Genetic and multi-omics analyses reveal BnaA07.PAP2In-184-317 as the key gene conferring anthocyanin

遗传学和多组学分析揭示BnaA07.PAP2In-184-317是赋予油菜花朵花青素颜色的关键基因。

Abstract

The molecular mechanisms underlying anthocyanin-based flower coloration remain unknown in Brassica napus. To identify the key genes and metabolites associated with apricot and pink flower colors, metabolome, BSA-seq, and RNA-seq analyses were conducted on apricot-, pink-, yellow-, and white-flowered F2B. napus. Yellow carotenoids and red anthocyanins were abundant in apricot petals, while colorless carotenoids and red anthocyanins accumulated in pink petals. Most carotenoid genes were not differentially regulated between apricot and yellow or between pink and white petals. Three regulator genes, BnaMYBL2, BnaA07.PAP2, and BnaTT8, and structural genes in anthocyanin biosynthesis were dramatically enhanced in apricot and pink petals in comparison with yellow and white petals. Map-based cloning revealed that BnaA07.PAP2 is responsible for anthocyanin-based flower color and encodes a nucleus-localized protein predominantly expressed in apricot and pink flowers. Two insertions in the promoter region are responsible for the transcriptional activation of BnaA07.PAP2 in flowers. Introducing the BnaA07.PAP2In-184-317 allele broadly activated the expression of anthocyanin-related genes and promoted anthocyanin accumulation in flowers, yielding color change from yellow to apricot. These findings illustrate the genetic basis of anthocyanin-based flower coloration and provide a valuable genetic resource for breeding varieties with novel flower colors in B. napus.

油菜花朵基于花青素的颜色分子机制仍未明了。为了鉴定与杏色和粉色花朵颜色相关的关键基因和代谢物，研究者对杏色、粉色、黄色和白色花朵的油菜F2群体进行了代谢组学、BSA-seq和RNA-seq分析。杏色花瓣中富含黄色类胡萝卜素和红色花青素，而粉色花瓣中积累了无色类胡萝卜素和红色花青素。大多数类胡萝卜素基因在杏色与黄色花瓣、粉色与白色花瓣之间未表现出差异调控。与黄色和白色花瓣相比，杏色和粉色花瓣中与花青素生物合成相关的三个调控基因BnaMYBL2、BnaA07.PAP2和BnaTT8以及结构基因的表达显著增强。基于图谱的克隆研究发现，BnaA07.PAP2基因是花青素基础花色的关键基因，编码一种主要在杏色和粉色花朵中表达的、定位于细胞核的蛋白质。该基因启动子区域的两个插入突变负责其在花朵中的转录激活。引入BnaA07.PAP2In-184-317等位基因广泛激活花青素相关基因的表达，促进花朵中花青素的积累，从而使花色由黄色变为杏色。这些发现阐明了基于花青素的花朵着色的遗传基础，并为油菜品种培育提供了具有新花色的宝贵遗传资源。

Introduction

Flower color is of paramount importance in pollinator attraction and ornamental breeding programs. The generation of new colors is an area where progress is being actively pursued. Many factors, including pigment composition, pH, cell shape, and co-pigmentation, are related to petal coloration (Grotewold, 2006; Iwashina, 2015). Carotenoids and flavonoids are the two major groups of pigments responsible for the attractive natural display of flower colors. Yellow and orange flower colors are mostly attributed to carotenoid pigments, a class of plastid-synthesized and localized isoprenoid compounds universally distributed in plant species. They provide important photoprotective functions during photosynthesis and serve as the precursors of abscisic acid and strigolactones (Seto and Yamaguchi, 2014; Nisar et al., 2015). Significant progress has been made in deciphering the synthesis and participation of carotenoids in flower and fruit pigmentation (Ruiz-Sola and Rodríguez-Concepción, 2012). The coordinated transcriptional regulation of core structural genes involved in carotenoid biosynthesis is largely responsible for changes in carotenoid content and composition, affecting the coloration of flowers and fruits. Additionally, the activity of enzymes controlling carotenoid degradation, such as carotenoid cleavage dioxygenases (CCDs), which cause the breakdown of C40 carotenoids into apocarotenoids, is critical for carotenoid content modulation. Growing evidence shows that enhanced transcription of CCD4 is associated with a color change from yellow to white in flowers and fruits (Nisar et al., 2015; Zhang et al., 2015).

花色在吸引传粉者和观赏育种项目中具有至关重要的作用。创造新的花色是一个正在积极追求的领域。许多因素，包括色素成分、pH值、细胞形状和共色素现象，都会影响花瓣的着色（Grotewold, 2006; Iwashina, 2015）。类胡萝卜素和黄酮类化合物是负责花色自然吸引力的两大主要色素群体。黄色和橙色的花色主要归因于类胡萝卜素色素，这是一类在植物中普遍分布的、由质体合成并局部化的异戊二烯化合物。它们在光合作用过程中提供重要的光保护功能，并且是脱落酸和小分子激素的前体（Seto and Yamaguchi, 2014; Nisar et al., 2015）。在解密类胡萝卜素的合成及其在花朵和果实着色中的参与方面，已经取得了显著进展（Ruiz-Sola and Rodríguez-Concepción, 2012）。类胡萝卜素生物合成过程中核心结构基因的协调转录调控在类胡萝卜素含量和组成变化中起着至关重要的作用，从而影响花卉和果实的着色。此外，控制类胡萝卜素降解的酶的活性，如类胡萝卜素裂解二氧化酶（CCDs），它们通过将C40类胡萝卜素分解为脱类胡萝卜素， 对类胡萝卜素的含量调节也至关重要。越来越多的证据表明，CCD4的转录增强与花朵和果实由黄色变白色的颜色变化相关（Nisar et al., 2015; Zhang et al., 2015）。

Flavonoids are secondary metabolites performing a wide range of functions, including pollinator attraction, antioxidant activity, UV-light protection, auxin transport regulation, and defense against biotic and abiotic stresses (Tohge et al., 2017). Although many flavonoids are colorless or extremely pale, certain flavonoids, including anthocyanins, chalcones, aurones, and some flavonols, act as flower pigments (Tohge et al., 2017). Anthocyanins contribute approximately 88% of flower coloration in angiosperms, ranging from orange to pink, red, purple, and blue (Grotewold, 2006; Iwashina, 2015). The genes and enzymes involved in flavonoid biosynthesis and regulation are well conserved and characterized in plants (Grotewold, 2006; Tohge et al., 2017). In Arabidopsis, the flavonoid pathway begins with the synthesis of naringenin chalcone by the first committed enzyme, chalcone synthase. Other committed enzymes, namely chalcone isomerase (CHI), flavonoid 3-hydroxylase (F3H), and flavonoid 3ʹ-hydroxylase, convert naringenin chalcone into the common precursors (dihydroflavonols). The genes encoding these enzymes are called early biosynthetic genes (EBGs). Downstream genes of the pathway, namely flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonoid 3-O-glycosyltransferase (UFGT), which lead to the production of different flavonoids, are called late biosynthetic genes (LBGs). DFR, ANS, and UFGT are key genes for anthocyanin biosynthesis. The anthocyanins produced are transported into vacuoles for storage through several mechanisms, including glutathione S-transferases (GST) (Luo et al., 2018). Besides of these structural genes, transcription factors (TFs) that induce the expression of structural genes have also been identified to be involved in anthocyanin biosynthesis. In Arabidopsis, anthocyanins are mainly regulated by a transcriptional complex (MBW complex) consisting of a WD-repeat protein, TRANSPARENT TESTA GLABRA1 (TTG1); a bHLH protein, GLABRA3 (GL3), ENHANCER OF GLABRA3 (EGL3), or TRANSPARENT TESTA (TT8); and an R2R3-MYB protein, PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1), PAP2, MYB113, MYB114, or MYBL2 (Xu et al., 2015).

类黄酮是次级代谢物，具有广泛的功能，包括吸引传粉者、抗氧化活性、紫外线保护、Auxin运输调节以及对生物和非生物胁迫的防御（Tohge 等，2017）。尽管许多类黄酮是无色或极为淡色的，但某些类黄酮，包括花青素、查尔酮、金黄色素和一些黄烷醇，作为花朵的色素（Tohge 等，2017）。花青素在被子植物的花朵着色中贡献了大约88%，其颜色从橙色、粉色、红色、紫色到蓝色（Grotewold，2006；Iwashina，2015）。参与类黄酮生物合成和调控的基因和酶在植物中是高度保守和表征的（Grotewold，2006；Tohge 等，2017）。在拟南芥中，类黄酮途径从第一步专一酶——查尔酮合酶合成橙皮素查尔酮开始。其他专一酶，即查尔酮异构酶（CHI）、类黄酮3-羟化酶（F3H）和类黄酮3ʹ-羟化酶，将橙皮素查尔酮转化为常见的前体（双氢黄烷醇）。编码这些酶的基因称为早期生物合成基因（EBGs）。该途径的下游基因，即黄烷醇合酶（FLS）、双氢黄烷醇4-还原酶（DFR）、花青素合成酶（ANS）和类黄酮3-O-糖基转移酶（UFGT），它们最终生成不同的类黄酮，称为晚期生物合成基因（LBGs）。DFR、ANS 和 UFGT 是花青素生物合成的关键基因。花青素通过包括谷胱甘肽S-转移酶（GST）等几种机制被转运到液泡中储存（Luo 等，2018）。除了这些结构基因外，已经鉴定出能够诱导结构基因表达的转录因子（TFs）也参与花青素的生物合成。在拟南芥中，花青素的主要调控是由一个转录复合体（MBW复合体）实现的，该复合体由一个WD重复蛋白TRANSPARENT TESTA GLABRA1（TTG1）、一个bHLH蛋白（GLABRA3（GL3）、ENHANCER OF GLABRA3（EGL3）或TRANSPARENT TESTA（TT8））和一个R2R3-MYB蛋白（PRODUCTION OF ANTHOCYANIN PIGMENT1（PAP1）、PAP2、MYB113、MYB114或MYBL2）组成（Xu 等，2015）。

In China, Brassica napus flowers have gained much attention recently owing to numerous reports of their contributions to the ornamental tourism industry. However, these flowers are not polymorphic with respect to petal color. Brassica napus typically produces yellow corollas, pigmented by the lipid-soluble carotenoids accumulated in the chromoplasts of epidermal cells. Several studies have reported white- and orange-flowered B. napus germplasms or mutants (Zhang et al., 2015; Liu et al., 2020). The white flower trait of the B. napus line 2127 is controlled by BnaC3.CCD4, a homolog of carotenoid cleavage dioxygenase 4 (CCD4), which encodes an enzyme that breaks down colored carotenoids into colorless volatile compounds. Disruption of this gene by a transposable element insertion produces a yellow-flowered phenotype (Zhang et al., 2015). Furthermore, BnaA09.ZEP and BnaC09.ZEP (zeaxanthin epoxidase) dysfunction in yellow-flowered accessions changed the carotenoid profile of petals, resulting in color change from yellow to orange (Liu et al., 2020). To date, few reports on the production and characterization of anthocyanin-conferred colors in B. napus flowers are available. Ectopic expression of Orychophragmus violaceus PAP2 in B. napus petals produced flowers with a slightly red blush only in the basal parts of the corolla (Fu et al., 2018). More recently, Yin et al. (2019) analysed the flavonoid profiles of red-, yellow-, pink-, and white-flowered lines of B. napus with different genetic backgrounds. However, the specific pigments and molecular mechanisms responsible for red or pink colors are still unknown. Therefore, there is a need to extend studies on pigment regulation in B. napus flowers in order to create new flower colors.

在中国，油菜花（Brassica napus）近年来受到广泛关注，主要因为其对观光旅游产业的贡献。然而，这些花卉在花瓣颜色上并无多态性。油菜通常开黄花，其花色由积累在表皮细胞色素体中的脂溶性类胡萝卜素所赋予。有几项研究报告了白色和橙色花朵的油菜种质或突变体（Zhang et al., 2015; Liu et al., 2020）。油菜2127号品种的白花性状由BnaC3.CCD4基因控制，该基因是类胡萝卜素裂解双加氧酶4（CCD4）的同源基因，编码一种将有色类胡萝卜素分解为无色挥发性化合物的酶。该基因由于可转移元素插入的破坏而产生黄色花朵表型（Zhang et al., 2015）。此外，BnaA09.ZEP和BnaC09.ZEP（玉米黄烯环氧化酶）在黄色花朵类型中的功能失常，改变了花瓣的类胡萝卜素谱，导致花色从黄色变为橙色（Liu et al., 2020）。迄今为止，关于油菜花中由花青素赋予的花色的研究报告较少。Orychophragmus violaceus PAP2基因的异位表达在油菜花瓣中仅在花冠基部产生了轻微的红色晕染（Fu et al., 2018）。最近，Yin等人（2019）分析了不同遗传背景的红色、黄色、粉色和白色油菜花品系的类黄酮谱。然而，导致红色或粉色花色的具体色素及其分子机制仍然未知。因此，亟需扩展对油菜花中色素调控的研究，以创造新的花色。

In this study, we report an apricot-flowered germplasm in B. napus. Interestingly, crosses between apricot-flowered B. napus and white-flowered lines led to progeny with flowers of apricot, yellow, white, and a novel flower color, pink. Phenotypic, histological and pigment characters of these colored flowers were studied. We then conducted metabolome, bulked segregant sequencing (BSA-seq) and RNA-seq analyses to identify the key genes and metabolites associated with apricot and pink flower color. A TF gene, BnaA07.PAP2, was then map-based cloned and functionally verified by transformation analysis. Two insertions in the promoter region cause the transcriptional activation of BnaA07.PAP2, which in turn launches the whole anthocyanin pathway to produce the reddish hue in B. napus flowers. These findings will improve our understanding of the molecular mechanism underlying anthocyanin-based flower color and lay the foundation for future attempts to engineer novel flower color in B. napus.

在这项研究中，我们报告了一个杏花色的油菜（B. napus）种质资源。有趣的是，杏花色油菜与白花油菜的杂交产生了杏色、黄色、白色以及一种新型花色——粉色的后代。我们对这些不同花色的表型、组织学和色素特征进行了研究。接着，我们进行了代谢组学、 bulked segregant sequencing (BSA-seq) 和 RNA-seq 分析，以鉴定与杏色和粉色花色相关的关键基因和代谢物。通过图位克隆和转化分析，我们鉴定出一个转录因子基因 BnaA07.PAP2，并进行了功能验证。我们发现该基因启动子区域的两个插入导致了 BnaA07.PAP2 的转录激活，从而启动了整个花青素合成通路，产生了油菜花中的红色色调。这些发现将有助于我们更好地理解基于花青素的花色分子机制，并为未来在油菜中创造新型花色奠定基础。

Materials and methods

Plant materials and sampling

Five inbred B. napus lines, yellow-flowered Y2346 (Supplementary Fig. S1A) and ZS11, white-flowered W2347 (Supplementary Fig. S1B) and 2127, and apricot-flowered A7603 (Supplementary Fig. S1C), were used as parents to construct segregating populations. A7603 was provided by Zhejiang Academy of Agricultural Sciences. Y2346 and W2347 are near-isogenic lines with the same genetic background but different flower colors. An allelism test comparing white-flowered W2347 and 2127 (Zhang et al., 2015) resulted in F1, F2, and BC1 individuals with only white flowers, revealing that W2347 also carries a dominant allele in the BnaC3.CCD4 locus. For genetic analysis of the apricot flower trait, F1 plants were produced by reciprocally crossing apricot-flowered A7603 with the yellow- or white-flowered lines. Four F2 populations, YA-1, WA-1, YA-2, and WA-2, were generated by self-pollination of F1 plants from the crosses Y2346×A7603, W2347×A7603, ZS11×A7603, and 2127×A7603, respectively. A BC1 population was constructed by backcrossing F1 plants of cross ZS11×A7603 with ZS11. WA-2 was grown in Mingle, Gansu Province, while the other populations were grown in Wuhan, Hubei Province.

使用五个自交系的油菜（B. napus）品系，分别为黄色花的Y2346（补充图S1A）和ZS11、白色花的W2347（补充图S1B）和2127，以及杏色花的A7603（补充图S1C），作为亲本构建分离群体。A7603由浙江省农业科学院提供。Y2346和W2347是近等基因系，具有相同的遗传背景，但花色不同。通过对白色花的W2347和2127（Zhang等，2015）进行等位基因检测，得到的F1、F2和BC1个体均为白色花，表明W2347在BnaC3.CCD4位点上也携带一个显性等位基因。为了对杏色花性状进行遗传分析，通过交叉杏色花的A7603与黄色或白色花的品系产生F1植物。通过自交F1植物，分别由交配Y2346×A7603、W2347×A7603、ZS11×A7603和2127×A7603生成四个F2群体：YA-1、WA-1、YA-2和WA-2。通过将F1植物Z2346×A7603的回交个体与ZS11回交，构建了一个BC1群体。WA-2群体在甘肃省民乐县种植，其他群体在湖北省武汉市种植。

Since the parents of the YA-1 and WA-1 populations had the same genetic background, yellow- and apricot-flowered individuals in the YA-1 population, and white- and pink-flowered individuals in the WA-1 population were used for the metabolomics, BSA-seq, and RNA-seq analyses. A total of 20 individuals with apricot, yellow, or pink flowers and 12 individuals with white flowers were used for leaf sampling (Fig. 1A–D). Petals were sampled from four individuals of each phenotype at three different stages of flower development (Fig. 1E–H), with three biological replicates (a total of 12 individuals). The stages of flower development were defined in terms of bud length and petal color as follows: stage P1, 3–4 mm-long buds with pale green petals (Fig. 1I–L); stage P2, 7–8 mm-long buds with strongly pigmented petals (Fig. 1M–P); and stage P3, newly opened flowers with colored petals. For convenience, we designated the petals from apricot-, yellow-, pink-, and white-flowered plants at all stages as apricot, yellow, pink, and white petals, respectively. All samples were stored at −80 °C for subsequent metabolome, BSA-seq, and RNA-seq analysis.

由于YA-1和WA-1种群的亲本具有相同的遗传背景，因此在YA-1种群中选择了黄色和杏色花朵的个体，在WA-1种群中选择了白色和粉色花朵的个体，用于代谢组学、BSA-seq和RNA-seq分析。共选择了20个杏色、黄色或粉色花朵的个体和12个白色花朵的个体用于叶片采样（图1A–D）。在花发育的三个不同阶段，从每种表型的四个个体中采集了花瓣样本（图1E–H），并进行了三次生物重复（共12个个体）。花发育的各个阶段通过花蕾长度和花瓣颜色进行定义，具体为：P1阶段，花蕾长度为3–4毫米，花瓣呈浅绿色（图1I–L）；P2阶段，花蕾长度为7–8毫米，花瓣明显着色（图1M–P）；P3阶段，刚开放的花朵，花瓣已着色。为了方便起见，我们将杏色、黄色、粉色和白色花朵植物在各个阶段的花瓣分别命名为杏色花瓣、黄色花瓣、粉色花瓣和白色花瓣。所有样本均存储在-80°C，用于后续的代谢组、BSA-seq和RNA-seq分析。

图1A–D图1E–H图1I–L图1M–P  图1. WA-1 F2种群中黄色（A, E, I, M, Q）、杏色（B, F, J, N, R）、白色（C, G, K, O, S）和粉色（D, H, L, P, T）花朵个体的代表性表型。（A–D）黄色、杏色、白色和粉色花朵个体的花序。比例尺：2厘米。（E–H）黄色、杏色、白色和粉色花朵个体的花发育阶段。P1，花蕾长3–4毫米，花瓣呈浅绿色；P2，花蕾长7–8毫米，花瓣颜色深；P3，完全成熟，开放并着色的花朵。比例尺：2厘米。（I–L）P1阶段解剖花蕾的视图，显示四片萼片（第一行）和四片花瓣及其余的雄蕊和雌蕊（第二行）的颜色。比例尺：1厘米。ÿ