R2R3-MYB转录因子GaPC调控棉花花瓣着色

The R2R3-MYB transcription factor GaPC controls petal coloration in cotton

Abstract

Although a few cases of genetic epistasis in plants have been reported, the combined analysis of genetically phenotypic segregation and the related molecular mechanism remains rarely studied. Here, we have identified a gene (named GaPC) controlling petal coloration in Gossypium arboreum and following a heritable recessive epistatic genetic model. Petal coloration is controlled by a single dominant gene, GaPC. A loss-of-function mutation of GaPC leads to a recessive gene Gapc that masks the phenotype of other color genes and shows recessive epistatic interactions. Map-based cloning showed that GaPC encodes an R2R3-MYB transcription factor. A 4814-bp long terminal repeat retrotransposon insertion at the second exon led to GaPC loss of function and disabled petal coloration. GaPC controlled petal coloration by regulating the anthocyanin and flavone biosynthesis pathways. Expression of core genes in the phenylpropanoid and anthocyanin pathways was higher in colored than in white petals. Petal color was conferred by flavonoids and anthocyanins, with red and yellow petals rich in anthocyanin and flavonol glycosides, respectively. This study provides new insight on molecular mechanism of recessive epistasis, also has potential breeding value by engineering GaPC to develop colored petals or fibers for multi-functional utilization of cotton.

摘要
尽管在植物中已经报道了一些基因表观遗传的案例，但基因型表型分离与相关分子机制的联合分析仍然是一个较少研究的领域。在本研究中，我们鉴定了一个控制棉花 (Gossypium arboreum) 花瓣着色的基因（命名为 GaPC），并发现其遵循可遗传的隐性表观遗传遗传模型。花瓣着色由单一的显性基因 GaPC 控制。GaPC 的功能丧失突变导致出现一个隐性基因 Gapc，该基因掩盖了其他颜色基因的表型并表现出隐性表观遗传相互作用。基于图谱的克隆分析显示，GaPC 编码一个 R2R3-MYB 转录因子。在第二外显子的 4814-bp 长末端重复反转录转座子插入导致 GaPC 功能丧失，并使花瓣失去着色能力。GaPC 通过调控花青素和黄酮类物质的合成途径来控制花瓣着色。苯丙氨酸和花青素途径的核心基因在有色花瓣中的表达高于白色花瓣。有色花瓣的颜色由黄酮类物质和花青素决定，其中红色花瓣富含花青素，而黄色花瓣则富含黄酮糖苷。该研究为隐性表观遗传的分子机制提供了新的见解，同时通过工程化 GaPC 来开发具有多功能利用潜力的有色花瓣或纤维，具有潜在的育种价值。

1. Introduction

Following Mendel’s genetic laws, two or more genes may interact with each other to influence their phenotypic expression. Among such non-allelic gene interactions, epistasis refers to masking by one gene of the phenotypes of other genes. The epistasis can be divided into dominant and recessive epistasis according to whether a dominant gene or recessive gene plays the role of standing over [1]. Although a few cases of genetic epistasis analysis based on phenotypic investigation are known, the simultaneous analysis of non-allelic gene epistasis genetic segregation and the associated molecular mechanism remains rarely reported.

1. 引言
   根据孟德尔遗传规律，两个或多个基因可能相互作用，影响它们的表型表现。在这种非等位基因相互作用中，表观遗传指的是一个基因掩盖其他基因表型的现象。表观遗传可以根据是显性基因还是隐性基因起主导作用分为显性表观遗传和隐性表观遗传[1]。尽管已知有一些基于表型调查的基因表观遗传分析案例，但关于非等位基因表观遗传遗传分离与相关分子机制的同时分析仍然很少报道。

Flower color is an important agronomic trait, and pigments are widely studied. Flowers, the major reproductive organs of plants, are composed of sepals, stamens, pistils, and petals. Bright colors of flower petals attract insect pollination, promoting pollination efficiency and increasing yields in cross-pollinated crops [2]. Flower petals also have ornamental or edible value. Petal colors are determined by the pigments in petal cells. The pigments responsible for petal colors are mainly flavonoids, anthocyanins, and carotenoids. Flavonoids are secondary metabolites that are present in plants mostly in the form of glycosides. Depending on the chemical substituents on their skeletons, they are divided into anthocyanins, flavonols, and other classes [3], [4], [5], [6]. The four main kinds of flavonol glycosides are kaempferol, quercetin, myricetin, and isorhamnetin, most of which are yellow. Six main anthocyanin glycosides (pelargonidin, cyanidin, delphinidin, peonidin, petunidin, and malvidin) can present red, pink, blue, or purple colors [4], [5].

花色是一个重要的农艺性状，且色素是广泛研究的对象。花朵是植物的主要生殖器官，通常由萼片、雄蕊、雌蕊和花瓣组成。花瓣的鲜艳颜色能够吸引昆虫授粉，促进授粉效率并增加异花授粉作物的产量[2]。花瓣还具有观赏或食用价值。花瓣的颜色由花瓣细胞中的色素决定。负责花瓣颜色的色素主要是类黄酮、花青素和胡萝卜素。类黄酮是植物中的次级代谢产物，主要以糖苷形式存在于植物中。根据其骨架上的化学取代基，它们被分为花青素、黄酮醇及其他类别[3]，[4]，[5]，[6]。四种主要的黄酮醇糖苷分别是槲皮素、槲皮素、杨梅素和异鼠李素，其中大多数呈黄色。六种主要的花青素糖苷（天竺葵素、花青素、龙胆紫素、凤仙花素、矢车菊素和美德紫素）可以呈现红色、粉色、蓝色或紫色[4]，[5]。

The flavonoid/anthocyanin biosynthetic pathway is well understood, and is related to the formation of pigments. In the general phenylpropanoid pathway, phenylalanine generates 4-coumaroyl-CoA under the catalysis of phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumaryol CoA ligase (4CL). The flavonoid/anthocyanin pathway starts from one molecule of p-coumaroyl-CoA and three molecules of malonyl-CoA to generate naringenin chalcone by the action of chalcone synthase (CHS). CHS and other enzymes including chalcone isomerase (CHI), flavonoid 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′5′ hydroxylase (F3′5′H), dihydroflavonol reductase (DFR), flavonol synthase (FLS), anthocyanidin synthase (ANS), and UDP-flavonoid glucosyl transferase (UFGT) function in the flavonoid and anthocyanin biosynthetic pathway. All flavonol and anthocyanin glycosides are transferred and stored in vacuoles under glutathione-s-transferase (GST) and multidrug and toxic compound extrusion (MATE), showing multifarious colors [4], [5], [6], [7]. CHS, CHI, F3H, F3′H and F3′5′H are early enzymes or genes in the flavonoid/anthocyanin pathway, and FLS, DFR, ANS and UFGT are late enzymes or genes. Previous studies [6], [8] showed that MBW ternary complexes (containing R2R3-MYB, bHLH transcription factors, and WD40 protein) were regulators that activate the core enzymes or genes of the flavonoid and anthocyanin pathway. Among MBW ternary complexes, subgroup 6 R2R3-MYB of Arabidopsis are central components. Subgroup 6 R2R3-MYB (AtMYB75, AtMYB90, AtMYB113 and AtMYB114) and their gene homologs have been reported to be key factors determining the various anthocyanin glycoside pigmentation patterns of flowers by regulating the anthocyanin biosynthetic pathway in crops and ornamental plants such as Medicago truncatula [8], Brassica juncea [9], Xanthoceras sorbifolium [10]. NiorMYB113-1 and NiorMYB113-2 function cooperatively to specify formation of complex petal color patterns in Nigella orientalis [11]. Ectopic expression of Eutrema salsugineum EsMYB90 in tobacco and Arabidopsis increased pigmentation and anthocyanin accumulation in various organs [12].

类黄酮/花青素生物合成途径已经得到了充分理解，并且与色素的形成密切相关。在一般的酚丙烷途径中，苯丙氨酸在苯丙氨酸氨基裂解酶（PAL）、肉桂酸4-羟化酶（C4H）和4-香豆酰辅酶A连接酶（4CL）的催化作用下生成4-香豆酰辅酶A。类黄酮/花青素途径从1分子p-香豆酰辅酶A和3分子马龙酰辅酶A开始，通过查尔酮合酶（CHS）的作用生成槲皮素查尔酮。CHS和其他酶，包括查尔酮异构酶（CHI）、类黄酮3-羟化酶（F3H）、类黄酮3′-羟化酶（F3′H）、类黄酮3′5′-羟化酶（F3′5′H）、二氢黄酮醇还原酶（DFR）、黄酮醇合成酶（FLS）、花青素合成酶（ANS）和UDP-类黄酮葡萄糖转移酶（UFGT）在类黄酮和花青素生物合成途径中发挥作用。所有的黄酮醇和花青素糖苷都通过谷胱甘肽-S-转移酶（GST）和多药耐药及毒物外排蛋白（MATE）转运并储存于液泡中，呈现出多样的颜色[4]，[5]，[6]，[7]。CHS、CHI、F3H、F3′H和F3′5′H是类黄酮/花青素途径中的早期酶或基因，而FLS、DFR、ANS和UFGT是晚期酶或基因。先前的研究[6]，[8]表明，MBW三元复合体（包括R2R3-MYB、bHLH转录因子和WD40蛋白）是激活类黄酮和花青素途径核心酶或基因的调控因子。在MBW三元复合体中，阿拉伯芥的6亚群R2R3-MYB是核心组成部分。6亚群R2R3-MYB（AtMYB75、AtMYB90、AtMYB113和AtMYB114）及其基因同源物已被报道为决定花卉中各种花青素糖苷着色模式的关键因素，作用于农作物和观赏植物如蚕豆（Medicago truncatula）[8]、芥菜（Brassica juncea）[9]、黄皮树（Xanthoceras sorbifolium）[10]的花青素生物合成途径。NiorMYB113-1和NiorMYB113-2在Nigella orientalis中协同作用，指定复杂的花瓣颜色模式的形成[11]。Eutrema salsugineum的EsMYB90在烟草和阿拉伯芥中的异位表达增加了各种器官的着色和花青素的积累[12]。