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植物花青素合成与调控研究进展

花匠小妙招
2024-11-13 04:33

0 引言

花青素是糖基化的多酚类化合物,又称花青素苷,是植物类黄酮化合物中的重要一类。花青素广泛存在于植物叶、花和果实等器官中,是植物呈现五颜六色的基础。花青素的基本结构是3,5,7-羟基-2-苯基苯并吡喃,根据结构不同可分为6大类:天竺葵素(Pelargonidin)、矢车菊素(Cyanidin)、芍药花素(Peonidin)、飞燕草素(Delphinidin)、矮牵牛素(Petunidin)和锦葵素(Malyidin)[1]。以上6类花青素的羟基与不同的糖类结合进而产生不同种类的花色苷。目前自然界中已经发现650多种花色苷,约92%的花色苷由这6类花青素衍生而来[2]。不同种类的花色苷所呈现的颜色也略有差异,天竺葵素及其衍生物呈红色或橙色,矢车菊素及其衍生物主要呈砖红或洋紫色,飞燕草素及其衍生物呈蓝色或者紫色[3,4]。花青素的颜色还与植物液泡的pH值有关,液泡pH值为1.0~2.0时,花青苷表现为红色;液泡pH值为6.0~6.5时,花青苷表现为蓝色;液泡pH>7.0时,花青苷表现为浅黄色[5]。

对植物来说,花青素具有多种多样的生物学功能。花青素在植物器官中积累,使植物呈现多种颜色赋予植物观赏价值,同时鲜艳的色彩为植物吸引昆虫授粉和传播种子,对植物繁殖具有重要意义[6,7]。花青素能提高植物抗性,帮助植物抵御多种生物与非生物胁迫。花青素可以吸收多余的可见光和帮助植物抵抗紫外线,并清除氧自由基保护植物不受强光灼伤,是植物天然的光保护剂[8]。对人类来说,花青素具有丰富的营养价值和医疗保健功能,对人类身体健康有着极大益处。花青素具有抗氧化能力,在防癌变、抗衰老、预防心血管疾病以及降血脂等方面有重要医疗价值[9]。食用花青素还能修复记忆损伤,缓解近视[10],因此富含花青素的食品蓝莓、紫薯和黑枸杞等越来越受到人们重视。

基于花青素的应用前景和研究潜力,剖析花青素的生物合成途径具有经济学和科学的重要意义。本研究从花青素生物合成的内外影响因子入手,重点综述了调控观赏植物和园艺作物花青素合成的环境因子、结构基因和调节基因。旨在通过对前人研究成果的总结,以期为利用分子生物学开展花青素代谢工程的研究提供参考。

1 环境因子对花青素的生物合成及植物呈色的调控

研究表明,植物通过感知外界环境因子的变化,调控体内花青素合成通路中结构基因和调节基因的表达,从而合成积累花青素,并确定花青素的种类[11]。花青素合成后转运至液泡储存,环境因子会影响花青素的稳定性,对花青素苷的降解产生加速或减速作用。植物所处的环境不同,其叶色和花色也会产生一定程度的变化,更加说明植物颜色受环境因子调控。影响植物体内花青素苷含量变化的环境因子主要有光、温度、植物激素以及糖类等[12]。

1.1 光

光通过激活花青素合成通路中的相关基因,促使植物体内合成并积累花青素[13]。研究表明,植物在强光条件下更易形成花青素。高光强可以同时诱发结构基因和调节基因的表达,而弱光或者黑暗会对结构基因和调节基因的表达产生抑制,从而减少花青素的合成,使植物呈现浅色甚至白色[14]。例如将茄子进行不透光的套袋处理发现茄子最终表现为白色[15]。李虹等[16]对月季的研究结果表明,光照是影响月季变色的关键因素,遮光处理阻碍了月季花瓣中花青素的合成,抑制了CHS和DFR的表达, 最终导致月季花冠不能着色。

高等植物通过不同的光受体接收和传递信号来实现对光的应答反应,从而完成自身的生长发育[17]。高等植物主要有5类光受体,它们分别是:光敏色素、隐花色素、向光素、UVR8和ZTLs家族[18]。感应光信号的顺式作用元件在结构基因的启动子序列中,转录因子在感受到光信号后,与顺式作用元件结合,进一步调控结构基因[12]。叶片是大多数植物感受光信号的重要器官。在开花植物中,叶片感受光信号并将光信号传递到花冠中,从而打开花青素合成通路,最终使花冠呈现出特定颜色[19]。此外,在对非洲菊的研究中发现,遮光处理花序后,舌状花内色素含量减少,基因CHS和DFR的表达受到抑制,说明部分植物的花冠在植物感受光信号的过程中也起到重要作用[20]。

在各种光照因素中,光质对花青素苷的合成起关键作用[12]。不同的光质对花青素合成影响不同,其中UV-A和UV-B通过加强花青素合成通路中关键基因的表达来积累花青素,是花青素合成的重要条件[21,22]。在拟南芥中,UV-A和UV-B可以上调CHS的表达[23];在芜菁中,UV-A上调PAL、CHS、F3H、ANS、DFR和GST等一系列基因的表达进而调控花青素的合成[24];在莴苣叶片,UV-B上调CHS、F3H和DFR等基因的表达,促进花青素合成[25]。李梦灵[26]通过光质对菊花呈色的研究发现,蓝光处理使基因CHS、F3H、ANS和3GT的表达量上升,而红光处理使基因CHS、F3H和转录因子MYB4的表达量下降。

1.2 温度

温度是影响植物体内花青素苷合成的另一重要因素。低温可以增强花青素合成通路中CHS、CHI和DFR等关键基因的表达,促进花青素在植物体内的积累;高温则抑制相关基因的合成,降低花青素的含量[27]。高温可以使李子果实中花青素苷降解,导致花青素含量降低[28]。温度不仅调控花青素合成通路中的相关基因,还影响花青素的稳定性。高温情况下,花青素合成速率减慢,降解速率加快。例如,当温度大于30℃时,菊花较难着色或者出现褪色情况[29]。此外,研究发现温度主要通过影响酶的活性来影响花青素的稳定性[30],高温下,花青素合成通路中相关酶的活性降低,因此花青素含量会降低。

1.3 激素

对植物施加外源激素可以促进或抑制植物花青素合成通路中部分基因的表达,植物内源激素通过影响调节基因来控制花青素的合成[31]。生长素在植物愈伤组织花青素合成过程中起重要作用。将红肉苹果使用浓度大于0.15 mg/L的2,4-D和浓度大于1.5 mg/L的NAA处理后,愈伤组织中的花青素积累受到明显抑制[32]。而经IAA处理的拟南芥幼苗,其花青素合成通路上的结构基因CHS、CHI和F3H以及调节基因MYB12、TTG1和PAP1的表达量有所上升,植株体内花青素的含量提高[33]。细胞分裂素对植物体内花青素的积累也具有一定影响,使用6-BA处理荔枝可以抑制荔枝果皮花青素的积累[34]。外源赤霉素处理矮牵牛,可以诱导花冠CHS基因的表达,促进花青素苷的积累[35]。拟南芥ga1-5突变体的内源赤霉素含量较低而激活了花青素生物合成的部分基因,使其体内花青素含量增加[36]。乙烯在花青素合成途径中也发挥重要作用。使用乙烯处理葡萄果实后,结构基因CHS、F3H和UFGT的表达量显著升高,花青素含量也随之升高[37]。此外乙烯对花青素合成的抑制作用也是显著的,乙烯可以通过激活负转录因子MYBL2实现抑制作用[38],牡丹切花要使用乙烯处理,再次证实了乙烯对结构基因和调节基因的抑制[39]。

1.4 糖类

糖类对花青素的合成调控主要有两方面原因:其一是糖参与花青素的合成,花青苷元经过糖基化后形成稳定的花青素;其二是花青素苷合成所需前体经由莽草酸产生,而莽草酸的形成依赖于旺盛的戊糖呼吸,充足的糖是戊糖呼吸的必要条件[40]。Lotkowska[41]等证实高浓度的糖分可以促进葡萄果实中的花青素积累。研究发现蔗糖不仅可以调控花青素合成通路中的结构基因,还能特异性地调控部分调节基因。使用外源蔗糖、果糖和葡萄糖处理的拟南芥,部分结构基因CHS、CHI、F3H和DFR以及调节基因PAP1的表达量显著上升[42]。

2 植物花青素的合成通路

2.1 花青素的合成途径

花青素合成通路是类黄酮合成通路中的一个重要分支,在高等植物中较为保守,其过程涉及多个复杂的酶促反应（

图1

所示）,主要分为3个阶段:花青素基本骨架的形成、花青素前体的形成以及将花青素前体修饰成各种不同花青素苷[22]。花青素在细胞质表面合成,后在内质网膜经过不同的甲基化、糖基化、羟基化和酰基化等修饰后形成了稳定的花青素苷[43]。最后花青素苷在转运蛋白和转运囊泡的协助下进入液泡中汇集并积累[44]。花青素苷的积累和转运对植物的呈色具有一定影响,目前已报道4类蛋白可能参与花青素苷向液泡的转运,分别是谷胱甘肽转移酶(glutathione S-transferase, GST)、多药耐药抗性相关蛋白(multidrug resistance-associated protein, MRP)、多药和有毒化合物排出家族(multidrug and toxic compound extrusion, MATE)和BTL-homologue[40]。

第一阶段是许多植物次生代谢的共有途径,主要由苯丙氨酸(phenylalanine)经过三次酶促反应合成4-香豆酰A(4-coumaroyl-CoA)。苯丙氨酸(phenylalanine)由苯丙氨酸解氨酶(phenylalanine ammonia lyase, PAL)催化形成肉桂酸(cinnamic acid),肉桂酸羟化酶(cinnamate 4-hydroxylase, C4H)将肉桂酸(cinnamic acid)催化成香豆酸(p-coumaric acid),最后香豆酸(p-coumaric acid)在4-香豆酰CoA连接酶(4-coumarate CoA ligase, 4CL)的作用下合成4-香豆酰A(4-coumaroyl-CoA)。

第二阶段是4-香豆酰A合成二氢黄酮醇(dihydrokaempferol)的过程。4-香豆酰A(4-coumaroyl-CoA)和丙二酰A(malonyl-CoA)在查尔酶合成酶(chalcone synthase, CHS)催化下形成通路中第一个类黄酮物质查尔酮(Chalcone),为后续化合物的合成提供了基本的碳骨架。后在查尔酮异构酶(chalcone isomerase, CHI)和黄烷酮-3-羟化酶(flavanone 3-hydroxylase,F3H)的作用下合成二氢黄酮醇(dihydrokaempferol)。

第三阶段即各种不同种类花青苷元的形成。二氢黄酮醇(dihydrokaempferol)合成之后产生三条支路,类黄酮3-羟化酶(flavonol 3'hydroxylase,F3'H)和类黄酮3,5-羟化酶(flavonol 3',5'hydroxylase, F3',5'H)以二氢黄酮醇 (dihydrokaempferol)为共同底物,分别催化生成双氢槲皮素(dihydroquercetin)和二氢杨梅黄酮(dihydromyricetin),之后处于三条支路上的二氢黄酮醇(dihydrokaempferol)、二氢杨梅黄酮(dihydro.myricetin)和双氢槲皮素(dihydroquercetin)在二氢黄酮醇-4-还原酶(dihydroflavonol 4-reductase, DFR)的催化下合成无色花青素,最后在花青素合成酶(anthocyanidin synthase, ANS)的氧化脱水作用下,分别生成显色但不稳定的天竺葵色素(pelargonidin)、矢车菊色素(cyanidin)和翠雀色素(delphinidin)[45,46,47]。

2.2 花青素生物合成通路中的主要结构基因

花青素合成通路中的相关酶由结构基因编码而成,这些结构基因分为早期生物合成基因(EBG):CHS、CHI、F3H和F3'H;晚期生物合成基因(LBG):F3',5'H、DFR、ANS和UFGT等[48]。这些结构基因的表达受环境因子和转录因子的共同控制,转录因子主要包括MYB、bHLH和WD40三大类[7]。

2.2.1 查尔酮合成酶基因查尔酮合成酶基因CHS是类黄酮合成通路中的第一个关键基因,CHS催化香豆酰辅酶A(4-coumaroyl-CoA)和丙二酰辅酶A(malonyl-CoA)形成类黄酮合成通路中的第一个有色物质查尔酮(Chalcone),并为后续类黄酮物质的合成提供碳骨架。研究表明,CHS是一个超家族基因,目前已从多种植物中鉴定并克隆,如拟南芥、玉米、大豆、矮牵牛和金鱼草。柑橘中有3个CHS基因,分别是CitCHS1、CitCHS2和CitCHS3。CitCHS2和CitCHS3的表达量与柑橘根、茎和子叶中的类黄酮含量呈正相关,CitCHS1可以加速叶片中类黄酮的积累[49]。CHS处于花青素合成的上游关键位置,因此使CHS基因沉默或使其过量表达对改变花色具有重要意义,例如矮牵牛中,CHS表达量减少,花色由紫色变为白色。番茄果实成熟的过程中CHS的活性显著增加,利用VIGS (Virus induced gene silencing)技术下调SICHS的表达量,果皮中类黄酮含量减少的同时颜色也会变淡[50]。

2.2.2 查尔酮异构酶基因查尔酮异构酶基因CHI催化查尔酮(Chalcone)形成柚皮素,是类黄酮化合物生物合成的上游关键基因之一,其表达量对植物体内类黄酮含量具有重要影响。Mehdy[51]最早从法国豌豆中分离出CHI,后陆续从矮牵牛等植物中克隆出来。CHI是多基因家族,其表达和积累具有时间和空间特异性。例如CHI-A和CHI-B,二者的表达具有组织特异性,CHI-A在成熟的花药中表达,CHI-B主要在未成熟的花药中表达[52]。在葡萄果实中,CHI主要分布在外果皮的细胞质、细胞核和叶绿体,中果皮维管束细胞的细胞质中;在葡萄根、茎、叶中,CHI分布在细胞质、细胞壁和细胞核中[53]。徐靖等[54]检测了黄、白和紫肉3个品种甘薯的花青素含量,只在紫肉品种中检测出花青素,经过定量PCR结果显示,IbCHIL1在紫肉甘薯中大量表达,在黄肉甘薯中微量表达,不在白肉甘薯中表达。杭菊中,CmCHI1和CmCHI3是参与类黄酮合成的主要结构基因,而CmCHI2是辅助基因[55]。

2.2.3 黄烷酮3-羟化酶基因黄烷酮3-羟化酶基因F3H是花青素代谢途径上游的又一个关键结构基因,该基因催化黄烷酮C3位上添加一个羟基形成二氢黄酮醇。F3H的催化反应依赖Fe2+、氧、2-酮戊二酸等作为辅助因子[56]。目前,F3H在金鱼草、拟南芥、玉米和胡萝卜等多种植物中克隆出来。F3H在植物中的表达具有组织特异性,例如在豆科植物中,F3H在根瘤维管束中表达,在根中不表达;在苜蓿中,该基因在花、根和根瘤中表达,在叶中不表达。F3H对花色差异形成具有一定影响,绣球花中HmF3H基因在不同组织器官中均能表达,但是对比盛花期的红色品种‘粉黛’和蓝色品种‘无尽夏’发现,HmF3H在‘粉黛’盛花期时表达量较高[57]。冯志熙等[58]对金凤花IuF3H进行克隆并分析,证实IuF3H参与花青素合成并起到关键作用。

2.2.4 二氢黄酮醇还原酶基因二氢黄酮醇还原酶基因DFR是花青素合成后期的基因,是无色翠雀素、无色天竺葵素以及无色矢车菊素合成的关键基因,对植物显色具有重要作用。Beld等[59]从玉米中克隆出第一个DFR基因,后相继从金鱼草、拟南芥、玫瑰、葡萄和苹果等植物中克隆出。甘霖鑫等对比丹凤牡丹的白色期、变色期、褐色期和黑色期,发现变色期PoDFR的表达量最高。证实DFR表达量与花青素合成密切相关。Zhuang等[60]通过对比紫色萝卜和绿色萝卜的代谢组与转录组发现,紫色萝卜中DFR的表达量上调导致类黄酮物质合成向花青素方向偏移,此外绿色萝卜中DFR基因产生突变,给绿色萝卜积累花青素造成障碍。

2.2.5 花青素合成酶基因花青素合成酶基因ANS将无色花青苷转化为有色花青素,其产物是花青素代谢途径中的第一个有色化合物,因此其表达对植物花、叶和果实等器官的呈色极为重要。抑制ANS基因的表达或使其活性降低会使植物花色变淡甚至变白。此外,将ANS导入植物中使其超量表达,花青素苷在植物体内的积累量会改变,植株的抗氧化能力增强。前人研究发现2个ANS等位基因变异可以造成洋葱花青素的缺失[61,62]。ANS基因的表达具有特异性,在桑树的特定部位叶片尖端和茎生长点能检测到MaANS的表达。

3 花青素合成通路中的转录调控

花青素的合成、转运和积累受到多种转录因子的特异性和协同性调控。目前花青素合成通路中被研究最多也是最重要的三类转录因子分别是MYB转录因子家族、bHLH转录因子家族和WDR转录因子家族。三类转录因子可以单独调控结构基因或者MYB、bHLH和WDR相互结合组成MBW复合体共同调控结构基因[45,63]。这些转录因子通过识别结构基因启动子特定区域,并与结构基因启动子结合,从而达到促进或抑制花青素生物合成的作用[7,64-65]。研究发现,单子叶植物和双子叶植物的调控模式也有差异[66],单子叶植物花青素合成调控由MBW复合物完成。双子叶植物例如拟南芥中,R2R3-MYB与bHLH和WDR构成MBW复合物激活晚期生物合成基因DFR、ANS和UFGT的表达最终导致花青素在植物体内的积累[67],而早期生物合成基因主要由R2R3-MYB类转录因子MYB11、MYB12、MYB111以及MYB75/PAP1进行调控[68]。此外,还报道了一些其他转录因子比如COP1、JAZ、NAC、SPL和 WRKY都可以和MBW复合物互相作用调控花青素和合成[69]。

3.1 MYB转录因子

MYB是最大的转录因子家族,通过分析被子植物6个不同亚群的MYB蛋白发现,尽管它们的表达强度和作用强度不同,但是都保留着激活花青素生物合成的能力。MYB转录因子是一类DNA结合蛋白,具有高度保守的DNA结合域—MYB结构域,由51~52个氨基酸构成。根据MYB结构域达到个数可将MYB转录因子分为4类:R1-MYB、R2R3-MYB、R1R2R3-MYB和4R-MYB[45]。Cone等[70]从玉米中分离出调控胚乳糊粉层中花青素合成的MYB转录因子ZmC1,是植物体内研究报道的第一个调控花青素生物合成的MYB转录因子。R3-MYB和R2R3-MYB两类转录因子作为调节蛋白广泛参与苯丙烷类代谢途径的调控,在植物中发挥巨大作用。拟南芥花青素合成过程涉及6个R2R3-MYB转录因子亚群,分别是:AtMYB75(PAP1,At1g56650)、AtMYB90 (PAP2、At1g66390)、AtMYB113 (At1g66370)和AtMYB114 (At1g66380)[71,72]。其中PAP1被证实是拟南芥花青素合成通路中重要的转录因子[73],过量表达PAP1使得转基因植物的根、茎、叶和花中积累了大量花青素。PAP2、MYB113和MYB114在特定的环境或者植物某一发展阶段也可以激活DFR和ANS等结构基因的表达,但是激活程度不如PAP1[71]。

MYB转录因子广泛参与重要花卉和园艺作物中花青素的生物合成。在苹果中分离出多个与花青素合成有关的MYB转录因子,包括与果皮花青素合成有关的MdMYBA和MdMYB1,以及与叶和果肉花青素合成有关的MdMYB10和MdMYB110a等[74]。MdMYB10及其同源基因广泛存在与蔷薇科植物的果实中并参与果实花青素的合成,比如在樱桃、桃子、梨子、梨、覆盆子、草莓中均鉴定出MdMYB10[75]。Ravaglia等[76]从桃中分离出PpMYB10。Lin-Wang等从草莓中分离出FaMYB10,过量表达FaMYB10使草莓中的花青素含量增加70%。Jin等[77]从甜樱桃中克隆出PavMYB10。对葡萄的研究表明,至少有3个MYB类转录因子VvMYBA1、VvMYBA2和VvMYB5b参与浆果成熟过程中花青素的积累,并且对UFGT、GST和OMT等结构基因进行调控。血橙体内的花青素主要在内果皮中积累而外果皮缺少花青素,主要因为R2R3-MYB类转录因子RUBY无法在果肉中表达[78]。SmMYB1在茄子的花青素合成通路中起到关键作用,该基因的表达上调使得花青素在叶片、花瓣、雄蕊以及其他内部组织中大量积累[79]。

在矮牵牛中,MYB类转录因子AN1、JAF13、PhMYB27和AN2先后被发现参与调控花青素的合成[80]。对月季‘红胜利’中与花青素合成有关的转录因子MYB4-1和MYB6-1检测可知,二者在花瓣中高水平表达,而叶片和花药中表达量较低。通过分析不同颜色的月季花瓣发现,花青素含量与两转录因子的表达量呈正相关,说明MYB4-1和MYB6-1在月季花青素苷合成和花瓣着色的过程中发挥重要作用[81]。王雪霁[82]分析了小兰屿蝴蝶兰花、叶、茎、根4种组织中的PeMYB71表达量,发现该转录因子在花中的表达量最高,花器官中的花青素含量也最多。郭亚飞等[83]研究发现CsMYB123正调控茶树花青素的生物合成。毛白杨中分离出R2R3-MYB类转录因子MYB6,并证实该因子主要在幼叶中表达。MYB6过表达使转基因毛白杨中的花青素和原花青素积累量显著增加[84]。

相比MYB转录因子,MBW复合体对靶细胞的激活能力更强,可以更加有效的对花青素合成通路中合成、修饰和转运等相关基因进行调控[40]。MBW复合体主要依靠其中的MYB和bHLH对靶基因进行调控,而WD40有可能在增强MBW复合体稳定性的同时,为二者形成转录复合体提供平台[85]。MYB转录因子被认为是MBW复合体中起主要作用的转录因子[86]。在拟南芥中,MYB家族的PAP1和PAP2可以与bHLH家族的TT8以及WD40家族的TTG1结合,形成复合体激活花青素合成过程中CHS和DFR的表达[87]。茄子中的SmGL3、SmTT8以及SmTTG1形成MBW复合体进而调控花青素合成[88]。MBW复合蛋白中,按WD40-bHLH-MYB的顺序,转录因子特异性逐渐提高,所涉生物学过程逐渐专一[89]。

R3-MYB和R2R3-MYB转录因子不仅能促进花青素的合成,也能抑制花青素的合成进而减少花青素的积累,正调控和负调控相互结合,维持植物体内花青素的平衡。尤其是R2R3-MYB的第四亚族成员大多为转录抑制因子[90]。研究发现的第一个具有负调控作用的转录因子是金鱼草中的R2R3-MYB类转录因子AmMYB308,AmMYB308的过量表达可以抑制苯丙烷的代谢[91]。R3-MYB转录因子通过阻碍MBW复合物和晚期生物合成基因的联系来抑制花青素的合成[92]。

研究报道,MYB4在调节拟南芥类黄酮生物合成时起到双重作用,MYB4与其同源转录因子MYB7是苯丙烷合成的阻遏物。MYB4、MYB7、MYB32与bHLH类转录因子TT8、GL3、EGL3相互作用,干扰MBW复合物的转录活性[46]（

图2

）。此外,MYB4还可以通过抑制ADT6 (Arogenate Dehydratase 6)基因的表达来抑制类黄酮的积累[46]。目前报道的拟南芥中的负转录因子还有MYBL2、MYB2、SPL9、GL2和CPC。例如MYBL2转录因子可以与TT8结合抑制DFR和TT8的表达来抑制植物体内花青素的合成[93,94]。Albert等[95]研究发现,MYB27与AN11互作,结合bHLH类转录因子破坏MBW复合蛋白,以此实现MYB27在花青素合成通路中的负调控功能。由此可见,MYB转录因子抑制花青素合成的调控机制大致可分为3类:一是通过破坏MBW复合体的稳定性,进而发挥其抑制功能;二是通过抑制结构基因尤其是DFR基因的表达;三是与其他转录因子互相作用来抑制正向转录因子的表达,从而减少花青素含量[45]。

图2 MYB4调控类黄酮合成通路的模型

Full size|PPT slide

3.2 bHLH转录因子

碱性螺旋-环-螺旋(Basichelix-loop-helix, bHLH)转录因子是植物第二大转录因子家族,约含有50~60个氨基酸残基。该蛋白结构包含两个功能不同的区域:碱性区域和螺旋-环-螺旋(HLH)区域。碱性区域在N端为DNA识别区,HLH区域在C端形成同源或异源二聚体,因此bHLH在植物的生长发育、抵抗胁迫和传导信号等方面以二聚体的形式发挥重要作用[96]。bHLH家族转录因子有Myc、R、Sn和Lc等。在MBW复合物中,bHLH转录因子能够特异性识别并激活靶基因的启动子[3]。Ludwig等[97]发现植物中第一个bHLH转录因子是玉米R1(RED1)蛋白。

在模式植物拟南芥中bHLH蛋白TT8、GL3、EGL3、MYC1通过和MYB蛋白结合形成MBW复合体,参与调控花青素的合成[98,99]。通过对模式植物的还研究发现,bHLH转录因子对花青素合成通路的调控主要体现在两方面,一是与MYB转录因子互作;二是对DFR和ANS的调控,尤其是对DFR的调控[45]。比如烟草中与花青素合成有关的bHLH蛋白是NtAn1a和NtAn1b,NtAn1a与NtAn2相互作用激活CHS和DFR的表达[100]。过表达拟南芥MYC3和MYC4均能促进植物中的花青素积累。

观赏植物和园艺作物的bHLH调控机制与模式植物中基本相同,仍是与MYB转录因子的互作和对DFR表达量的调控。例如Espley[101]发现苹果中的MdbbHLH3和MdbbHLH33均与MdbMYB10互作,促使果实变红。荔枝中LcbHLH1、LcbHLH3与LcMYB1协同作用,调控荔枝中花青素的合成与积累[102]。CsbHLHs和CsMYBs转录因子在茶树中相互作用促进了茶叶中类黄酮的积累[103]。茄科植物中,参与花青素合成调控的bHLH转录因子主要有两个分支,分别是矮牵牛PhAN1和PhJAF13。茄子和辣椒中,已经显色的果实和未成熟的果实相比,PhAN1的同源基因CabHLH和SmbHLH的表达量明显较高[104]。Li等[105]通过分析光诱导的茄子花青素合成的分子机理发现,转录因子SmTT8参与茄子花青素的合成。Zong等[106]证实TsMYC2诱导小麦胚芽鞘中花青素的合成,是培育具有新性状蓝色糊粉层小黑麦的重要基因。猕猴桃的内果皮中积累着大量花青素,证实基因ACMYB123和ACBHLH42的协同作用是使内果皮中ACANS的表达上调的原因[107]。

bHLH转录因子也能抑制花青素的合成,抑制花青素合成的机制推测为:bHLH负转录因子通过破坏bHLH-MYB复合体,阻止bHLH-MYB复合体发挥作用;或不与MYB蛋白结合,干扰具有活性的bHLH-MYB复合体形成。

3.3 WD40转录因子

WD40转录因子又称WDR蛋白,是一类高度保守的蛋白,一般含有4~16个串联重复的WD基元序列,每个WD基元由44-60个氨基酸残基组成。WD40主要功能是促进蛋白相互作用,为MYB和bHLH蛋白形成MBW复合物提供一个稳定的平台。WD40转录因子家族主要有AN11、TTG1、PFWD40和PAC1。第一个被发现的WD40蛋白是矮牵牛的AN11（ANTHOCYANIN11）蛋白。目前多种WD40蛋白已从植物中被分离鉴定出来,主要包括玉米PAC1蛋白、拟南芥TTG1蛋白、紫苏PFWD40蛋白、茄子SmWD40蛋白和辣椒CaWD40蛋白等,它们的转录水平几乎不随结构基因的表达水平和花青素的含量而改变。WD40的表达也具有特异性,冯如[108]在银杏叶片中发现3个WD40蛋白,分别是GbWD401、GbWD402和GbWD403,并证实3个蛋白只在茎和叶中表达,在根中不表达。

WD40转录因子的稳定表达也是花青素稳定积累不可或缺的一环。马铃薯中的StAN11基因属于WD40转录因子,该基因过量表达能调控DFR基因表达,进而加深块茎表皮颜色。矮牵牛PhAN11的突变导致矮牵牛出现白色花[109]。辣椒中的CaMYBA和CaWD40保持沉默,结构基因的表达和花青素的含量也会对应的下降[110]。小苍兰中的WD40类转录因子FhTTG1的表达与植株内花青素和原花青素的积累同步。FhTTG1与MYB和bHLH形成复合体后,能够高度激活花青素或原花青素生物合成的相关结构基因,这说明FhTTG1作为MBW复合体中的重要一员,调控小苍兰花青素合成[111]。此外,TTG1能与bHLH蛋白互作并转移至细胞核中,TTG1进入细胞核后,能提高MBW复合物的稳定性,从而使MBW复合物更易与相关结构基因的启动子结合,进而对花青素的生物合成进行调控。

4 展望

植物体内花青素的含量对植物呈色具有重要意义,而花青素的合成与积累同环境因子、结构基因和调节基因有密不可分的关系。因此,改变环境因子,激活或抑制结构基因和调节基因,诱导关键基因突变对修饰花色、调控叶色、丰富果色,提高园林植物的观赏价值,改良园艺作物的关键性状具有重要作用。目前,花青素代谢途径中一些关键问题仍未阐明,例如环境因子、结构基因和转录因子之间如何互作,花青苷如何进一步修饰成颜色不同的花青素,花青素的合成与降解如何实现动态平衡。利用分子育种技术对植物颜色进行调节是目前研究的热门,但是还缺乏非常有用的外源基因,找到并克隆有效的基因也是漫长而艰巨的任务。此外,尽管利用基因工程对月季、菊花、香石竹等花色的改良已基本成功,但是仍有许多植物的再生及转化体系较难建立,转基因体系仍需要进一步完善。综上所述,在未来的研究中应重点解决以下问题:（1）深入研究花青素代谢过程中各类影响因子的互相作用模式;（2）通过多组学联合分析,挖掘花青素通路中关键有效的基因;（3）加大对建立植物遗传转化体系的研究力度,为基因育种奠定基础。

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UV and blue light control the expression of flavonoid biosynthesis genes in a range of higher plants. To investigate the signal transduction processes involved in the induction of chalcone synthase (CHS) gene expression by UV-B and UV-A/blue light, we examined the effects of specific agonists and inhibitors of known signaling components in mammalian systems in a photomixotrophic Arabidopsis cell suspension culture. CHS expression is induced specifically by these wavelengths in the cell culture, in a manner similar to that in mature Arabidopsis leaf tissue. Both the UV-B and UV-A/blue phototransduction processes involve calcium, although the elevation of cytosolic calcium is insufficient on its own to stimulate CHS expression. The UV-A/blue light induction of CHS expression does not appear to involve calmodulin, whereas the UV-B response does; this difference indicates that the signal transduction pathways are, at least in part, distinct. We provide evidence that both pathways involve reversible protein phosphorylation and require protein synthesis. The UV-B and UV-A/blue light signaling pathways are therefore different from the phytochrome signal transduction pathway regulating CHS expression in other species.

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Sugars act as signaling molecules, whose signal transduction pathways may lead to the activation or inactivation of gene expression. Whole-genome transcript profiling reveals that the flavonoid and anthocyanin biosynthetic pathways are strongly up-regulated following sucrose (Suc) treatment. Besides mRNA accumulation, Suc affects both flavonoid and anthocyanin contents. We investigated the effects of sugars (Suc, glucose, and fructose) on genes coding for flavonoid and anthocyanin biosynthetic enzymes in Arabidopsis (Arabidopsis thaliana). The results indicate that the sugar-dependent up-regulation of the anthocyanin synthesis pathway is Suc specific. An altered induction of several anthocyanin biosynthetic genes, consistent with in vivo sugar modulation of mRNA accumulation, is observed in the phosphoglucomutase Arabidopsis mutant accumulating high levels of soluble sugars.

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宋雪薇, 魏解冰, 狄少康, 等. 花青素转录因子调控机制及代谢工程研究进展[J]. 植物学报, 2019,54(1):133-156.

花青素是广泛存在于植物中的一类重要的类黄酮化合物, 在植物生长发育和人类营养保健方面具有重要价值。花青素的生物合成途径已经解析得比较清楚, 但花青素的代谢调控网络还在不断完善。调控花青素生物合成的转录因子主要包括MYB、bHLH和WD40三大类, 这些转录因子通过激活或抑制CHS、ANS和DFR等花青素途径关键结构基因的表达水平, 进而决定花青素积累的部位与水平。该文结合国内外花青素生物合成与转录调控方面的研究进展, 简要介绍了花青素的生物合成途径, 归纳总结了模式植物中花青素代谢调控的分子机理, 尤其是MYB、bHLH和WD40三类主要转录因子的调控机理, 以及这些转录因子在观赏植物和水果等经济作物花青素代谢工程中的应用。该文将为系统阐明花青素的转录调控机制和利用代谢工程改良花青素的相关研究提供有益参考。

Wu J

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Higashi Y

Nakabayashi R

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During their evolution, plants have acquired the ability to produce a huge variety of compounds. Unlike the specialized metabolites that accumulate in limited numbers of species, flavonoids are widely distributed in the plant kingdom. Therefore, a detailed analysis of flavonoid metabolism in genomics and metabolomics is an ideal way to investigate how plants have developed their unique metabolic pathways during the process of evolution. More comprehensive and precise metabolite profiling integrated with genomic information are helpful to emerge unexpected gene functions and/or pathways. The distribution of flavonoids and their biosynthetic genes in the plant kingdom suggests that flavonoid biosynthetic pathways evolved through a series of steps. The enzymes that form the flavonoid scaffold structures probably first appeared by recruitment of enzymes from primary metabolic pathways, and later, enzymes that belong to superfamilies such as 2-oxoglutarate-dependent dioxygenase, cytochrome P450, and short-chain dehydrogenase/reductase modified and varied the structures. It is widely accepted that the first two enzymes in flavonoid biosynthesis, chalcone synthase, and chalcone isomerase, were derived from common ancestors with enzymes in lipid metabolism. Later enzymes acquired their function by gene duplication and the subsequent acquisition of new functions. In this review, we describe the recent progress in metabolomics technologies for flavonoids and the evolution of flavonoid skeleton biosynthetic enzymes to understand the complicate evolutionary traits of flavonoid metabolism in plant kingdom.

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Three Chrysanthemum-chalcone-isomerase genes( CmCHI) were successfully cloned by PCR from the database of Chrysanthemum transcriptome and named CmCHI1,CmCHI2 and CmCHI3,respectively. Bioinformatics analysis showed that the base numbers of CmCHI1-3 open reading frame were 708,633 and 681 bp,encoding 235,210 and 226 amino acids,respectively. Three fusion proteins of about 30 kDa were successfully induced by prokaryotic expression technology,and the corresponding recombinant fusion proteins were isolated and purified by Ni-NTA resin column. Clustering analysis showed that the 3 CmCHI were homologous with Compositae plants,and CmCHI1 and CmCHI3 belonged to type Ⅰ CHI. CmCHI2 belongs to type Ⅳ CHI. Using β-actin as an internal reference gene,RT-qPCR was used to detect and analyze the expression of CmCHI1-3 genes in Hangju. The results showed that the expression levels of CmCHI1 and CmCHI3 were higher,while the expression levels of CmCHI2 were lower. It was concluded that CmCHI1 and CmCHI3 were the main chalcone isomerase genes involved in the synthesis of flavonoids in Hangju,and CmCHI2 was a helper gene. Flooding treatment significantly promoted the expression of CmCHI1 and CmCHI3 genes,but had no regulatory effect on CmCHI2. The above results provided a basis for further study of the molecular regulation mechanism of CHI gene in the metabolism of flavonoids in Hangju,which laid a foundation for improving the content of flavonoids in Hangju and finally improving the medicinal quality of Hangju.

贾赵东, 马佩勇, 边小峰, 等. 植物花青素合成代谢途径及其分子调控[J]. 西北植物学报, 2014,34(07):1496-1506.

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冯志熙, 刘应丽, 朱佳鹏, 等. 滇水金凤黄烷酮3-羟化酶基因(IuF3H)的克隆及表达分析[J]. 分子植物育种, 2021,19(1):65-71.

甘林鑫, 李厚华, 李果, 等. 凤丹牡丹二氢黄酮醇-4-还原酶基因克隆及表达特性分析[J]. 江苏农业科学, 2020,48(10):73-79.

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Robin Arif Hasan Khan

Natarajan Sathishkumar

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Kohlrabi (Brassica oleracea var. gongylodes L.) is an important vegetable of the Brassicaceae family. The main edible part of kohlrabi is the swollen stem. The purple cultivars make anthocyanin mainly in the peel of the swollen stem, while in the leaf, it is limited to the midrib, but green cultivars do not. Anthocyanins are advantageous for both plants as well as humans. Two anthocyanin compounds were detected by high pressure liquid chromatography (HPLC) only in the peel of the purple kohlrabi cultivar. Three MYBs, three bHLHs, and one WD40 TF were identified as the candidate regulatory genes in kohlrabi. There was an abundance of transcript levels for the late biosynthetic genes more specifically for BoF3'H, BoDFR, BoLDOX, and BoGST in the purple peel while scarcely detectable in other tissues for both cultivars. The expression of BoPAP2 and BoTT8 was higher in the peel of the purple cultivar than the green cultivar. The expression of BoMYBL2.2 orthologue of Arabidopsis MYBL2, a negative regulator of anthocyanins, was dramatically decreased in the purple peel. The expression of BoACO1, a key gene for ethylene biosynthesis, and BoNCED3, an important gene of the ABA pathway, was down- and upregulated, respectively, in the peel of purple kohlrabi.

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Cavallini E

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Bolitho K

Grafton K

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Background: The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all the commercially important rosaceous species. Results: We use gene specific primers to show that the three MYB activators of apple anthocyanin (MYB10/MYB1/MYBA) are likely alleles of each other. MYB transcription factors, with high sequence identity to the apple gene were isolated from across the rosaceous family (e. g. apples, pears, plums, cherries, peaches, raspberries, rose, strawberry). Key identifying amino acid residues were found in both the DNA-binding and C-terminal domains of these MYBs. The expression of these MYB10 genes correlates with fruit and flower anthocyanin levels. Their function was tested in tobacco and strawberry. In tobacco, these MYBs were shown to induce the anthocyanin pathway when co-expressed with bHLHs, while over-expression of strawberry and apple genes in the crop of origin elevates anthocyanins. Conclusions: This family-wide study of rosaceous R2R3 MYBs provides insight into the evolution of this plant trait. It has implications for the development of new coloured fruit and flowers, as well as aiding the understanding of temporal-spatial colour change.

Adolfo Luís dos Santos

Antonio Chalfun-Júnior

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Anthocyanins are naturally occurring flavonoids derived from the phenylpropanoid pathway. There is increasing evidence of the preventative and protective roles of anthocyanins against a broad range of pathologies, including different cancer types and metabolic diseases. However, most of the fresh produce available to consumers typically contains only small amounts of anthocyanins, mostly limited to the epidermis of plant organs. Therefore, transgenic and non-transgenic approaches have been proposed to enhance the levels of this phytonutrient in vegetables, fruits, and cereals. Here, were review the current literature on the anthocyanin biosynthesis pathway in model and crop species, including the structural and regulatory genes involved in the differential pigmentation patterns of plant structures. Furthermore, we explore the genetic regulation of anthocyanin biosynthesis and the reasons why it is strongly repressed in specific cell types, in order to create more efficient breeding strategies to boost the biosynthesis and accumulation of anthocyanins in fresh fruits and vegetables.Copyright © 2018. Published by Elsevier Ltd.

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Hu Z

, et al. Genetically engineered anthocyanin pathway for high health-promoting pigment production in eggplant[J]. Mol Breed, 2016,36:1-14.

赵恩鹏, 成玉富, 杨旭. 茄科植物转录因子MYB基因家族的研究进展[J]. 分子植物育种, 2021,19(5):1522-1530.

严倩, 赵佳, 刘荣, 等. 月季花青素苷相关R2R3-MYB蛋白基因的克隆和表达分析[J]. 中国农业科学, 2015,48(7):1392-1404.

王雪霁, 梁立雄, 李潞滨, 等. 小兰屿蝴蝶兰R2R3-MYB转录因子分析[J]. 林业科学研究, 2018,31(3):104-113.

郭亚飞, 马煜明, 水刘媛, 等. 茶树Cs MYB123转录因子的克隆及表达特性研究[J]. 西北植物学报, 2018(1):9-16.

Lu W X

Ran L Y

, et al. R2R3-MYB transcription factor MYB6 promotes anthocyanin and proanthocyanidin biosynjournal but inhibits secondary cell wall formation in Populus tomentosa[J]. The Plant Journal: for cell and molecular biology, 2019,99(4):733-751.

祝志欣, 鲁迎青. 花青素代谢途径与植物颜色变异[J]. 植物学报, 2016,51(1):107-119.

Wang H

Li M

, et al. The R2R3 MYB transcription factor PavMYB10.1 involves in anthocyanin biosynjournal and determines fruit skin colour in sweet cherry (Prunus avium L.)[J]. Plant Biotechnol, 2016,14:2120-2133.

Xia Y J

Blount J

, et al. Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynjournal[J]. Plant Cell, 2000,12:2383-2393.

Plants produce a wide array of natural products, many of which are likely to be useful bioactive structures. Unfortunately, these complex natural products usually occur at very low abundance and with restricted tissue distribution, thereby hindering their evaluation. Here, we report a novel approach for enhancing the accumulation of natural products based on activation tagging by Agrobacterium-mediated transformation with a T-DNA that carries cauliflower mosaic virus 35S enhancer sequences at its right border. Among approximately 5000 Arabidopsis activation-tagged lines, we found a plant that exhibited intense purple pigmentation in many vegetative organs throughout development. This upregulation of pigmentation reflected a dominant mutation that resulted in massive activation of phenylpropanoid biosynthetic genes and enhanced accumulation of lignin, hydroxycinnamic acid esters, and flavonoids, including various anthocyanins that were responsible for the purple color. These phenotypes, caused by insertion of the viral enhancer sequences adjacent to an MYB transcription factor gene, indicate that activation tagging can overcome the stringent genetic controls regulating the accumulation of specific natural products during plant development. Our findings suggest a functional genomics approach to the biotechnological evaluation of phytochemical biodiversity through the generation of massively enriched tissue sources for drug screening and for isolating underlying regulatory and biosynthetic genes.

刘新宇, 韩洪强, 葛海燕, 等. 茄子花青素合成中SmTTG1、SmGL3和SmTT8的表达及其蛋白质间的相互作用[J]. 园艺学报, 2014,41(11):2241-2249.

Gonzalez A

Zhao M

, et al. A network of redundant bHLH proteins functions in all TTG1 dependent pathways of Arabidopsis[J]. Development, 2000,130:4859-4869.

王玉, 杨雪, 杨蕊菁, 等. 调控苯丙烷类生物合成的MYB类转录因子研究进展[J]. 安徽农业大学学报, 2019,46(5):859-864.

Merida A

Parr A

, et al. The AmMYB308 and AmMYB330 transcription factors from Antirrhinum regulate phenylpropanoid and lignin biosynjournal in transgenic tobacco[J]. Plant Cell, 1998,10:135-154.

Olafsdottir S M

Heidari B

, et al. Nitrogen depletion and small R3-MYB transcription factors affecting anthocyanin accumulation in Arabidopsis leaves[J]. Phytochemistry, 2014,98:34-40.

Ternary complexes consisting of a R2R3-MYB, a bHLH and a WD40 protein (MBW complexes) regulate trichome formation and anthocyanin synthesis in plants. Small R3-MYBs interact with the MBW complexes to exert a negative feedback, and thereby participate in regulation of epidermal cell fate, for example trichome numbers and clustering in leaves. In Arabidopsis thaliana, GL3, a bHLH transcription factor, is important in the MBW complex regulating trichome formation as well as in the MBW complex induced by nitrogen depletion and promoting anthocyanin formation. The small R3-MYBs: CPC, TRY, ETC1, ETC2, ETC3/CPL3, TCL1, MYBL2, are all known to interact with GL3. We here investigated these R3-MYBs in leaves of Arabidopsis rosette stage plants under nitrogen depletion to examine if the small MYBs would interfere with anthocyanin accumulation in plants under normal (autotrophic) growth conditions. CPC expression was enhanced two-fold in response to nitrogen depletion, and ETC3/CPL3 expression was enhanced by almost an order of magnitude (9×). Knockout of ETC3/CPL3 did not influence anthocyanin accumulation, but the results establish ETC3/CPL3 as a nitrate regulated gene and a putative candidate for being involved in nitrate status signaling and root development. Other R3-MYBs tested were not significantly influenced by nitrogen depletion. In conclusion, only CPC expression increased and clearly exerted a negative feedback on anthocyanin accumulation during nitrogen starvation in rosette leaves. Copyright © 2013 Elsevier Ltd. All rights reserved.

Umemura Y

Ohme-Takagi M

. AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynjournal in Arabidopsis[J]. Plant, 2008,55:954-967.

Wang X

Hu Q

, et al. Characterization of an activation-tagged mutant uncovers a role of GLABRA2 in anthocyanin biosynjournal in Arabidopsis[J]. Plant, 2015,83:300-311.

Davies K M

Lewis D H

, et al. A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots[J]. Plant Cell, 2014,26:962-980.

杨梦婷, 张春, 王作平, 等. 玉米ZmbHLH161基因的克隆及功能研究[J]. 作物学报, 2020,46(12):2008-2016.

Habera L F

Dellaporta S L

, et al. Lc, a member of the maize R gene family responsible for tissue-specific anthocyanin production, encodes a protein similar to transcriptional activators and contains the MYC homology region[J]. Proc Natl Acad Sci USA, 1989,86:7092-7096.

Zhang F

Lloyd A M

. GL3 encodes a bHLH protein that regulates trichome development in Arabidopsis through interaction with GL1 and TTG1[J]. Ge- netics, 2000,156:1349-1362.

Debeaujon I

Jond C

, et al. The TT8 gene encodes a basic helix- loop-helix domain protein required for expression of DFR and BAN genes in Arabidopsis siliques[J]. Plant Cell, 2000,12:1863-1878.

The TRANSPARENT TESTA8 (TT8) locus is involved in the regulation of flavonoid biosynthesis in Arabidopsis. The tt8-3 allele was isolated from a T-DNA-mutagenized Arabidopsis collection and found to be tagged by an integrative molecule, thus permitting the cloning and sequencing of the TT8 gene. TT8 identity was confirmed by complementation of tt8-3 and sequence analysis of an additional allele. The TT8 gene encodes a protein that displays a basic helix-loop-helix at its C terminus and represents an Arabidopsis ortholog of the maize R transcription factors. The TT8 transcript is present in developing siliques and in young seedlings. The TT8 protein is required for normal expression of two flavonoid late biosynthetic genes, namely, DIHYDROFLAVONOL 4-REDUCTASE (DFR) and BANYULS (BAN), in Arabidopsis siliques. Interestingly, TRANSPARENT TESTA GLABRA1 (TTG1) and TT2 genes also control the expression of DFR and BAN genes. Our results suggest that the TT8, TTG1, and TT2 proteins may interact to control flavonoid metabolism in the Arabidopsis seed coat.

Pattanaik S

Patra B

, et al. Flavonoid-related basic helix-loop-helix regulators, NtAn1a and NtAn1b of tobacco have originatedfrom two ancestors and are functionally active[J]. Planta, 2001,234:363-375.

Hellens R P

Putterill J

, et al. Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10[J]. Plant, 2007,49:414-427.

Du L

Liu R

, et al. Two LcbHLH transcription factors interacting with LcMYB1 in regulating late structural genes of anthocyanin biosynjournal in Nicotiana and Litchi chinensis during anthocyanin accumulation[J]. Front Plant Sci, 2016,7:166.

Qingshan X

Shiqi Z

, et al. Comprehensive co-expression analysis provides novel insights into temporal variation of flavonoids in fresh leaves of the tea plant (Camellia sinensis)[J]. Plant Science, 2020,290:110306.

Dumm J M

Prohens J

, et al. A spontaneous eggplant (Solanum melongena L.) color mutant conditions anthocyanin free fruit pigmentation[J]. Hortscience, 2016,51:793-798.

Qiu J

Ding L

, et al. Anthocyanin biosynjournal regulation of DhMYB2 and DhbHLH1 in Dendrobium hybrids petals[J]. Plant Physiol Biochem, 2017,112:335-345.

Guomin L

Xingyuan X

, et al. bHLH Transcription Factor TsMYC2 Is Associated With the Blue Grain Character in Triticale (Triticum × Secale)[J].Plant Cell Rep, 2019 Oct, 38(10):1291-1298.

Key message RNA-Seq was employed to compare the transcriptome differences between the triticale lines and to identify the key gene responsible for the blue aleurone trait. The accumulation of anthocyanins in the aleurone of triticale results in the formation of the blue-grained trait, but the identity of the genes associated with anthocyanin biosynthesis in the aleurone has not yet been reported. In this manuscript, RNA-Seq was employed to compare the transcriptome differences between the triticale lines HM13 (blue aleurone) and HM5 (white aleurone), and to identify the key genes responsible for the blue aleurone trait. There were 32,406 differentially expressed genes between HM13 and HM5. Seventy-three unigenes were homologous to the structural genes related to anthocyanin biosynthesis, and the average transcript level of the structural genes was higher in HM13 than in HM5, so that quantitative differences between the two lines in transcription rates could be the cause of the blue aleurone. The MYB and bHLH transcription factors had two homologous unigenes, but contained only one differentially expressed unigene each. The relative transcript level of bHLH Unigene5672_All (TsMYC2) in HM13 was 42.71 times that in HM5, while the relative transcript level of the MYB transcription factor Unigene12228_All in HM13 was 2.20 times that in HM5. qPCR experiments determined the relative transcript level of TsMYC2 in developing grain, with the expression of TsMYC2 in grain being the highest compared with that in root, stem or leaf tissue. TsMYC2 was homologous to the bHLH transcription factor regulating anthocyanin biosynthesis and contained three entire functional domains: bHLH-MYC_N, HLH and ACT-like, which were important for exercising regulation of anthocyanin biosynthesis as a bHLH transcription factor. Transient expression of ZmC1 and TsMYC2 could induce anthocyanin biosynthesis in white wheat coleoptile cells, demonstrating that TsMYC2 was a functional bHLH transcription factor. These results indicated that TsMYC2 was associated with the blue aleurone trait and could prove to be a valuable gene with which to breed new triticale cultivars with the blue aleurone trait.

Wei T

Yawen H

, et al. A MYB/bHLH complex regulates tissue specific anthocyanin biosynjournal in the inner pericarp of red-centered kiwifruit Actinidia chinensis cv. Hongyang[J]. The Plant Journal, 2019,99(2):359-378.

Many Actinidia cultivars are characterized by anthocyanin accumulation, specifically in the inner pericarp, but the underlying regulatory mechanism remains elusive. Here we report two interacting transcription factors, AcMYB123 and AcbHLH42, that regulate tissue-specific anthocyanin biosynthesis in the inner pericarp of Actinidia chinensis cv. Hongyang. Through transcriptome profiling analysis we identified five MYB and three bHLH transcription factors that were upregulated in the inner pericarp. We show that the combinatorial action of two of them, AcMYB123 and AcbHLH42, is required for activating promoters of AcANS and AcF3GT1 that encode the dedicated enzymes for anthocyanin biosynthesis. The presence of anthocyanin in the inner pericarp appears to be tightly associated with elevated expression of AcMYB123 and AcbHLH42. RNA interference repression of AcMYB123, AcbHLH42, AcF3GT1 and AcANS in 'Hongyang' fruits resulted in significantly reduced anthocyanin biosynthesis. Using both transient assays in Nicotiana tabacum leaves or Actinidia arguta fruits and stable transformation in Arabidopsis, we demonstrate that co-expression of AcMYB123 and AcbHLH42 is a prerequisite for anthocyanin production by activating transcription of AcF3GT1 and AcANS or the homologous genes. Phylogenetic analysis suggests that AcMYB123 or AcbHLH42 are closely related to TT2 or TT8, respectively, which determines proanthocyanidin biosynthesis in Arabidopsis, and to anthocyanin regulators in monocots rather than regulators in dicots. All these experimental results suggest that AcMYB123 and AcbHLH42 are the components involved in spatiotemporal regulation of anthocyanin biosynthesis specifically in the inner pericarp of kiwifruit.© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.

冯如, 李泽宏, 袁红慧, 等. 银杏两个WD40转录因子基因克隆及序列分析[J]. 北方园艺, 2017(18):100-108.

Verweij W

Kroon A

, et al. PH4 of petunia is an R2R3 MYB protein that activates vacuolar acidification through interactions with Basic-Helix-Loop-Helix transcription factors of the anthocyanin pathway[J]. The Plant Cell, 2006,18(5):1274-1291.

Ochoa-Alejo N

. Virus-induced silencing of MYB and WD40 transcription factor genes affects the accumulation of anthocyanins in chilli pepper fruit[J]. Biol Plant, 2014,58:567-574.

Li Y Q

Yang S

, et al. A functional homologue of Arabidopsis TTG1 from Freesia interacts with bHLH proteins to regulate anthocyanin and proanthocyanidin biosynjournal in both Freesia hybrida and Arabidopsis thaliana[J]. Plant Physiology and Biochemistry, 2019,141:60-72.

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