转录组和化学分析揭示了Rosa Rugosa中花色形成的机制

Transcriptome and chemical analyses revealed the mechanism of flower color formation in Rosa rugosa

Rosa rugosa is a famous Chinese traditional flower with high ornamental value and well environmental adapt ability. The cultivation of new colorful germplasms to improve monotonous flower color could promote its landscape application. However, the mechanism of flower color formation in R. rugosa remains unclear. In this study, combined analyses of the chemical and transcriptome were performed in the R. rugosa germplasms with representative flower colors. Among the identified anthocyanins, cyanidin 3,5-O-diglucoside (Cy3G5G) and peonidin 3,5-O-diglucoside (Pn3G5G) were the two dominant anthocyanins in the petals of R. rugosa. The sum content of Cy3G5G and Pn3G5G was responsible for the petal color intensity, such as pink or purple, light- or dark- red. The ratio of Cy3G5G to Pn3G5G was contributed to the petal color hue, that is, red or pink/purple. Maintaining both high relative and high absolute content of Cy3G5G may be the precondition for forming red-colored petals in R. rugosa. Cyanidin biosynthesis shunt was the dominant pathway for anthocyanin accumulation in R. rugosa, which may be the key reason for the presence of monotonous petal color in R. rugosa, mainly pink/purple. In the upstream pathway of cyanidin biosynthesis, 35 differentially expressed structural genes encoding 12 enzymes co-expressed to regulate the sum contents of Cy3G5G and Pn3G5G, and then determined the color intensity of petals. RrAOMT, involved in the downstream pathway of cyanidin biosynthesis, regulated the ratio of Cy3G5G to Pn3G5G via methylation and then determined the color hue of petals. It was worth mentioning that significantly higher delphinidin-3,5-O-diglucoside content and RrF3’5’H expression were detected from deep purple-red-flowered 8-16 germplasm with somewhat unique and visible blue hue. Three candidate key transcription factors identified by correlation analysis, RrMYB108, RrC1, and RrMYB114, might play critical roles in the control of petal color by regulating the expression of both RrAOMT and other multiple structural genes. These results provided novel insights into anthocyanin accumulation and flower coloration mechanism in R. rugosa, and the candidate key genes involved in anthocyanin biosynthesis could be valuable resources for the breeding of ornamental plants in future.

玫瑰蔷薇（Rosa rugosa）是一种具有高观赏价值和良好环境适应能力的中国传统名花。培育新的多彩种质以改善其单一的花色，能够促进其在景观应用中的推广。然而，目前对于玫瑰蔷薇花色形成机制的研究仍不清楚。本研究对具有代表性花色的玫瑰蔷薇种质进行了化学分析与转录组联合分析。在鉴定出的花青素中，矢车菊素-3,5-二葡萄糖苷（Cy3G5G）和芍药素-3,5-二葡萄糖苷（Pn3G5G）是玫瑰蔷薇花瓣中的两种主要花青素。Cy3G5G和Pn3G5G的总含量决定了花瓣的颜色强度，例如粉色或紫色、浅红或深红。Cy3G5G与Pn3G5G的比例则决定了花瓣的颜色色调，即红色或粉色/紫色。在玫瑰蔷薇中，维持Cy3G5G的高相对含量和高绝对含量可能是形成红色花瓣的先决条件。矢车菊素生物合成分支是玫瑰蔷薇花青素积累的主要途径，这可能是导致玫瑰蔷薇花瓣颜色单一，主要为粉色/紫色的关键原因。在矢车菊素生物合成上游途径中，有35个差异表达的结构基因编码12种酶，它们共同表达以调节Cy3G5G和Pn3G5G的总含量，从而决定了花瓣的颜色强度。RrAOMT参与矢车菊素生物合成下游途径，通过甲基化调节Cy3G5G与Pn3G5G的比例，进而决定了花瓣的颜色色调。值得注意的是，在具有独特且可见蓝色调的深紫红色花的8-16种质中，检测到了显著更高的飞燕草素-3,5-二葡萄糖苷含量和RrF3'5'H的表达。通过相关性分析鉴定出的三个候选关键转录因子RrMYB108、RrC1和RrMYB114，可能通过调节RrAOMT以及其他多个结构基因的表达，在花瓣颜色调控中发挥关键作用。这些结果为玫瑰蔷薇中花青素积累和花色形成机制提供了新的见解，而参与花青素生物合成的候选关键基因可能成为未来观赏植物育种的宝贵资源。

介绍Introduction  

Flower color is one of the main quality traits of ornamental plants, which is determined by the content and type of anthocyanins. Anthocyanins, a class of secondary metabolites with the different substituents on the B ring of flavonoid basic skeleton, are important water-soluble pigments accumulated in vascular plants widely (Tanaka et al., 2008; Landi et al., 2015). So far, more than 700 kinds of anthocyanidins have been found in plants, which mainly derive from six anthocyanidin aglycones, i.e., pelargonidin, cyanidin, peonidin, delphinidin, petunidin, and malvidin (Jaakola, 2013). Generally, pelargonidin and cyanidin provide red pigment to flowers and fruits, peonidin makes great contributions to purple-red color of plant tissues, while delphinidin, petunidin and malvidin are responsible for blue and bluish violet color (Khoo et al., 2017). However, the relationship between anthocyanin accumulation and petal coloration varies among different species. The anthocyanin coloration can be consistent among different plant species. For example, cyanidins are the main anthocyanins responsible for the pink and red petals of Camellia japonica and Prunus persica (Cheng et al., 2014; Fu et al., 2021). In addition, the anthocyanin coloration can also show species specificity. For instance, delphinidins are the dominant anthocyanins in most plants with pure blue petals, whereas the petals of transgenic R. hybrida with a high percentage of delphinidins (up to 95%) are not as pure blue as any other plants (Katsumoto et al., 2007).

花色是观赏植物的主要品质性状之一，由花青素的含量和种类决定。花青素是一类在黄酮类基本骨架的B环上具有不同取代基的次生代谢产物，是广泛存在于维管植物中的重要水溶性色素（Tanaka et al., 2008; Landi et al., 2015）。迄今为止，植物中已发现700多种花青素，它们主要来源于六种花青素苷元，即牻牛儿素、矢车菊素、芍药素、飞燕草素、牵牛花素和锦葵素（Jaakola, 2013）。一般来说，牻牛儿素和矢车菊素为花和果实提供红色色素，芍药素对植物组织的紫红色贡献最大，而飞燕草素、牵牛花素和锦葵素则负责蓝色和蓝紫色（Khoo et al., 2017）。然而，花青素积累与花瓣着色之间的关系在不同物种之间存在差异。花青素的着色在不同植物物种之间可能是一致的。例如，矢车菊素是山茶花（Camellia japonica）和油桃（Prunus persica）粉色和红色花瓣的主要花青素（Cheng et al., 2014; Fu et al., 2021）。此外，花青素的着色也可以表现出物种特异性。例如，飞燕草素是大多数具有纯蓝色花瓣植物中的主要花青素，然而，即使转基因杂交玫瑰（Rosa hybrida）的花瓣中飞燕草素含量高达95%，其花瓣的颜色也不如其他植物那样纯蓝（Katsumoto et al., 2007）。

Anthocyanins biosynthesis process is relatively conserved and have been studied in many seed plants (Shoeva et al., 2017; Nabavi et al., 2020). The biosynthetic pathways, using phenylalanine as substrate, requires a series of catalytic enzymes. Firstly, phenylalanine is catalyzed to form cinnamic acid under the action of phenylalanine ammonia-lyase (PAL, EC:4.3.1.24). Secondly, dihydroflavonols were generated by a series of enzymes, such as cinnamate-4-hydroxylase (C4H, EC:1.14.14.91), 4-coumarate-CoA ligase (4CL, EC:6.2.1.12), chalcone synthase (CHS, EC:2.3.1.74), chalcone isomerase (CHI, EC:5.5.1.6), flavanone 3-hydroxylase (F3H, EC:1.14.11.9), flavonoid-3’-hydroxylase (F3’H, EC:1.14.13.21) and flavonoid-3’5’-hydroxylase (F3’5’H, EC:1.14.14.81), which is a key step in the metabolism of flavonoids. The performance of F3’H and F3’5’H leads to form dihydroquercetin and dihydromyricetin with different hydroxylation pattern of dihydrokaempferol and then promote the biosynthesis of cyanidin and delphinidin (Zhuang et al., 2019). Subsequently, colored anthocyanins are formed by dihydroflavonol 4-reductase (DFR, EC:1.1.1.219), anthocyanin synthase (ANS, EC:1.14.20.4), and modified by glycosylation, methylation, and acetyltransferase under the actions of glycosyl transferases (GT, EC:2.4.1.-), methyl transferase (MT, EC:2.1.1.-), and acyl transferase (AT, EC:2.3.1.-), respectively (Koes et al., 2005; Jaakola, 2013; Iaria et al., 2016).

花青素的生物合成过程相对保守，并已在许多种子植物中得到研究（Shoeva et al., 2017; Nabavi et al., 2020）。这一生物合成途径以苯丙氨酸为底物，需要一系列催化酶的参与。首先，苯丙氨酸在苯丙氨酸解氨酶（PAL，EC:4.3.1.24）的作用下被催化生成桂皮酸。其次，一系列酶（如桂皮酸-4-羟化酶（C4H，EC:1.14.14.91）、4-香豆酸辅酶A连接酶（4CL，EC:6.2.1.12）、查尔酮合成酶（CHS，EC:2.3.1.74）、查尔酮异构酶（CHI，EC:5.5.1.6）、黄烷酮-3-羟化酶（F3H，EC:1.14.11.9）、黄酮类-3'-羟化酶（F3'H，EC:1.14.13.21）和黄酮类-3',5'-羟化酶（F3'5'H，EC:1.14.14.81））将生成二氢黄酮醇，这是黄酮类代谢的关键步骤。F3'H和F3'5'H的作用导致形成具有不同二氢山柰酚羟基化模式的二氢槲皮素和二氢杨梅素，并进一步促进矢车菊素和飞燕草素的生物合成（Zhuang et al., 2019）。随后，花青素在二氢黄酮醇-4-还原酶（DFR，EC:1.1.1.219）和花青素合成酶（ANS，EC:1.14.20.4）的作用下形成，并通过糖基转移酶（GT，EC:2.4.1.-）、甲基转移酶（MT，EC:2.1.1.-）和酰基转移酶（AT，EC:2.3.1.-）的作用进行糖基化、甲基化和酰基化修饰（Koes et al., 2005; Jaakola, 2013; Iaria et al., 2016）。

By binding to the promoter regions of structural genes, some transcription factors (TFs) are studied the effect on the synthesis of anthocyanins, such as MYB, bHLH, WD, bZIP, and MADS-box (An et al., 2017; Lu et al., 2018; Jian et al., 2019; Khan et al., 2022). Among them, the regulatory function of MYB, bHLH, and WD40 families are well stablished, which can play regulatory roles alone or by consisting the MBW (MYB-bHLH-WD) protein complex (Ramsay and Glover, 2005; Feng et al., 2020). MYB TFs are generally considered to be the most critical TFs for the synthesis of plant anthocyanins, with a large number of R2R3-MYB TFs being isolated. Most of these TFs play positive regulatory roles in anthocyanin biosynthesis, but few of them play negative regulatory roles (Espley et al., 2009; Butelli et al., 2012; Ni et al., 2020).

通过与结构基因的启动子区域结合，一些转录因子（TFs）被研究以了解其对花青素合成的影响，例如MYB、bHLH、WD、bZIP和MADS-box（An et al., 2017; Lu et al., 2018; Jian et al., 2019; Khan et al., 2022）。其中，MYB、bHLH和WD40家族的调控功能已被广泛确立，它们可以单独发挥作用，也可以通过组成MBW（MYB-bHLH-WD）蛋白复合体来发挥作用（Ramsay and Glover, 2005; Feng et al., 2020）。MYB转录因子通常被认为是植物花青素合成中最关键的转录因子，已有大量R2R3-MYB转录因子被分离出来。这些转录因子中的大多数在花青素生物合成中起正向调控作用，但也有少数发挥负向调控作用（Espley et al., 2009; Butelli et al., 2012; Ni et al., 2020）。

Rosa rugosa is a famous Chinese traditional flower with aromatic, cold resistance, drought resistance, pest resistance, salt and alkali resistance (Chen et al., 2021). But so far, R. rugosa has not been widely used as an ornamental plant because of its monotonous color. Except four white-flowered R. rugosa cultivars, more than 40 other cultivars and all wild germplasms are pink- and purple-flowered, and there is a lack of excellent cultivars with novel flower colors such as red, orange, and blue. Therefore, it is important to elucidate the mechanism of flower color formation in R. rugosa and breed new cultivars with novel flower colors on this basis. At present, there are few reports on the mechanism of flower color formation in R. rugosa, only involving three cultivars with pink and white flowers. Zhang et al. (2015) studied that peonidins were the main composition that determined the petal color of R. rugosa ‘Zi zhi’ (deep pink-flowered cultivar). Sheng et al. (2018) found that there was almost no anthocyanin in the petals of R. rugosa ‘Bai Zizhi’ (white-flowered cultivar), and there were large amount of peonidins in the petals of R. rugosa ‘Fen Zizhi’ (light pink-flowered cultivar) and ‘Zi zhi’. Moreover, 172 R. rugosa germplasms were clustered into seven categories by the petal chroma values and the anthocyanin composition analysis of 21 germplasms from different groups showed that the anthocyanin types were similar but the contents were different (in process). Overall, it’s hard to explore the mechanism of flower color formation in R. rugosa comprehensively by the previous studies, especially for the new colored germplasms.

玫瑰蔷薇（Rosa rugosa）是一种著名的中国传统花卉，具有香气浓郁、耐寒、耐旱、抗虫、耐盐碱等特性（Chen et al., 2021）。然而，由于花色单一，迄今为止玫瑰蔷薇尚未被广泛用作观赏植物。除了四种白色花的玫瑰蔷薇品种外，其他40多个品种以及所有野生种质均为粉色和紫色花，缺乏红色、橙色和蓝色等新颖花色的优良品种。因此，阐明玫瑰蔷薇花色形成的机制，并在此基础上培育具有新颖花色的新品种显得尤为重要。

目前，关于玫瑰蔷薇花色形成机制的研究较少，仅涉及粉色和白色花的三个品种。Zhang等人（2015）研究发现，芍药素是决定玫瑰蔷薇‘紫枝’（深粉色花品种）花瓣颜色的主要成分。Sheng等人（2018）发现，玫瑰蔷薇‘白紫枝’（白色花品种）的花瓣中几乎不含花青素，而‘粉紫枝’（浅粉色花品种）和‘紫枝’的花瓣中含有大量的芍药素。此外，通过对172份玫瑰蔷薇种质的花瓣色度值进行聚类分析，将其分为七个类别，并对不同类群中的21份种质进行花青素组成分析，结果显示花青素类型相似，但含量不同（正在进行中）。总体而言，仅通过以往的研究很难全面探讨玫瑰蔷薇花色形成的机制，尤其是对于新颖花色种质的研究。

In this study, we explored changes in anthocyanin type, anthocyanin content and related gene expression in the petals of different R. rugosa germplasms with representative flower colors. The aims of the study were to: (1) confirm the type and content of anthocyanin in the petals of R. rugosa; (2) identify functional structural genes and TFs involved in anthocyanin biosynthesis; (3) explore the metabolic pathways and the mechanism of color formation in R. rugosa petals. This study can lay an important foundation for further comprehensive and in-depth interpretation of the flower coloration mechanism in R. rugosa.

在本研究中，我们探讨了具有代表性花色的不同玫瑰蔷薇（Rosa rugosa）种质花瓣中花青素类型、花青素含量及相关基因表达的变化。研究目标是：（1）确认玫瑰蔷薇花瓣中的花青素类型和含量；（2）鉴定参与花青素生物合成的功能性结构基因和转录因子（TFs）；（3）探索玫瑰蔷薇花瓣中花青素的代谢途径及颜色形成的机制。本研究可以为进一步全面深入地阐释玫瑰蔷薇花色形成机制奠定重要基础。

材料和方法  Materials and methods  

植物材料  Plant materials  

The plants of R. rugosa and R. hybrida used in this study were planted in Rose Germplasm Nursery, Forestry Experimental Station of Shandong Agricultural University, Tai’an, China (36°10′15″ N, 117°09′25″ E), where they grew under nature conditions. In 2021, three R. rugosa germplasms with representative flower colors (red: 7-23; pink: 8-37; purple-red: 8-16) were selected for ultra-performance liquid chromatography tandem mass spectrometry system (UPLC-MS/MS) and transcriptome analysis. At 7:00 a.m. to 7:30 a.m. on May 2nd, the petals at half-opening stage from three individual plants of each germplasm were sampled and pooled, immediately frozen in liquid nitrogen and then stored at –80°C. Three replicates were set for each germplasm. In 2022, six R. rugosa germplasms (red: 7-23, 9-12; pink: 8-37,6-30; purple-red: 8-16, 4-50) and six R. hybrida cultivars (red: ‘smile’, ‘La Sevillana Plus’, ‘Alcantara’; pink: ‘Carefree Wonder’, ‘Rhapsody in Blue’, ‘Pretty Sunrise’) with representative flower colors were selected for high performance liquid chromatography (HPLC) and gene expression patterns analysis. At 7:00 a.m. to 7:30 a.m. on May 4th, the petals were sampled, pooled, frozen and stored by the same way as in 2021.

本研究中使用的玫瑰蔷薇（R. rugosa）和杂种蔷薇（R. hybrida）植株种植于中国泰安山东农业大学林业实验站的玫瑰种质苗圃（北纬36°10′15″，东经117°09′25″），在自然条件下生长。2021年，选取了三种具有代表性花色的玫瑰蔷薇种质（红色：7-23；粉色：8-37；紫红色：8-16）用于超高效液相色谱串联质谱（UPLC-MS/MS）和转录组分析。在5月2日早上7:00到7:30期间，从每种种质的三株植物上采集半开放阶段的花瓣，混合后立即放入液氮中速冻，并储存在-80°C。每种种质设置三个重复。2022年，选取了六种玫瑰蔷薇种质（红色：7-23、9-12；粉色：8-37、6-30；紫红色：8-16、4-50）和六种具有代表性花色的杂种蔷薇品种（红色：‘smile’、‘La Sevillana Plus’、‘Alcantara’；粉色：‘Carefree Wonder’、‘Rhapsody in Blue’、‘Pretty Sunrise’）用于高效液相色谱（HPLC）和基因表达模式分析。在5月4日早上7:00到7:30期间，按照2021年相同的方法采集、混合、速冻并储存花瓣样本。

UPLC-MS/MS分析  UPLC-MS/MS analysis  

The anthocyanin types and contents in the petals of three R. rugosa germplasms were performed via an UPLC-MS/MS by Metware Biotechnology Co., Ltd. (Wuhan, China). A total of 50 mg grounded petal samples were extracted with 0.5 mL methanol/water/hydrochloric acid (500:500:1, v/v/v). The extract was vortexed for 5 min and ultrasound for 5