plant Journal,2008,55(6):954-967.
[25] AHARONI A,DE VOS C H,WEIN M,SUN Z,GRECO R,KROON A,MOL J N,O'CONNELL A P. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco[J]. The Plant Journal,2001,28(3):319-332.
[26] HUANG D,WANG X,TANG Z,YUAN Y,XU Y,HE J,JIANG X,PENG S-A,LI L,BUTELLI E,DENG X,XU Q. Subfunctionalization of the Ruby2–Ruby1 gene cluster during the domestication of citrus[J]. Nature Plants,2018,4(11):930-941.
[27] JIN H,COMINELLI E,BAILEY P,PARR A,MEHRTENS F,JONES J,TONELLI C,WEISSHAAR B,MARTIN C. Transcriptional repression by AtMYB4 controls production of UV‐protecting sunscreens in Arabidopsis[J]. The EMBO Journal,2000,19(22):6150-6161.
[28] YOSHIDA K,MA D,CONSTABEL C P. The MYB182 protein downregulates proanthocyanidin and anthocyanin biosynthesis in poplar by repressing both structural and regulatory flavonoid genes[J]. Plant Physiology,2015,167(3):693-710.
[29] KRANZ H D,DENEKAMP M,GRECO R,JIN H,LEYVA A,MEISSNER R C,PETRONI K,URZAINQUI A,BEVAN M,MARTIN C,SMEEKENS S,TONELLI C,PAZ-ARES J,WEISSHAAR B. Towards functional characterisation of the members of the R2R3-MYB gene family from Arabidopsis thaliana[J].The Plant Journal,1998,16(2):263-276.
[30] LIN-WANG K,BOLITHO K,GRAFTON K,KORTSTEE A,KARUNAIRETNAM S,MCGHIE T,ESPLEY R,HELLENS R,ALLAN A. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae[J]. BMC Plant Biology,2010,10(1):50.
[31] GU C,LIAO L,ZHOU H,WANG L,DENG X,HAN Y. Constitutive activation of an anthocyanin regulatory gene PcMYB10.6is related to red coloration in purple-foliage plum[J]. PLoS ONE,2015,10(8):e0135159.
[32] FANG Z Z,ZHOU D R,YE X F,JIANG C C,PAN S L. Identification of candidate anthocyanin-related genes by transcriptomic analysis of‘Furongli’plum(Prunus salicina Lindl.)during fruit ripening using RNA-Seq[J]. Frontiers in Plant Science,2016,7:1338.
[33] FANG Z Z,LAI C C,ZHANG Y L,LIN Y L,LAI Z X. Identification of a PTC-containing DlRan transcript and its differential expression during somatic embryogenesis in Dimocarpus longan[J]. Gene,2013,529(1):37-44.
[34] LIN-WANG K,MICHELETTI D,PALMER J,VOLZ R,LOZANO L,ESPLEY R,HELLENS R P,CHAGNèD,ROWAN D D,TROGGIO M,IGLESIAS I,ALLAN A C. High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex[J]. Plant,Cell and Environment,2011,34(7):1176-1190.
[35] DUBOS C,STRACKE R,GROTEWOLD E,WEISSHAAR B,MARTIN C,LEPINIEC L. MYB transcription factors in Arabidopsis[J]. Trends in Plant Science,2010,15(10):573-581.
[36] CAVALLINI E,MATUS J T,FINEZZO L,ZENONI S,LOYOLA R,GUZZO F,SCHLECHTER R,AGEORGES A,ARCE-JOHNSON P,TORNIELLI G B. The phenylpropanoid pathway is controlled at different branches by a set of R2R3-MYB C2 repressors in grapevine[J]. Plant Physiology,2015,167(4):1448-1470.
[37] DUBOS C,LEGOURRIEREC J,BAUDRY A,HUEP G,LANET E,DEBEAUJON I,ROUTABOUL J M,ALBORESI A,WEISSHAAR B,LEPINIEC L. MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana[J].The Plant Journal,2008,55(6):940-953.
[38] ALBERT N W,LEWIS D H,ZHANG H,SCHWINN K E,JAMESON P E,DAVIES K M. Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning[J]. The Plant Journal,2011,65(5):771-784.
[39] P REZ-D AZ J R,P REZ-DAZ J,MADRID-ESPINOZA J,GONZ LEZ-VILLANUEVA E,MORENO Y,RUIZ-LARA S.New member of the R2R3-MYB transcription factors family in grapevine suppresses the anthocyanin accumulation in the flowers of transgenic tobacco[J]. Plant Molecular Biology,2016,90(1):63-76.
[40] ALBERT N W. Subspecialization of R2R3-MYB repressors for anthocyanin and proanthocyanidin regulation in forage legumes[J]. Frontiers Plant Science,2015,6:1165.
[41] MA D,REICHELT M,YOSHIDA K,GERSHENZON J,CONSTABEL C P. Two R2R3-MYB proteins are broad repressors of flavonoid and phenylpropanoid metabolism in poplar[J]. The Plant Journal,2018,96(5):949-965.
[2] CHENG Y,LIU L,YUAN C,GUAN J. Molecular characterization of ethylene-regulated anthocyanin biosynthesis in plums during fruit ripening[J]. Plant Molecular Biology Reporter,2016,34(4):777-785.
[3] BAI S,SUN Y,QIAN M,YANG F,NI J,TAO R,LI L,SHU Q,ZHANG D,TENG Y. Transcriptome analysis of bagging-treated red Chinese sand pear peels reveals light-responsive pathway functions in anthocyanin accumulation[J]. Scientific Reports,2017,7(1):63.
[4] TAO R,BAI S,NI J,YANG Q,ZHAO Y,TENG Y. The blue light signal transduction pathway is involved in anthocyanin accumulation in‘Red Zaosu’pear[J]. Planta,2018,248(1):37-48.
[5] WANG N,QU C,JIANG S,CHEN Z,XU H,FANG H,SU M,ZHANG J,WANG Y,LIU W,ZHANG Z,LU N,CHEN X. The proanthocyanidin-specific transcription factor MdMYBPA1 initiates anthocyanin synthesis under low-temperature conditions in red-fleshed apples[J]. The Plant Journal,2018,96(1):39-55.
[6] PAN H,WANG R,LI L,WANG J,CAO J,JIANG W. Manipulation of ripening progress of different plum cultivars during shelf life by post-storage treatments with ethylene and 1-methylcyclopropene[J]. Scientia Horticulturae,2016,198:176-182.
[7] ZHAO J,DIXON R A. The‘ins’and‘outs’of flavonoid transport[J]. Trends in Plant Science,2010,15(2):72-80.
[8] ALLAN A C,HELLENS R P,LAING W A. MYB transcription factors that colour our fruit[J]. Trends in Plant Science,2008,13(3):99-102.
[9] ESPLEY R V,HELLENS R P,PUTTERILL J,STEVENSON D E,KUTTY-AMMA S,ALLAN A C. Red colouration in apple fruit is due to the activity of the MYB transcription factor,MdMYB10[J].The Plant Journal,2007,49(3):414-427.
[10] ZHANG Y,FANG Z,YE X,PAN S. Identification of candidate genes involved in anthocyanin accumulation in the peel of jaboticaba(Myrciaria cauliflora)fruits by transcriptomic analysis[J]. Gene,2018,676:202-213.
[11] WANG N,LIU W,ZHANG T,JIANG S,XU H,WANG Y,ZHANG Z,WANG C,CHEN X. Transcriptomic analysis of redfleshed apples reveals the novel role of MdWRKY11 in flavonoid and anthocyanin biosynthesis[J]. Journal of Agricultural and Food Chemistry,2018,66(27):7076-7086.
[12] LIU C C,CHI C,JIN L J,ZHU J,YU J Q,ZHOU Y H. The bZip transcription factor HY5 mediates CRY1a-induced anthocyanin biosynthesis in tomato[J]. Plant,Cell and Environment,2018,41(8):1762-1775.
[13] AN J P,YAO J F,XU R R,YOU C X,WANG X F,HAO Y J.Apple bZIP transcription factor MdbZIP44 regulates ABA-promoted anthocyanin accumulation[J]. Plant,Cell and Environment,2018,41(11):2678-2692.
[14] ZHOU H,WANG L K,WANG H,GU C,DARE A P,ESPLEY R V,HE H,ALLAN A C,HAN Y. Molecular genetics of bloodfleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors[J]. The Plant Journal,2015,82(1):105-121.
[15] PREZ-DAZ J,PREZ-DAZ J,MADRID-ESPINOZA J,GONZ LEZ-VILLANUEVA E,MORENO Y,RUIZ-LARA S. New member of the R2R3-MYB transcription factors family in grapevine suppresses the anthocyanin accumulation in the flowers of transgenic tobacco[J]. Plant Molecular Biology,2015,90(1):63-76.
[16] HUANG Y F,VIALET S,GUIRAUD JL,TORREGROSA L,BERTRAND Y,CHEYNIER V,THIS P,TERRIER N. A negative MYB regulator of proanthocyanidin accumulation,identified through expression quantitative locus mapping in the grape berry[J]. New Phytologist,2014,201(3):795-809.
[17] ZHOU H,LIN-WANG K,WANG F,ESPLEY R V,REN F,ZHAO J,OGUTU C,HE H,JIANG Q,ALLAN A C,HAN Y.Activator-type R2R3-MYB genes induce a repressor-type R2R3-MYB gene to balance anthocyanin and proanthocyanidin accumulation[J]. New Phytologist,2019,221(4):1919-1934.
[18] KOES R,VERWEIJ W,QUATTROCCHIO F. Flavonoids:a colorful model for the regulation and evolution of biochemical pathways[J]. Trends in Plant Science,2005,10(5):236-242.
[19] ZHANG X Y,ZHANG S L,LUO J,YE Z W,GAO Q H,DU J H,WANG X Q. Developmental changes in anthocyanin biosynthesis in plum skins and the effects of ethephon[J]. The Journal of Horticultural Science and Biotechnology,2009,84(5):495-498.
[20] JUN J H,LIU C,XIAO X,DIXON R A. The transcriptional repressor MYB2 regulates both spatial and temporal patterns of proanthocyandin and anthocyanin pigmentation in Medicago truncatula[J]. The Plant Cell,2015,27(10):2860-2879.
[21] TOMINAGA R,IWATA M,OKADA K,WADA T. Functional analysis of the epidermal-specific MYB genes CAPRICE and WEREWOLF in Arabidopsis[J]. The Plant Cell,2007,19(7):2264-2277.
[22] WANG S,HUBBARD L,CHANG Y,GUO J,SCHIEFELBEIN J,CHEN J G. Comprehensive analysis of single-repeat R3 MYB proteins in epidermal cell patterning and their transcriptional regulation in Arabidopsis[J]. BMC Plant Biology,2008,8(1):81.
[23] ZHU H F,FITZSIMMONS K,KHANDELWAL A,KRANZ R G. CPC,a single-repeat R3 MYB,is a negative regulator of anthocyanin biosynthesis in Arabidopsis[J]. Molecular Plant,2009,2(4):790-802.
[24] MATSUI K,UMEMURA Y,OHME-TAKAGI M. AtMYBL2,a protein with a single MYB domain,acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis[J]. The Plant Journal,2008,55(6):954-967.
[25] AHARONI A,DE VOS C H,WEIN M,SUN Z,GRECO R,KROON A,MOL J N,O'CONNELL A P. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco[J]. The Plant Journal,2001,28(3):319-332.
[26] HUANG D,WANG X,TANG Z,YUAN Y,XU Y,HE J,JIANG X,PENG S-A,LI L,BUTELLI E,DENG X,XU Q. Subfunctionalization of the Ruby2–Ruby1 gene cluster during the domestication of citrus[J]. Nature Plants,2018,4(11):930-941.
[27] JIN H,COMINELLI E,BAILEY P,PARR A,MEHRTENS F,JONES J,TONELLI C,WEISSHAAR B,MARTIN C. Transcriptional repression by AtMYB4 controls production of UV‐protecting sunscreens in Arabidopsis[J]. The EMBO Journal,2000,19(22):6150-6161.
[28] YOSHIDA K,MA D,CONSTABEL C P. The MYB182 protein downregulates proanthocyanidin and anthocyanin biosynthesis in poplar by repressing both structural and regulatory flavonoid genes[J]. Plant Physiology,2015,167(3):693-710.
[29] KRANZ H D,DENEKAMP M,GRECO R,JIN H,LEYVA A,MEISSNER R C,PETRONI K,URZAINQUI A,BEVAN M,MARTIN C,SMEEKENS S,TONELLI C,PAZ-ARES J,WEISSHAAR B. Towards functional characterisation of the members of the R2R3-MYB gene family from Arabidopsis thaliana[J].The Plant Journal,1998,16(2):263-276.
[30] LIN-WANG K,BOLITHO K,GRAFTON K,KORTSTEE A,KARUNAIRETNAM S,MCGHIE T,ESPLEY R,HELLENS R,ALLAN A. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae[J]. BMC Plant Biology,2010,10(1):50.
[31] GU C,LIAO L,ZHOU H,WANG L,DENG X,HAN Y. Constitutive activation of an anthocyanin regulatory gene PcMYB10.6is related to red coloration in purple-foliage plum[J]. PLoS ONE,2015,10(8):e0135159.
[32] FANG Z Z,ZHOU D R,YE X F,JIANG C C,PAN S L. Identification of candidate anthocyanin-related genes by transcriptomic analysis of‘Furongli’plum(Prunus salicina Lindl.)during fruit ripening using RNA-Seq[J]. Frontiers in Plant Science,2016,7:1338.
[33] FANG Z Z,LAI C C,ZHANG Y L,LIN Y L,LAI Z X. Identification of a PTC-containing DlRan transcript and its differential expression during somatic embryogenesis in Dimocarpus longan[J]. Gene,2013,529(1):37-44.
[34] LIN-WANG K,MICHELETTI D,PALMER J,VOLZ R,LOZANO L,ESPLEY R,HELLENS R P,CHAGNèD,ROWAN D D,TROGGIO M,IGLESIAS I,ALLAN A C. High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex[J]. Plant,Cell and Environment,2011,34(7):1176-1190.
[35] DUBOS C,STRACKE R,GROTEWOLD E,WEISSHAAR B,MARTIN C,LEPINIEC L. MYB transcription factors in Arabidopsis[J]. Trends in Plant Science,2010,15(10):573-581.
[36] CAVALLINI E,MATUS J T,FINEZZO L,ZENONI S,LOYOLA R,GUZZO F,SCHLECHTER R,AGEORGES A,ARCE-JOHNSON P,TORNIELLI G B. The phenylpropanoid pathway is controlled at different branches by a set of R2R3-MYB C2 repressors in grapevine[J]. Plant Physiology,2015,167(4):1448-1470.
[37] DUBOS C,LEGOURRIEREC J,BAUDRY A,HUEP G,LANET E,DEBEAUJON I,ROUTABOUL J M,ALBORESI A,WEISSHAAR B,LEPINIEC L. MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana[J].The Plant Journal,2008,55(6):940-953.
[38] ALBERT N W,LEWIS D H,ZHANG H,SCHWINN K E,JAMESON P E,DAVIES K M. Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning[J]. The Plant Journal,2011,65(5):771-784.
[39] P REZ-D AZ J R,P REZ-DAZ J,MADRID-ESPINOZA J,GONZ LEZ-VILLANUEVA E,MORENO Y,RUIZ-LARA S.New member of the R2R3-MYB transcription factors family in grapevine suppresses the anthocyanin accumulation in the flowers of transgenic tobacco[J]. Plant Molecular Biology,2016,90(1):63-76.
[40] ALBERT N W. Subspecialization of R2R3-MYB repressors for anthocyanin and proanthocyanidin regulation in forage legumes[J]. Frontiers Plant Science,2015,6:1165.
[41] MA D,REICHELT M,YOSHIDA K,GERSHENZON J,CONSTABEL C P. Two R2R3-MYB proteins are broad repressors of flavonoid and phenylpropanoid metabolism in poplar[J]. The Plant Journal,2018,96(5):949-965.