整合生理学和解剖学见解以揭示西伯利亚杏的颜色形成机制-动脉网

Integrating physiological and anatomical insights to unveil the mechanism of coloration in Prunus sibirica

Nature 等信源发布 2025-02-28 14:09

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可切换为仅中文

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Abstract

摘要

Pink-flowered

粉红花的

Prunus sibirica

西伯利亚杏

, of the genus

，属

Prunus

李属

, is an exceptional germplasm resource with high ornamental value. Understanding the mechanism behind petal coloration is crucial for cultivating ornamental

，是一种具有高观赏价值的特殊种质资源。了解花瓣着色背后的机制对于培育观赏性品种至关重要。

P. sibirica

西伯利亚松

varieties. This study utilized pink-flowered and white-flowered

品种。本研究利用了粉红花和白花

P. sibirica

西伯利亚松

petals at different stages of flowering to explore the relationship between various physiological indicators, anatomical structures of petals, and flower coloration during flowering. Results indicated that anthocyanins, key pigment indicators in pink-flowered

不同开花阶段的花瓣，以探讨各种生理指标、花瓣解剖结构与开花过程中花色之间的关系。结果表明，花青素是粉红色花中关键的色素指标。

P. sibirica

, directly influenced the

，直接影响了

values (redness). Increased activity of phenylalanine deaminase (4.43–29.69 U/g), chalcone isomerase (9.80–46.67 U/g), and soluble sugar content (29.25–35.28 mg/g) promoted anthocyanin synthesis and accumulation. These substances indirectly affected flower color by influencing anthocyanin content through physiological processes related to petal coloration.

值（红色度）。苯丙氨酸脱氨酶（4.43–29.69 U/g）、查尔酮异构酶（9.80–46.67 U/g）和可溶性糖含量（29.25–35.28 mg/g）的活性增加促进了花青素的合成与积累。这些物质通过与花瓣颜色相关的生理过程影响花青素含量，从而间接影响花色。

Structural changes in epidermal cells of pink and white flower petals during flowering were similar, with differences in pigment content and distribution impacting petal light absorption. Correlation analysis revealed that .

粉色和白色花瓣的表皮细胞在开花过程中的结构变化相似，但色素含量和分布的差异影响了花瓣的光吸收。相关性分析表明。

values were significantly and positively correlated with five factors, one of which was anthocyanin content, and significant negative correlations with soluble protein content and cytosol pH. This study examined the factors influencing petal coloration in pink-flowered

数值与五个因素显著正相关，其中之一为花青素含量，并与可溶性蛋白含量和细胞质pH显著负相关。本研究考察了影响粉红花花瓣颜色的因素。

P. sibirica

西伯利亚松

from both physiological and anatomical perspectives, providing a theoretical foundation for breeding new varieties of ornamental flowering plants.

从生理和解剖学的角度，为培育新的观赏性开花植物品种提供了理论基础。

Introduction

简介

Prunus sibirica

西伯利亚杏

, a wild plant belonging to the genus

，一种属于该属的野生植物

Prunus

李属

of the Rosaceae family, is primarily found in three northern regions of China. Known for its cold and drought resistance, adaptability, and soil conservation properties

蔷薇科植物，主要分布在中国的三个北部地区。以其耐寒、耐旱、适应性强和保土特性而闻名。

, it is a key species for afforestation and ecological restoration projects. Besides its ecological significance,

它是造林和生态恢复项目的关键物种。除了其生态意义之外，

P. sibirica

西伯利亚松

, particularly the pink-flowered variety, is valued for its ornamental qualities. The pink-flowered

，尤其是开粉红花的品种，因其观赏品质而受到重视。粉红花的

P. sibirica

exhibits distinct physiological characteristics, making it more preferable to the common variety. When in full bloom, its petals (5–10), veins, and stamens are a bright pink, enhancing its ornamental appeal

表现出独特的生理特性，使其比普通品种更受欢迎。盛开时，其花瓣（5-10）、叶脉和雄蕊呈明亮的粉红色，增强了其观赏吸引力。

. Current research on

. 当前研究

P. sibirica

blossoms focus on its cold tolerance

花朵专注于其耐寒性

, regulation of its flowering genes

，对其开花基因的调控

, and pistil abortion

，以及雌蕊败育

. However, the underlying mechanisms governing the coloration of the pink-flowered

然而，控制粉红花颜色的潜在机制

P. sibirica

remain unexplored.

保持未被探索。

Flower coloration, regulated by various factors, is a crucial characteristic of ornamental plants

花色是由多种因素调控的观赏植物的重要特性

. Recent research highlights the significant role of pigments in determining flower color

最近的研究强调了色素在决定花色方面的重要作用。

. These pigments, classified as flavonoids, carotenoids, and betaines

这些色素被归类为黄酮类、胡萝卜素类和甜菜碱类。

, influence flower color through their chemical structure, cellular localization, and biochemical synthesis pathways. Among these, anthocyanins are key secondary metabolites of flavonoids that can impart red to blue-violet hues to petals. Modifications to anthocyanin, such as methylation, glycosylation, and acylation, can alter petal color.

，通过其化学结构、细胞定位和生化合成途径影响花的颜色。其中，花青素是黄酮类化合物的关键次生代谢产物，可赋予花瓣红色至蓝紫色的色调。花青素的修饰，如甲基化、糖基化和酰化，可以改变花瓣的颜色。

. Research has demonstrated that studies on flower color in plants such as

研究表明，对植物如花卉颜色的研究已经证明

Rosa rugosa

皱叶玫瑰

，

Dahlia pinnata

大丽花

, and

，以及

Nelumbo nucifera

莲属植物

have identified anthocyanins as the primary influencing factor in the coloration of their petals. Variations in the intrinsic pigment content of petals at different developmental stages can result in differences in extrinsic petal color. One study indicated that in rose flower, increased flavonoids and anthocyanins have caused petal blueing.

已经确定花青素是影响花瓣颜色的主要因素。花瓣在不同发育阶段内在色素含量的变化可能导致外在花瓣颜色的差异。一项研究表明，在玫瑰花中，黄酮类化合物和花青素的增加导致了花瓣的蓝色加深。

. Physicochemical factors during flowering, such as the activities of enzymes like phenylalaninammo nialyase (PAL) and chalcone isomerase (CHI), the presence of precursors like soluble sugars and proteins, and cytosol pH, indirectly influence flower color by affecting pigment synthesis. Moreover, the epidermal structure of petal cells plays a role in light absorption by pigmented substances, impacting petal color.

开花期间的理化因素，如苯丙氨酸解氨酶（PAL）和查尔酮异构酶（CHI）等酶的活性、可溶性糖和蛋白质等前体的存在以及细胞质pH值，通过影响色素合成间接影响花色。此外，花瓣细胞的表皮结构在有色物质的光吸收中起作用，从而影响花瓣颜色。

. The epidermal cells of the petal can be categorized into conical and flat cells based on the ratio of their thickness to width. Typically, conical cells enhance the proportion of incident light entering the cells, resulting in greater light absorption and a deeper color of the petal. In contrast, light that strikes the flat cells perpendicularly is predominantly reflected, leading to a lighter coloration of the petal.

花瓣的表皮细胞根据其厚度与宽度的比例可以分为锥形细胞和平坦细胞。通常，锥形细胞增加入射光进入细胞的比例，从而增强光吸收，使花瓣颜色更深。相反，垂直照射到平坦细胞上的光主要被反射，导致花瓣颜色较浅。

. However, variations exist among different families of plants. In the case of

然而，不同植物科之间存在差异。在

Phalaenopsis

石斛兰

, it was observed that there is no significant correlation between the epidermal structure of the petal cells and the color of the flowers.

观察到花瓣细胞的表皮结构与花的颜色之间没有显著相关性。

To enhance our understanding of the coloring mechanism in pink-flowered

为了加深我们对粉红花着色机制的理解

P. sibirica

petals, this research analyzed petals from pink- and white-flowered

花瓣，本研究分析了来自粉红色和白色花朵的花瓣

P. sibirica

specimens sourced from the National Forest Germplasm Resource Preservation Repository for

来自国家森林种质资源保存库的样本

Prunus

李属

species at Shenyang Agricultural University (Kazuo, Liaoning, China), and aimed to provide a theoretical basis for new variety cultivation and enriching ecological and ornamental plant resources in northeast China and the northern region. The sampling date and sample size were determined based on preliminary investigations on phenological stages, floral organ traits, and tree traits of pink-flowered .

沈阳农业大学（中国辽宁喀左）的物种，旨在为新品种培育提供理论依据，丰富东北及北方地区生态观赏植物资源。根据对粉花表型期、花器官性状和树体性状的初步调查，确定了取样日期和样本量。

P. sibirica

. In particular, the study focused on measuring and analyzing parameters such as floral color, pigmentation, nutrient content, cytosol pH, and cell morphology during the blossoming process.

特别是，该研究集中在测量和分析开花过程中的花色、色素、营养成分、细胞质pH值和细胞形态等参数。

Materials and methods

材料与方法

Plant materials

植物材料

Pink-flowered

粉红色花朵的

P. sibirica

西伯利亚松

(Appraisal of improved varieties of forest tree, S-SV-PS-001–2023, Liaoning Provincial Department of Forest and Grassland) and white-flowered

（森林树木改良品种的评估，S-SV-PS-001–2023，辽宁省林业和草原局）和白花型

P. sibirica

petals (Appraisal of improved varieties of forest tree, S-SC-AS-005–2014, Liaoning Provincial Department of Forest and Grassland) were selected as experimental materials from the National Forest Germplasm Resource Preservation Repository for

花瓣（森林树改良品种评估，S-SC-AS-005–2014，辽宁省林业和草原局）选自国家森林种质资源保存库作为实验材料。

Prunus

李属

species at Shenyang Agricultural University (Kazuo, Liaoning, China) (Fig.

沈阳农业大学（中国辽宁喀左）的物种 (图。

). Each sample, aged 6–7 years with stable genetic traits, was carefully chosen. Three single plants of uniform vigor, robust growth, and free from pests and diseases were selected for each clone. These plants were classified into five stages based on petal color and morphological characteristics: ruddy stage (S1), exposure stage (S2), early flowering stage (S3), blooming stage (S4), and late flowering stage (S5).

）。每个样本都经过精心挑选，年龄为6-7年，具有稳定的遗传特性。每个克隆选择三株生长均匀、健壮、无病虫害的单株植物。这些植物根据花瓣颜色和形态特征分为五个阶段：微红阶段（S1）、露色阶段（S2）、初花阶段（S3）、盛开阶段（S4）和末花阶段（S5）。

Petals from each stage were collected between March–April of 2022 and 2023. Some petals were used for observing flower color parameters and anatomical structures, while the remaining petals were promptly frozen in liquid nitrogen and stored at − 80 °C in an ultra-low-temperature refrigerator upon return to the laboratory..

2022年和2023年3月至4月期间收集了各阶段的花瓣。一部分花瓣用于观察花色参数和解剖结构，其余花瓣在返回实验室后立即用液氮冷冻，并保存在-80°C的超低温冰箱中。

Fig. 1

图1

Petals of

花瓣

P. sibirica

西伯利亚松

at different stages of blooming. The petals were categorized as: ruddy stage (S1), exposure stage (S2), early flowering stage (S3), blooming stage (S4), and late flowering stage (S5). The top row represents the pink-flowered

在不同的开花阶段。花瓣被分为：初开期（S1）、露色期（S2）、初花期（S3）、盛开期（S4）和末花期（S5）。顶行代表粉红色花朵。

P. sibirica

西伯利亚松

(P1–P5) and the bottom row represents the white-flowered

(P1–P5) 底行代表白色花的

P. sibirica

西伯利亚松

(W1–W5). Bar = 5 mm.

(W1–W5)。标尺=5毫米。

Full size image

全尺寸图像

Observation indicators and methods

观察指标与方法

Determination of color parameters

颜色参数的确定

For the quantitative analysis of the pink- and white-flowered

对于粉红色和白色花朵的定量分析

P. sibirica

西伯利亚松

petal colors the CIELab color system introduced by the International Commission on Illumination in 1970 was used as a reference

花瓣颜色以国际照明委员会1970年引入的CIELab颜色系统为参考

. In this study, flower color

。在本研究中，花色

values (brightness),

值（亮度），

values (redness), and

值（红色度），以及

values (yellowness) were determined by using a colorimeter (CR-10PLUS, Konica minolta/ Japan) aimed at the middle of the petals.

使用色差仪（CR-10PLUS，柯尼卡美能达/日本）对花瓣中部进行测量以确定其黄色值。

Determination of pigment contents

色素含量的测定

The flavonoid content of petals was determined using the plant flavonoid test kit from Nanjing Jiancheng Bioengineering Institute. The petals were washed with distilled water, the surface water was wiped dry, ground to powder in liquid nitrogen, weighed to 0.05 g, 2 mL of extraction solution was added, extracted by shaking at 60 °C for 2 h, 10,000 g, centrifuged at room temperature (around 15 °C) for 10 min, the supernatant was collected and the absorbance measured at 502 nm.

使用南京建成生物工程研究所的植物黄酮试剂盒测定花瓣的黄酮含量。用蒸馏水冲洗花瓣，擦干表面水分，于液氮中研磨成粉末，称取0.05克，加入2毫升提取液，在60°C下振荡提取2小时，以10,000g在室温（约15°C）下离心10分钟，收集上清液，并在502纳米处测量吸光度。

Three biological replicates were performed for each developmental stage. The calculation formula was as follows:.

每个发育阶段进行了三个生物学重复。计算公式如下：。

$${\text{Flavonoid content }}\left( {{\text{mg}}/{\text{g}}} \right) = \frac{{\left( {\Delta {\text{A}}_{{{5}0{2}}} - \, 0.0{141}} \right)}}{{{9}.0{771} \times {\text{V}}/{\text{M}}}}$$

黄酮含量（mg/g）= （ΔA502 - 0.0141）/（9.0771 × V/M）

△

502

, the difference between measurement tube and control tube under 502 nm; V, total volume of the extract (mL); M, sample quality (g).

`, 测量管与控制管在502 nm下的差异；V，提取物的总体积（mL）；M，样品质量（g）。`

The anthocyanin, carotenoid, and chlorophyll contents of the petals were determined following the method described by Piccaglia and Scrob

花瓣中的花青素、类胡萝卜素和叶绿素含量是按照Piccaglia和Scrob所述的方法测定的。

，

. Petals were weighed, cut into 0.3 g portions, and placed into two 25 mL stoppered glass test tubes. A mixture of 20 mL of 0.1 mol/L HCl extract and 15 mL of 95% alcohol solution was added to the test tubes, which were then inverted several times to mix. The test tubes were then placed in a thermostat at 32 °C for 16 h and 37 °C for 24 h, respectively.

花瓣被称重后切成0.3克的部分，并放入两个25毫升的带塞玻璃试管中。向试管中加入20毫升0.1摩尔/升的盐酸提取液和15毫升95%的酒精溶液，然后多次倒置以混合。随后，将试管分别置于32°C的恒温箱中16小时和37°C的恒温箱中24小时。

Once all the petals whitened, the test tubes were removed, cooled to room temperature (around 15 °C), and the absorbance was measured at 530 nm using 0.1 mol/L HCl solution as a control. Three biological replicates were performed for each developmental stage. The calculation formula was as follows:.

一旦所有花瓣变白，将试管取出，冷却至室温（约15°C），并使用0.1 mol/L HCl溶液作为对照，在530 nm处测量吸光度。每个发育阶段进行了三次生物学重复。计算公式如下：

$${\text{Relative content of anthocyanin }}\left( {{\text{mg}}/{\text{g}}} \right) = {\text{A}}_{{{53}0}} /0.{1} \times {\text{M}}$$

花色苷相对含量（mg/g）= A_530 / 0.1 × M

530

, the absorbance was measured at 530 nm; M, sample quality (g).

，吸光度是在530纳米处测量的；M，样品质量（克）。

The OD values were determined at 645 nm, 649 nm, and 470 nm using 95% alcohol solution as a control to calculate carotenoid and chlorophyll contents. Three biological replicates were performed for each developmental stage. The calculation formula was as follows:

使用95%酒精溶液作为空白对照，在645 nm、649 nm和470 nm处测定OD值，以计算类胡萝卜素和叶绿素含量。每个发育阶段进行了三个生物学重复。计算公式如下：

Chlorophyll a content (mg/g) = (13.95 A

叶绿素a含量（毫克/克）=（13.95 A

649

− 6.88 A

645

) × V × N/ M.

) × V × N / M。

Chlorophyll b content (mg/g) = (24.96 A

叶绿素 b 含量 (mg/g) = (24.96 A

645

− 7.32 A

649

) × V × N/ M.

) × V × N / M。

Carotenoid content (mg/g) =

类胡萝卜素含量（毫克/克）=

$\frac{1000\text{ A}470 - 2.05\text{ Ca }- 114.8\text{ Cb}}{245}$

$\frac{1000\text{ A}470 - 2.05\text{ 钙 }- 114.8\text{ 钴}}{245}$

× V × N/ M.

× V × N/ M。

A, the absorbance were measured at 645 nm, 649 nm, and 470 nm; V, volume of extract (L); N, dilution factor; M, sample quality (g).

A，吸光度是在645纳米、649纳米和470纳米处测量的；V，提取物的体积（升）；N，稀释因子；M，样品质量（克）。

Determination of PAL and CHI activities

PAL和CHI活性的测定

PAL and CHI activities were assayed using a modified version of the method described by La et al.

使用La等人描述的方法的改良版本对PAL和CHI活性进行了测定。

. PAL activity was determined as follows: for the extraction, 0.5 g of petals were ground in a mortar with 1 mL of 7 mmol/L β-mercaptoethanol, 1 mL of 0.1 mol/L boric acid retardant (containing an appropriate amount of polyvinylpyrrolidone), and 3 mL of the extract solution in an ice bath until homogenized.

如下测定PAL活性：提取时，将0.5克花瓣在研钵中与1毫升7毫摩尔/升的β-巯基乙醇、1毫升0.1摩尔/升的硼酸缓释剂（含适量聚乙烯吡咯烷酮）和3毫升提取液一起在冰浴中研磨至均质化。

The mixture was then centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatant was collected and stored at a low temperature. Subsequently, 5 mL test tubes were prepared by adding 1 mL of 0.02 mol/L phenylalanine, 2 mL of 0.1 mol/L boric acid buffer at pH 8.8, and 0.1 mL of the crude enzyme extract.

然后在4°C下以12,000 rpm离心该混合物15分钟，收集上清液并低温保存。随后，准备5 mL试管，加入1 mL 0.02 mol/L苯丙氨酸、2 mL 0.1 mol/L pH 8.8的硼酸缓冲液和0.1 mL粗酶提取物。

The contents of each test tube were mixed, and the absorbance at 290 nm was measured using a UV spectrophotometer (U-5100, Hitachi/ Japan). After incubating the tubes in a water bath at 30 °C for 30 min, the absorbance at 290 nm was measured again. The enzyme activity was calculated based on the change in OD value by 0.01 per 30 min, defined as one unit of enzyme activity.

将每个试管中的内容混合后，使用紫外分光光度计（U-5100，日立/日本）测量290 nm处的吸光度。在30°C水浴中孵育试管30分钟后，再次测量290 nm处的吸光度。酶活性根据OD值每30分钟变化0.01来计算，定义为一个酶活性单位。

Three biological replicates were performed for each developmental stage..

每个发育阶段进行了三个生物学重复。

CHI activity was determined as follows: 0.5 g of petals were placed in a mortar and ground with 5 mL of pH 7.0 buffer in an ice bath until homogenized. After which, it was centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatant was collected and stored at a low temperature. In a 5 mL test tube, 0.1 mL of crude enzyme extract was combined with 0.05 mL of chalcone, and 2.5 mL of bovine serum albumin.

CHI活性测定如下：将0.5克花瓣放入研钵中，加入5毫升pH 7.0的缓冲液，在冰浴中研磨至匀浆。然后在4°C下以12,000转/分钟离心15分钟，收集上清液并低温保存。在5毫升试管中，将0.1毫升粗酶提取物与0.05毫升查尔酮和2.5毫升牛血清白蛋白混合。

The absorbance was measured at 381 nm for 30 min in a water bath at 35 °C. The enzyme activity was calculated based on an increase in OD value of 0.1 per minute, defined as one unit of enzyme activity unit. Three biological replicates were performed for each developmental stage..

在35°C水浴中，于381 nm波长下测量吸光度30分钟。酶活性根据每分钟OD值增加0.1来计算，定义为一个酶活性单位。每个发育阶段进行了三个生物学重复。

Determination of nutrient contents

营养成分含量的测定

The soluble sugar content was analyzed following the method described by Liu et al.

可溶性糖含量按照Liu等人描述的方法进行分析。

. Petals weighing 0.5 g were ground and then diluted with 10 mL of distilled water. The mixture was extracted in boiling water for 30 min and then centrifuged at 12,000 rpm for 20 min. The resulting supernatant was collected and diluted to a final volume of 25 mL. Subsequently, 1.5 mL of distilled water, 0.5 mL of ethyl anthrone acetate reagent, and 5 mL of concentrated sulfuric acid were added in sequence.

称取0.5克花瓣，研磨后用10毫升蒸馏水稀释。将混合物在沸水中提取30分钟，然后以12,000转/分钟离心20分钟。收集上清液并稀释至最终体积25毫升。随后依次加入1.5毫升蒸馏水、0.5毫升乙基蒽酮乙酸酯试剂和5毫升浓硫酸。

The solution was then placed in a boiling water bath for 1 min after thorough shaking. After cooling to room temperature, the absorbance was measured at 630 nm. Three biological replicates were performed for each developmental stage..

将溶液剧烈摇匀后置于沸水浴中加热1分钟。冷却至室温后，在630纳米处测量吸光度。每个发育阶段进行了三个生物学重复。

The soluble protein content was assessed using the method described by Kučerová et al.

可溶性蛋白含量通过Kučerová等人描述的方法进行评估。

. Petals weighing 0.5 g were ground and then diluted with 10 mL of distilled water. The supernatant was obtained through centrifugation at 10,000 rpm for 15 min. Subsequently, 1 mL of the supernatant was pipetted, mixed with 5 mL of G-250 Coomassie Brilliant Blue solution, allowed to stand for 2 min, and the absorbance at 595 nm was measured.

花瓣称重0.5克，研磨后用10毫升蒸馏水稀释。通过以10,000转/分钟离心15分钟获得上清液。随后，吸取1毫升上清液，与5毫升G-250考马斯亮蓝溶液混合，静置2分钟，并测量其在595纳米处的吸光度。

Three biological replicates were performed for each developmental stage..

每个发育阶段进行了三个生物学重复。

Determination of cytosol pH

细胞质pH的测定

The method described by Zhang et al.

张等人描述的方法。

, with minor adjustments, was utilized to determine the pH of the petal cytosol. Specifically, 0.5 g of petals was ground into a homogenate using a mortar and pestle, with 2 mL of distilled water added during the process. The pH of the resulting homogenate was then measured using a PHS-3E pH meter (INESA/ China), which served as a proxy for the pH of the petal cytosol.

，经过微调后，被用于测定花瓣细胞溶胶的pH值。具体而言，将0.5克花瓣用研钵和杵研磨成匀浆，并在过程中加入2毫升蒸馏水。随后使用PHS-3E型pH计（INESA/中国）测量所得匀浆的pH值，该值作为花瓣细胞溶胶pH值的替代指标。

Three biological replicates were performed for each developmental stage..

每个发育阶段进行了三个生物学重复。

Observation of petal morphology and structure

花瓣形态与结构的观察

Fresh petals were taken after the flowers had fully opened and temporary water mounts were prepared by the freehand sectioning method to observe the upper epidermis of the petals. The mounts were photographed and observed under a light microscope (Primostar 3, ZEISS/ Germany) at 10× magnification.

鲜花完全开放后采集新鲜花瓣，并通过徒手切片法制备临时水装片，以观察花瓣的上表皮。装片在光学显微镜（Primostar 3，ZEISS/德国）下以10倍放大倍率进行拍照和观察。

Paraffin sections were prepared following conventional procedures

石蜡切片按照常规程序制备。

. The middle portion of the petals was cut and fixed in formaldehyde alcohol acetic acid (FAA) fixative, with section thickness ranging from 6–8 μm. These sections were then stained with Senna solid green and sealed with neutral gum. Observation of the petals was conducted using a light microscope (Olympus-BX51, Hitachi/ Japan), and data analysis was performed using Image J software.

花瓣的中间部分被切下并固定在甲醛酒精乙酸（FAA）固定剂中，切片厚度范围为6-8微米。这些切片随后用藏红固绿染色，并用中性树胶封片。使用光学显微镜（Olympus-BX51，日立/日本）观察花瓣，并使用Image J软件进行数据分析。

Parameters measured included upper epidermal cell thickness, width, thickness-to-width ratio, and cell pinch angle. Three transverse structural sections were obtained from each petal, with 10 fields of view analyzed per section..

测量的参数包括上表皮细胞厚度、宽度、厚宽比和细胞夹角。每个花瓣获取三个横向结构切片，每个切片分析10个视野。

Following the methodology outlined by Norikoshi et al.

按照Norikoshi等人提出的方法论

, fresh petals were carefully cleaned with distilled water to remove any impurities. Subsequently, 5 mm × 5 mm samples were extracted and immersed in glutaraldehyde fixative for preservation. The petals then underwent a process of gradual dehydration and were dried using a vacuum freeze dryer. These samples were affixed to aluminum trays and coated with gold using an MC1000 ion sputtering instrument before being examined with a Hitachi Regulus 8100 type cold-field emission scanning electron microscope.

，用蒸馏水仔细清洗新鲜花瓣以去除任何杂质。随后，提取出5毫米×5毫米的样本并浸入戊二醛固定剂中进行保存。花瓣接着经历了逐步脱水的过程，并使用真空冷冻干燥机进行干燥。这些样本被固定在铝制托盘上，并使用MC1000离子溅射仪镀金，然后用日立Regulus 8100型冷场发射扫描电子显微镜进行观察。

Epidermal cells on the petals were quantified and analyzed at 500× magnification, with cell morphology observed and captured at 1000× magnification. Data analysis was conducted using Image J software, with measurements repeated nine times for accuracy..

花瓣上的表皮细胞在500倍放大下进行量化和分析，并在1000倍放大下观察和捕捉细胞形态。数据分析使用Image J软件进行，为确保准确性，测量重复进行了九次。

Data analysis

数据分析

Statistical analysis on all experimental data was completed using SPSS Statistics V 22.0. Results are presented as the mean ± standard deviation (SD), using a significance threshold of

使用SPSS Statistics V 22.0完成所有实验数据的统计分析。结果以平均值±标准差（SD）表示，显著性阈值为

< 0.05. The correlation among color parameters, pigment contents, physiological responses, and petal morphology and structure was calculated by Pearson’s test at two significance levels,

< 0.05。颜色参数、色素含量、生理反应以及花瓣形态和结构之间的相关性通过皮尔逊检验在两个显著性水平上计算。

< 0.05 and

< 0.05 且

< 0.01. On the other hand, a stepwise regression analysis at

小于0.01。另一方面，逐步回归分析在

< 0.05 and

< 0.05 且

< 0.01 was done to determine the key factors affecting the petal coloration of pink-flowered

< 0.01是为了确定影响粉红花花瓣颜色的关键因素

P. sibirica.

西伯利亚松。

Plotting bar charts and heatmaps for correlation analysis were performed using OriginPro 2022 software.

使用OriginPro 2022软件绘制条形图和热图进行相关性分析。

Results

结果

Comparison of color parameters

颜色参数的比较

Pink-flowered

粉红色的花

P. sibirica

petals are dark pink at S1, gradually changing to light pink as the flowers open up; whereas, white-flowered

花瓣在S1阶段为深粉色，随着花朵开放逐渐变为浅粉色；而白色花的

P. sibirica

petals are white from S1 to S5. As shown in Fig.

花瓣从 S1 到 S5 均为白色。如图所示。

, the

，这个

values of both pink and white petals showed a trend of first increasing and then decreasing. The

粉色和白色花瓣的值都呈现出先增加后减少的趋势。

values of pink petals ranged from 66.73–76.90, while the

粉色花瓣的值范围为66.73–76.90，而

values of white petals ranged from 71.27–83.87. Notably, the

白色花瓣的值范围为71.27至83.87。值得注意的是，

values of pink flower petals were significantly lower than those of white flower petals across all five stages, being 93.64%, 89.28%, 89.51%, 93.40% and 95.64% of the

粉色花瓣的值在所有五个阶段均显著低于白色花瓣，分别为其93.64%、89.28%、89.51%、93.40%和95.64%。

values of white flower petals, respectively. The

白色花瓣的值，分别。

and

和

values of the petals of the two samples showed an opposite trend, with the

两个样品的花瓣值呈现相反的趋势，

values of pink petals ranging from 7.17–16.87, and for white petals from − 1.30 to − 0.50. The

粉色花瓣的值范围为 7.17 至 16.87，白色花瓣的值范围为 -1.30 至 -0.50。

values of pink flower petals were significantly higher than those of white flower petals across all five stages, being 33.73, 11.26, 12.36, 8.96, and 12.48 times higher, respectively. The

粉色花瓣的值在所有五个阶段都显著高于白色花瓣，分别是白色花瓣的33.73倍、11.26倍、12.36倍、8.96倍和12.48倍。

values of both pink and white flowers showed a gradually increasing trend, with the

粉红花和白花的数值均呈现逐渐增加的趋势，

values of pink flowers ranging from 2.33 to 8.07 and those of white flowers from 8.67 to 12.90. In all stages of flowering, the

粉红花的值范围为2.33到8.07，白花的值范围为8.67到12.90。在开花的所有阶段，

values of pink flower petals were significantly lower than those of white flower petals.

粉色花瓣的值显著低于白色花瓣的值。

Fig. 2

图2

Comparison of color parameters in pink- and white-flowered

粉色和白色花的颜色参数比较

P. sibirica

. The comparison of the lightness (

. 对亮度的比较 (

values) of the two samples (

两个样本的值 (

), the comparison of the redness (

)，红色度的比较（

values) of the two samples (

值）的两个样本（

), and the comparison of the yellowness (

），以及黄色度的比较（

values) of the two samples (

两个样本的值 (

). Different lowercase letters indicate significant differences at the

`).` 不同的小写字母表示在

< 0.05 level, the same letter means no significant difference. Error bars represent standard deviation.

< 0.05 水平，相同字母表示无显著差异。误差条代表标准偏差。

Full size image

全尺寸图像

Comparison of pigment contents

色素含量的比较

As shown in Table

如表所示

, the anthocyanin and total chlorophyll contents of both pink and white flower petals showed a trend of initially increasing and then decreasing, with the maximum values of anthocyanin and total chlorophyll of pink flower petals observed at S2, respectively. The chlorophyll a and chlorophyll b contents of pink flower petals reached a maximum at S2, whereas the chlorophyll a and chlorophyll b contents of white flower petals reached a maximum at S3 and S4, respectively.

，粉红和白色花瓣的花青素和总叶绿素含量均呈现先升高后降低的趋势，其中粉红色花瓣的花青素和总叶绿素含量在S2时期达到最大值，而粉红色花瓣的叶绿素a和叶绿素b含量在S2时期达到最大值，白色花瓣的叶绿素a和叶绿素b含量分别在S3和S4时期达到最大值。

The anthocyanin content of pink flower petals increased by 25.84% at S2 compared to the beginning and decreased to 2.22 mg/g at S5, which was 62.18% lower compared to S1. Across all five stages, the anthocyanin content of pink flower petals was significantly higher than that of white flower petals. In the pre-flowering stage, the total chlorophyll content of pink flower petals was significantly higher than that of white flower petals.

与初期相比，粉花花瓣在S2阶段的花青素含量增加了25.84%，到S5阶段下降至2.22 mg/g，比S1阶段降低了62.18%。在全部五个阶段中，粉花花瓣的花青素含量显著高于白花花瓣。在开花前阶段，粉花花瓣的总叶绿素含量显著高于白花花瓣。

However, no significant difference was observed between the total chlorophyll content of pink and white flower petals at S4 and S5..

然而，在S4和S5阶段，粉色和白色花瓣的总叶绿素含量之间没有观察到显著差异。

Table 1 Comparison of petal pigments in different

表1 不同花瓣色素的比较

Prunus sibirica

西伯利亚杏

samples.

样本。

Full size table

全尺寸表格

The flavonoid and carotenoid content of both pink and white flowers showed a decreasing trend, the flavonoid and carotenoid contents of pink petals decreased by 64.23% and 77.33%, respectively, at S5 compared to S1. Similarly, the flavonoid and carotenoid contents of white petals decreased by 80.23% and 70.51%, respectively, at S5 relative to S1.

粉色和白色花的类黄酮和类胡萝卜素含量均呈下降趋势，与S1相比，粉色花瓣在S5阶段的类黄酮和类胡萝卜素含量分别下降了64.23%和77.33%；同样，白色花瓣在S5阶段的类黄酮和类胡萝卜素含量较S1分别下降了80.23%和70.51%。

Notably, the flavonoid content and carotenoid content of pink petals were significantly higher than those of white petals at all five stages..

值得注意的是，在五个阶段中，粉红色花瓣的类黄酮含量和类胡萝卜素含量均显著高于白色花瓣。

Comparison of PAL and CHI activities

PAL和CHI活性的比较

As shown in Table

如表所示

, the PAL activities of both pink and white petals displayed a pattern characterized by an initial increase followed by a subsequent decrease. The highest activities were recorded at S2, reaching 29.69 U/g for pink petals and 8.39 U/g for white petals. The PAL activity decreased by 85.08% for pink petals and 68.65% for white petals during the S5, compared to S2.

，粉色和白色花瓣的PAL活性均呈现出先升高后降低的趋势。在S2时期活性最高，其中粉色花瓣为29.69 U/g，白色花瓣为8.39 U/g；到S5时期，粉色花瓣和白色花瓣分别比S2时期下降了85.08%和68.65%。

The PAL activity of pink flower petals was significantly higher than that of white flower petals, being 7.57, 3.54, 3.07, and 2.19 times higher at S1, S2, S3, and S4, respectively. The CHI activities of both pink and white flower petals showed a gradually decreasing trend. The CHI activities decreased by 79.00% and 68.15% at S5 of pink and white petals, respectively, compared to S1.

粉色花瓣的PAL活性显著高于白色花瓣，在S1、S2、S3和S4时期分别是白色花瓣的7.57倍、3.54倍、3.07倍和2.19倍。粉色和白色花瓣的CHI活性均呈逐渐下降趋势，与S1相比，粉色和白色花瓣在S5时期的CHI活性分别下降了79.00%和68.15%。

The CHI activity of pink flower petals was significantly higher than that of white flower petals across all five stages, being 2.10, 2.02, 2.20, 1.70, and 1.39 times higher, respectively..

粉色花瓣的CHI活性在五个阶段均显著高于白色花瓣，分别是白色花瓣的2.10、2.02、2.20、1.70和1.39倍。

Table 2 Comparison of petal PAL and CHI activities in different

表2 不同花瓣中PAL和CHI活性的比较

Prunus sibirica

西伯利亚杏

samples.

样本。

Full size table

全尺寸表格

Comparison of nutrient contents and cytosol pH

营养成分和细胞质pH值的比较

As shown in Table

如表所示

, the soluble sugar content of both pink and white petals showed a trend of initially increasing followed by then decreasing. The highest content of 35.28 mg/g and 32.54 mg/g was observed at S2 and S4 for pink and white petals, respectively. The soluble sugar content of pink petals increased by 7.07% at S2 but decreased by 11.23% at S5 when compared to the S1.

粉色和白色花瓣的可溶性糖含量均呈现先升高后降低的趋势，其中粉色花瓣在S2时期可溶性糖含量最高，为35.28 mg/g，白色花瓣在S4时期可溶性糖含量最高，为32.54 mg/g；与S1相比，粉色花瓣在S2时期的可溶性糖含量增加了7.07%，而在S5时期则下降了11.23%。

In contrast, the soluble sugar content of white petals decreased by 4.56% at S5 relative to the S1. The soluble sugar content of pink flower petals was significantly higher than that of white flower petals, being 1.06, 1.10 and 1.06 times higher at S1, S2 and S4, respectively. The trend of the soluble protein content of pink and white petals was different, with pink petals exhibiting a pattern of first decreasing and then increasing, while white petals showed the opposite trend of increasing and then decreasing.

相比之下，白花瓣在S5阶段的可溶性糖含量较S1阶段下降了4.56%。粉色花瓣的可溶性糖含量显著高于白色花瓣，在S1、S2和S4阶段分别高出1.06倍、1.10倍和1.06倍。粉色和白色花瓣的可溶性蛋白含量趋势不同，粉色花瓣表现为先下降后上升的趋势，而白色花瓣则呈现相反的先上升后下降的趋势。

The soluble protein content of the petals of pink and white flowers reached maximum values of 9.32 mg/g and 29.04 mg/g at S5 and S4, respectively. At S5, the soluble protein content was elevated by 54.82% and 105.60% in pink and white petals, respectively, compared to the S1. The soluble protein content within the petals of pink flowers was significantly lower than that of white flowers across all five stages, being 48.86%, 37.54%, 17.82%, 23.42%, and 36.79% of that of white flower petals, respectively.

粉色和白色花瓣的可溶性蛋白含量分别在S5和S4阶段达到最大值，分别为9.32 mg/g和29.04 mg/g。在S5阶段，与S1阶段相比，粉色和白色花瓣中的可溶性蛋白含量分别提高了54.82%和105.60%。粉色花瓣中的可溶性蛋白含量在五个阶段中均显著低于白色花瓣，分别为白色花瓣的48.86%、37.54%、17.82%、23.42%和36.79%。

The cytosol pH of the petals of both pink and white flowers showed a gradual increase. Furthermore, across all five stages, the cytosol pH of pink flower petals was significantly lower than that of white flower petals, being 97.35%, 93.83%, 93.90%, 94.36% and 88.47% of that of white flower petals, respectively..

粉色和白色花瓣的细胞溶胶 pH 值均逐渐增加。此外，在五个阶段中，粉色花花瓣的细胞溶胶 pH 值显著低于白色花花瓣，分别为白色花花瓣的 97.35%、93.83%、93.90%、94.36% 和 88.47%。

Table 3 Comparison of petal soluble sugars, soluble protein content, and cytosol pH in different

表3 不同花瓣可溶性糖、可溶性蛋白含量及细胞质pH的比较

Prunus sibirica

西伯利亚杏

samples.

样本。

Full size table

全尺寸表格

Comparison of the internal microstructure of petals

花瓣内部微观结构的比较

Observations on the upper epidermis of fully open petals by freehand sectioning revealed differences in epidermal cell morphology and pigment distribution among different colored petals (Fig.

通过徒手切片观察完全开放花瓣的上表皮，发现不同颜色花瓣之间表皮细胞形态和色素分布存在差异（图。

). The epidermal cell structure on the petals of pink flowers appeared irregularly shaped, accompanied by conspicuous pigment distribution (Fig.

）。粉红色花瓣上表皮细胞结构呈不规则形状，伴有明显的色素分布（图。

a). It was hypothesized the majority of these pigments might be anthocyanins. Conversely, the epidermal cell structure on the petals of white flowers exhibited nearly rectangular shapes, devoid of obvious pigment accumulation (Fig.

a). 据推测，这些色素中的大多数可能是花青素。相反，白花花瓣上的表皮细胞结构呈现出近乎矩形的形状，没有明显的色素积累（图。

b).

Fig. 3

图3

Morphology and pigment distribution of the upper epidermis of the petals of pink-flowered

粉红花花瓣上表皮的形态学和色素分布

P. sibirica

(

), morphology and pigment distribution of the upper epidermis of the petals of white-flowered

），白色花花瓣上表皮的形态学和色素分布

P. sibirica

(

）。

Full size image

全尺寸图像

The use of paraffin sectioning experiments allowed for better observation of changes in petal sections (Fig.

石蜡切片实验的使用能够更好地观察花瓣切片的变化（图。

). The upper epidermal cell thickness and width of both pink and white flowers petals exhibited an initial increase followed by a subsequent decrease (Fig.

）。粉色和白色花花瓣的上表皮细胞厚度和宽度均呈现先增加后减少的趋势（图。

a and b). Additionally, the thickness-to-width ratio of epidermal cells on petals is representative of the degree of conicity of the cells. During the flowering process, the thickness-to-width ratios of the epidermal cells on the petals of both pink and white flowers displayed a trend of decreasing initially, followed by an increase (Fig. .

a 和 b）。此外，花瓣上表皮细胞的厚宽比代表了细胞的锥度程度。在开花过程中，粉红花和白花花瓣上表皮细胞的厚宽比呈现出先降低后增加的趋势（图。

c). The highest values of the thickness-to-width ratios of the epidermal cells on the petals of both pink and white flowers were found at S1, while the lowest values were found at S4. At S2, the thickness-to-width ratios of the epidermal cells on the petals of pink flowers were significantly higher than those on the petals of white flowers, being 1.20 times as high as those on white flowers.

c). 粉色和白色花瓣上表皮细胞的厚度与宽度比值在S1时最高，而在S4时最低。在S2时，粉色花瓣上表皮细胞的厚度与宽度比值显著高于白色花瓣，是白色花瓣的1.20倍。

Conversely, at S3, the thickness-to-width ratios of the epidermal cells on the petals of pink flowers were significantly lower than those on the petals of white flowers, accounting for only 79.26% of those on white flowers..

相反，在S3阶段，粉红色花瓣上表皮细胞的厚宽比显著低于白色花瓣上的表皮细胞，仅为白色花瓣的79.26%。

Fig. 4

图4

Capital letters represent the cross-sectioned structure of the pink-flowered

大写字母代表粉红花的横截面结构

P. sibirica

西伯利亚松

petals, lowercase letters represent the cross-sectioned structure of the white-flowered

花瓣，小写字母代表白花的横截面结构

P. sibirica

西伯利亚松

petals; (

花瓣；（

) ruddy stage; (

) 赤色阶段；(

exposure stage; (

曝光阶段；（

) early flowering stage; (

）初花期；（

) blooming stage; (

）开花阶段；（

) late flowering stage; EC: epidermal cell; PC: parenchyma cell.

）晚花期；EC：表皮细胞；PC：薄壁细胞。

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The epidermal cell angle on the petals of both pink and white flowers followed a similar pattern, initially increasing and then decreasing (Fig.

花瓣上粉红和白色花朵的表皮细胞角度遵循相似的模式，先增加后减少（图。

d). In particular, the epidermal cell angle on the petals of both pink and white flowers was the largest at S4 and the smallest at S1. At S1, the angle of the epidermal cells on the petals of pink flowers was significantly lower than that on the petals of white flowers, accounting for only 54.84% of the petals of white flowers.

d). 特别是粉色花和白色花的花瓣上，表皮细胞角度在S4阶段最大，而在S1阶段最小。在S1阶段，粉色花花瓣上的表皮细胞角度显著低于白色花的花瓣，仅为白色花花瓣的54.84%。

However, at S3 and S5, the angle of the epidermal cells on the petals of pink flowers was significantly higher than that on the petals of white flowers, being 1.08 and 1.14 times as high as that on the petals of white flowers..

然而，在S3和S5阶段，粉红色花瓣上表皮细胞的角度显著高于白色花瓣，分别是白色花瓣的1.08倍和1.14倍。

Scanning electron microscope images showed changes in the upper epidermal cells of the petals of

扫描电子显微镜图像显示花瓣上表皮细胞发生了变化

P. sibirica

at five stages (Fig.

在五个阶段（图。

). Noticeable trends in the epidermal cell area on the petals of pink flowers showed a trend of increasing and then decreasing, whereas the number of cells per unit showed a continuous downward trend. Conversely, the epidermal cell area on the petals of white flowers showed a gradual upward trend, and the number of cells per unit showed a trend of initially decreasing followed by increasing (Fig. .

）。粉花花瓣上表皮细胞面积呈现出先增加后减少的趋势，而单位面积上的细胞数则持续下降；相反，白花花瓣上表皮细胞面积呈现逐渐上升的趋势，单位面积上的细胞数则呈现出先减少后增加的趋势（图。

e and f). At S1, the area of epidermal cells on pink flower petals was significantly lower than that on white flower petals, accounting for 83.62% of that on white flower petals. The number of epidermal cells on the petals of pink flowers was significantly higher than that on the petals of white flowers at S1, S2 and S4, being 1.54 1.61 and 1.13 times higher, respectively.

在S1阶段，粉红色花瓣上表皮细胞的面积显著低于白色花瓣，仅占白色花瓣的83.62%。粉红色花瓣表皮细胞的数量在S1、S2和S4阶段显著高于白色花瓣，分别是白色花瓣的1.54倍、1.61倍和1.13倍。

Additionally, at S5, the number of epidermal cells on the petals of pink flowers was significantly lower than that on the petals of white flowers, being 79.73% lower than that of the petals of white flowers..

此外，在S5阶段，粉色花花瓣上的表皮细胞数量显著低于白色花花瓣，比白色花花瓣的表皮细胞数量低79.73%。

Fig. 5

图5

Morphology of the epidermal cells on the petals of

花瓣上表皮细胞的形态学

P. sibirica

西伯利亚松

at different stages of blooming. (

在不同的开花阶段。 (

–

) are pink-flowered

）是粉红花的

P. sibirica

西伯利亚松

petals, (

花瓣，（

–

) are white-flowered

）是开白花的

P. sibirica

西伯利亚松

petals. All images are petal morphology at 500× magnification.

花瓣。所有图像均为500倍放大下的花瓣形态。

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Fig. 6

图6

Comparison of multiple comparisons of quantitative parameters of epidermal cells in pink- and white-flowered

粉花和白花表皮细胞定量参数的多重比较对比

P. sibirica

. The comparison of the upper epidermal cell thickness of the two samples (

. 对两个样品上表皮细胞厚度的比较 (

), the comparison of the upper epidermal cell width of the two samples (

），两个样品上表皮细胞宽度的比较（

), the comparison of the upper epidermal cell thickness and width ratio of the two samples (

)，两个样本的上表皮细胞厚度与宽度比的比较（

), the comparison of the upper epidermal cell angle of the two samples (

)，两个样品上表皮细胞角的比较（

), the comparison of the upper epidermal cell area of the two samples (

)，两个样品上表皮细胞面积的比较（

), and the comparison of the upper epidermal cell number of the two samples (

），以及两个样品上表皮细胞数量的比较（

）。

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Comprehensive analysis among indicators

指标间的综合分析

Pearson’s correlation matrix showed the correlation between the different variables (Fig.

皮尔逊相关矩阵显示了不同变量之间的相关性（图。

), pink flower petal

），粉色花瓣

values were significantly negatively correlated with

值与...显著负相关

values (

值（

< 0.05). However, there was no significant correlation between

<0.05）。然而，两者之间没有显著的相关性

values and

价值观和

values. However,

价值观。然而，

values were significantly negatively correlated with

值与...显著负相关

values. Furthermore, the

价值观。此外，

values showed highly significant positive correlations

数值显示出高度显著的正相关性

< 0.01) with upper epidermal cell width, cell pinch angle, and cell area, and significant positive correlations with total chlorophyll and chlorophyll b content. The

小于0.01）与上表皮细胞宽度、细胞夹角和细胞面积呈显著负相关，而与总叶绿素和叶绿素b含量呈显著正相关。

values showed highly significant negative correlations with flavonoid content, carotenoid content, upper epidermal cell thickness to width ratio and cell number, and significant negative correlations with CHI activity. Furthermore, the

数值与类黄酮含量、类胡萝卜素含量、上表皮细胞厚度与宽度比及细胞数量呈极显著负相关，并与CHI活性呈显著负相关。此外，

values showed a highly significant positive correlation with flavonoid content, carotenoid content, PAL activity, CHI activity, the thickness-to-width ratio of upper epidermal cells and cell number, and a significant positive correlation with anthocyanin content. Conversely, a highly significant negative correlation with cytosol pH, upper epidermal cell pinch angle and cell area, and significant negative correlation with soluble protein content.

数值与黄酮含量、类胡萝卜素含量、PAL活性、CHI活性、上表皮细胞的厚宽比及细胞数量呈极显著正相关，与花青素含量呈显著正相关。相反，与细胞质pH值、上表皮细胞夹角和细胞面积呈极显著负相关，与可溶性蛋白含量呈显著负相关。

In addition, The .

此外，The 。

values showed highly significant positive correlations with soluble protein and cytosol pH, significant positive correlations with upper epidermal cell pinch angle and cell area. In contrast,

数值与可溶性蛋白和细胞质pH值显示出高度显著的正相关，与上表皮细胞夹角和细胞面积呈现显著的正相关。相比之下，

values showed highly significant negative correlations with anthocyanin content, carotenoid content, PAL activity, CHI activity, soluble sugar content and upper epidermal cell thickness, and significant negative correlation with flavonoid content and upper epidermal cell number.

数值与花青素含量、类胡萝卜素含量、PAL活性、CHI活性、可溶性糖含量和上表皮细胞厚度呈极显著负相关，与黄酮含量和上表皮细胞数量呈显著负相关。

Fig. 7

图7

Correlation coefficients among the indicators of petals of pink-flowered

粉红花花瓣指标之间的相关系数

P. sibirica

. Fla. Flavonoid content; Ant. Anthocyanin content; Chl. Total chlorophyll content; Chl a. Chlorophyll a content; Chl b. Chlorophyll b content; Car. Carotenoid content; SS. Soluble sugar content; SP. Soluble protein content; PT. Petal thickness; UT. Upper epidermal thickness; UW. Upper epidermal width; TW.

类黄酮含量；花青素含量；总叶绿素含量；叶绿素a含量；叶绿素b含量；类胡萝卜素含量；可溶性糖含量；可溶性蛋白含量；花瓣厚度；上表皮厚度；上表皮宽度；TW。

Thickness and width ratio; CA. Upper epidermal cell angle; UA. Upper epidermal cell area; NU. Upper epidermal cell number..

厚度与宽度比；CA. 上表皮细胞角度；UA. 上表皮细胞面积；NU. 上表皮细胞数量。

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As shown in Table

如表所示

, the insignificant independent variables were excluded at the 5% significant level, and the indicators with partial regression coefficients reaching significant or highly significant levels were selected to enter into the regression equation for flower color, and the indicators with the most significant effects on the .

，不显著的自变量在5%显著性水平上被排除，选择偏回归系数达到显著或极显著水平的指标进入花色的回归方程，并且对影响最显著的指标进行筛选。

and

和

values of petal were further found from each indicator significantly correlated with flower color. In the stepwise regression equation for the

花瓣的值进一步从每个与花色显著相关的指标中发现。在逐步回归方程中，

value of pink petals, four indicators, namely anthocyanin content, CHI activity, upper epidermal cell area and soluble proteins, were retained and all the rest were excluded. In the stepwise regression equation for the

粉色花瓣的价值、四个指标，即花青素含量、CHI活性、上表皮细胞面积和可溶性蛋白被保留，其余全部排除。在逐步回归方程中

value, two indicators, namely anthocyanin content and PAL activity, were retained and all the rest were excluded. This result suggests that anthocyanins play an important role in the colouration of the petals of pink-flowered

值，保留了两个指标，即花青素含量和PAL活性，其余全部排除。该结果表明花青素在粉红花花瓣的着色中起重要作用。

P. sibirica

西伯利亚松

。

Table 4 Stepwise regression for flower color parameters of pink-flowered

表4 粉色系花色参数的逐步回归分析

P. sibirica

西伯利亚松

with various indicators.

具有各种指标。

Full size table

全尺寸表格

Discussion

讨论

Flower color is an important feature of ornamental plants, and the determination of flower color parameters to further explore the mechanism of flower color formation has been widely used in plants such as

花卉颜色是观赏植物的重要特征，测定花色参数以进一步探索花色形成的机制已广泛应用于如

Rhododendron

杜鹃花属

, Snapdragon

，骁龙

, and

，以及

Lagerstroemia

紫薇属

. The CIELab system is a commonly used method for determining the parameters of flower color as it quantifies the brightness, redness, and yellowness of the petals through the use of the

CIELab系统是一种常用于确定花色参数的方法，因为它通过使用量化花瓣的亮度、红度和黄度。

value, the

价值，这个

value, and the

价值，以及

value, respectively

值，分别

. Changes in the

. 变化

values, the

价值观，

values and the

价值观和

values during the opening process of

在打开过程中值

P. sibirica

flowers indicate that petals gradually unfolded, petals become increasingly pale. The

花朵表明花瓣逐渐展开，花瓣变得越来越淡。

and

和

values of pink petals were significantly lower than those of white petals, and the

粉红色花瓣的值显著低于白色花瓣的值，而且

value was significantly higher than that of white petals, which visually reflected that the color of pink petals was more intense than that of white petals at different stages of time. This indicates that the CIELab system was able to capture and quantify the subtle changes in petal color effectively to provide us with an accurate picture of the changes in flower color.

该值显著高于白色花瓣的值，这从视觉上反映了在不同时间阶段，粉色花瓣的颜色比白色花瓣更加浓烈。这表明CIELab系统能够有效捕捉和量化花瓣颜色的细微变化，为我们提供花卉颜色变化的准确图像。

. In this study, the

。在这项研究中，

values of the petals were negatively correlated with the

花瓣的价值与

values and not significantly correlated with the

值，并且与

values. Notably, pink flowers from

价值观。特别是，粉红色的花从

P. sibirica

varieties are better evaluated based on

品种最好根据以下方面进行评估

values rather than

价值观而不是

values for their color assessment.

用于颜色评估的值。

The type and concentration of pigments in the petals are the main factors affecting the petal coloration, with anthocyanins predominantly regulating the pink to blue-violet color of the petals

花瓣中的色素种类和浓度是影响花瓣颜色的主要因素，其中花青素主要调控花瓣的粉红色至蓝紫色。

，

。

In our study, correlation and stepwise regression analyses together showed that anthocyanins play an important role in pink-flowered

在我们的研究中，相关分析和逐步回归分析共同表明，花青素在粉红花中起着重要作用。

P. sibirica

西伯利亚松

petal coloration. We observed that the pink flower petals were not fully unfolded and had a dark color during the pre-flowering stage. The anthocyanin content peaked during the exposure stage, suggesting complete accumulation during the pre-flowering stage. A strong positive relationship between the .

花瓣颜色。我们观察到粉红色的花瓣在开花前阶段并未完全展开，且颜色较深。花青素含量在暴露阶段达到峰值，表明其在开花前阶段已完全积累。两者之间存在很强的正相关关系。

value of pink flower petals and anthocyanin content, indicating that changes in anthocyanin levels directly impacted the color intensity of pink flower petals at various stages

粉色花瓣和花青素含量的价值，表明花青素水平的变化直接影响了不同阶段粉色花瓣的颜色强度。

. Building on previous research

。基于之前的研究

，

, future studies could employ high-performance liquid chromatography-mass spectrometry (HPLC–MS) for the quantitative analysis of anthocyanins in pink-flowered

未来的研究可以采用高效液相色谱-质谱联用技术（HPLC-MS）对粉花中的花青素进行定量分析。

P. sibirica

. This approach could help identify unique compounds in

这种方法有助于识别独特的化合物在

P. sibirica

flower petals at a secondary metabolism level, shedding light on the pigment metabolism mechanism. The flavonoid and carotenoid contents of pink petals were highly significantly and positively correlated with the

花花瓣在次生代谢水平上，揭示了色素代谢机制。粉色花瓣的类黄酮和类胡萝卜素含量与

value, and changes in their contents to some extent aided the colouring of pink petals. The accumulation of large amounts of chlorophyll can mask the color of petals

价值，其内容的变化在某种程度上帮助了粉色花瓣的着色。大量叶绿素的积累可以掩盖花瓣的颜色。

, but the chlorophyll content in this study is lower than the other pigments and there is no significant correlation with the

，但本研究中的叶绿素含量低于其他色素，并且与

value.

价值。

The synthesis of anthocyanin, a key pigment influencing the color of pink flower petals, initiates with phenylalanine and involves various enzymes along the metabolic pathway. Notably, PAL and CHI act as upstream synthetases and play a crucial role in anthocyanin production, thereby impacting flower color expression.

花青素是影响粉红色花瓣颜色的关键色素，其合成始于苯丙氨酸，并在代谢途径中涉及多种酶。值得注意的是，PAL和CHI作为上游合成酶，在花青素的生产中起着关键作用，从而影响花色的表现。

. The findings from previous studies regarding the influence of individual enzymes on anthocyanin synthesis have yielded varied conclusions. For instance, in the examination of the coloration of

.以往关于单个酶对花青素合成影响的研究结果得出了不同的结论。例如，在研究

Nymphaea hybrid

杂交睡莲

, it was determined that PAL and CHI were extremely important for anthocyanin synthesis. Conversely, in the study on the coloration of

，研究确定PAL和CHI对花青素的合成非常重要。相反，在关于着色的研究中

Litchi

荔枝

’s pericarp

果皮

, it was determined that neither PAL nor CHI exhibited a significant correlation with anthocyanin synthesis, indicating that CHI was not closely related to anthocyanin synthesis. In this study, the anthocyanin content of pink flower petals showed a highly significant and significant positive correlation with PAL and CHI activities, respectively, and the .

，结果表明PAL和CHI与花色苷合成均无显著相关性，说明CHI与花色苷的合成关系不密切；本研究中，粉色花瓣中花色苷含量与PAL和CHI活性呈极显著和显著正相关。

values showed a highly significant positive correlation with the changes in the activities of both. These finding suggest that PAL and CHI activities both affect anthocyanin synthesis and hence flower color changes.

这些结果表明，PAL和CHI的活性变化与花青素合成及花色变化呈高度显著的正相关。

Sugars, as precursors of anthocyanin synthesis, can influence the synthesis of anthocyanins. In addition, soluble sugars, together with soluble proteins, serve as an important source of energy for the vital activities of petals

糖作为花青素合成的前体，能够影响花青素的合成。此外，可溶性糖与可溶性蛋白质一起为花瓣的生命活动提供重要的能量来源。

，

. In this study, there was a highly significant positive correlation between the content of soluble sugars and anthocyanins within the petals of pink flowers, indicating that the accumulation of soluble sugars promoted the production of anthocyanins. However, there was no significant correlation between the soluble sugar content and the .

在这项研究中，粉色花花瓣内可溶性糖含量与花青素含量之间存在极显著的正相关关系，表明可溶性糖的积累促进了花青素的产生。但是，可溶性糖含量与之间没有显著相关性。

value, indicating that soluble sugars are not a direct factor influencing petal coloration. The soluble protein content of pink flower petals and white flower petals exhibited opposite trends across the different stages of blooming, while the anthocyanin content remained consistent. In pink flower petals, the soluble protein content showed a highly significant negative correlation with the anthocyanin content.

值，表明可溶性糖不是直接影响花瓣颜色的因素。粉色花和白色花的可溶性蛋白含量在不同开花阶段呈现出相反的趋势，而花青素含量保持一致。在粉色花瓣中，可溶性蛋白含量与花青素含量呈现极显著的负相关关系。

Although some studies have shown that an increase in the soluble protein content inhibits anthocyanin synthesis.

尽管一些研究表明可溶性蛋白含量的增加会抑制花青素的合成。

, this conclusion may be applicable mainly to species that are rich in anthocyanins. The extent to which soluble proteins influence anthocyanins synthesis needs to be further explored to fully understand their role across various species and conditions.

，这个结论可能主要适用于富含花青素的物种。可溶性蛋白对花青素合成的影响程度需要进一步探索，以充分理解它们在不同物种和条件下的作用。

Numerous studies have shown that petals tend to be more red in color and anthocyanins can exist more stably when the pH of the petal cytosol is low, and more blue when the pH is high

许多研究表明，当花瓣细胞溶胶的 pH 值较低时，花瓣往往更呈红色，花青素能够更稳定地存在，而当 pH 值较高时则更呈蓝色。

. In this study, the cytosol pH of pink and white flower petals was weakly acidic at all stages of blooming, which was favorable for the preservation and accumulation of anthocyanins. In the correlation analysis, as the pH value of the cytosol increased, anthocyanins gradually degraded, and the color of pink flower petals gradually became lighter.

在本研究中，粉红和白色花瓣的细胞质pH值在开花的所有阶段均为弱酸性，这有利于花青素的保存和积累。在相关性分析中，随着细胞质pH值的升高，花青素逐渐降解，粉红色花瓣的颜色逐渐变浅。

This finding aligns with the results of a study on the mechanism of color presentation in .

这一发现与一项关于色彩呈现机制的研究结果一致。

Hibiscus sabdariffa

玫瑰茄

。

Petals are the carriers of flower color, and in this study, significant epidermal pigment accumulation was on the petals of pink flowers, while no significant pigment accumulation was noted on the epidermis of the petals of white flowers using the freehand sectioning method. This suggests that the presentation of the color of the white flowers is not due to pigmentation but is instead an optical phenomenon.

花瓣是花色的载体，在这项研究中，使用徒手切片法观察到粉红色花的花瓣表皮有显著的色素积累，而白色花的花瓣表皮没有显著的色素积累。这表明白色花的颜色呈现不是由于色素，而是一种光学现象。

，

四十三

. A variety of flowers, such as the white-flowered

各种各样的花，比如白花

P. sibirica

西伯利亚松

, take on a white hue due to the presence of numerous air bubbles within their petals, which causes incident light to enter the petals and refract multiple times.

，由于花瓣内存在大量气泡，入射光进入花瓣后会多次折射，从而使花朵呈现白色。

After observing the cross-section structure of petals using the paraffin section method, we found that the epidermal cells of the petals of pink and white flowers were closely arranged and regular in shape during the early stage of flowering. As the petals gradually unfolded, the epidermal cells appeared to be fragmented, and the thin-walled tissues became gradually loosened.

采用石蜡切片法观察花瓣横切面结构发现，粉红花和白花花瓣的表皮细胞在开花前期排列紧密，形状规则，随着花瓣逐渐展开，表皮细胞呈现破碎状，薄壁组织也逐渐疏松。

The epidermal cells of both the flower types exhibited similar structural changes. It was hypothesized that the differences in the content and distribution of pigments influence the light-absorbing characteristics of the petals, leading to the color differences between the petals of the pink flowers and white flowers.

两种类型花的表皮细胞表现出相似的结构变化。假设色素含量和分布的差异影响花瓣的吸光特性，导致粉红花和白花花瓣之间的颜色差异。

. The ratio of epidermal cell thickness and width to the angle of cell entrapment is commonly used to evaluate the degree of canonicalization of cells

表皮细胞厚度与宽度和细胞截留角度的比值常被用来评估细胞的规范程度。

. Correlation analysis indicates that the higher the degree of canonicalization of the epidermis of the powdered flower petals, the smaller the angle of the intercellular pinch, and the higher the proportion of incident light entering the epidermal cells, which could enhance the absorption of light by the epidermal pigments, thus resulting in denser colors of the petals.

相关性分析表明，粉末状花瓣表皮的角质化程度越高，细胞间夹角越小，进入表皮细胞的入射光比例越高，这可能增强了表皮色素对光的吸收，从而使花瓣的颜色更加浓密。

。

Observation of petal epidermal cells by scanning electron microscopy revealed that variations in the area of petal epidermal cells and the number of cells per unit affect the petal color

通过扫描电子显微镜观察花瓣表皮细胞发现，花瓣表皮细胞的面积变化和单位面积内的细胞数量会影响花瓣颜色。

，

. In this study, the

。在这项研究中，

value showed a highly significant negative correlation with the area of the upper epidermal cells of the petals and a highly significant positive correlation with the number of upper epidermal cells. This suggests that the rapid expansion of the petal area would result in a corresponding reduction in the number of cells per unit area, but the rate of pigment synthesis would be relatively slow, reducing the pigment content per unit area and resulting in a lighter petal color.

数值与花瓣上表皮细胞的面积呈极显著负相关，而与上表皮细胞数量呈极显著正相关。这表明花瓣面积的快速扩展会导致单位面积内的细胞数量相应减少，但色素合成的速度相对较慢，从而降低了单位面积内的色素含量，使花瓣颜色变浅。

。

During the dissection of petals at each stage of flowering, thinner petals were difficult to observe by freehand sectioning treatment due to variations in petal thickness. To explore the relationship between petal microstructure and coloration, this study used paraffin sectioning and scanning electron microscopy for the multi-dimensional observation of petal transverse structure and petal epidermal cells across the five different stages of flowering.

在开花各阶段的花瓣解剖过程中，由于花瓣厚度的变化，较薄的花瓣难以通过徒手切片处理进行观察。为探究花瓣微观结构与颜色的关系，本研究采用石蜡切片法和扫描电子显微镜对花瓣横截面结构及花瓣表皮细胞在开花的五个不同阶段进行了多维度观察。

We hope that this comprehensive approach will serve as a useful reference for future researchers..

我们希望这一综合方法能够为未来的研究人员提供有用的参考。

Conclusion

结论

In this study, we measured the flower color parameters and physiological indicators of pink-flowered and white-flowered

在本研究中，我们测量了粉红花和白花的花色参数和生理指标。

P. sibirica

petals, observed the changes in epidermal cells, and interpreted the mechanism of pink-flowered

花瓣，观察表皮细胞的变化，并解释粉红花的机制

P. sibirica

petal coloration from multiple perspectives. The results showed that the

从多个角度观察花瓣的颜色。结果表明，

value is the primary flower color parameter describing pink-flowered

value 是描述粉红色花朵的主要颜色参数

P. sibirica

西伯利亚松

petals, with anthocyanins being the main pigments influencing this value. The elevation of PAL and CHI activities, along with soluble sugar content, promoted the synthesis and accumulation of anthocyanins. During this process, the soluble protein content and cytosol pH were lower, and these parameters influenced the anthocyanin content by participating in the physiological processes related to petal coloring, indirectly affecting petal color changes.

花瓣中的花青素是影响该值的主要色素。PAL和CHI活性的提升以及可溶性糖含量的增加促进了花青素的合成与积累。在此过程中，可溶性蛋白含量和细胞质pH较低，这些参数通过参与与花瓣着色相关的生理过程间接影响花瓣颜色变化，从而影响花青素含量。

The thickness-to-width ratio, cell pinch angle, cell area, and number of cells in the upper epidermal of pink flower petals influenced the absorption of light by the petal pigments, resulting in different petal colors at different stages. The results of this study offer an initial insight into the mechanism underlying the coloration of pink-flowered .

粉红花花瓣的上表皮细胞的厚宽比、细胞夹角、细胞面积和细胞数量影响了花瓣色素对光的吸收，从而导致不同阶段花瓣颜色的差异。本研究的结果为粉红花颜色形成的机制提供了初步的认识。

P. sibirica

西伯利亚松

, providing a foundation for future studies to delve deeper into the petal color genetics, transcription factors, and other regulatory mechanisms, to comprehensively analyze the formation and regulation of flower color in pink-flowered

为今后深入研究花瓣颜色遗传、转录因子及其他调控机制奠定基础，全面分析粉花花色形成的调控机理。

P. sibirica

西伯利亚松

flower, and to provide a theoretical basis for the cultivation of new varieties of ornamental plant varieties with desired flower colors.

花卉，并为培育具有理想花色的新观赏植物品种提供理论依据。

Data availability

数据可用性

The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.

当前研究期间生成和分析的数据集可在合理要求下由通讯作者获取。

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Funding

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This research was funded by the Liaoning Province Wild apricot Germplasm Resource Preservation and Breeding National Permanent Scientific Research Base [Grant No. 2020132519], and the Innovation Team for Creating and Utilizing Non-wood Forest Germplasm Resources in Semi-arid Regions of National Forestry and Grassland Administration..

本研究由辽宁省山杏种质资源保存与育种国家长期科研基地[项目编号：2020132519]和国家林业和草原局半干旱地区非木质林木种质资源创制与利用创新团队资助。

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College of Forestry, Shenyang Agricultural University, Shenyang, 110866, Liaoning Province, China

中国辽宁省沈阳市沈北新区道义南大街沈阳农业大学林学院，邮编110866

Wenxuan Fan, Jianhua Chen, Junxin Feng, Jindi Yang, Yongqiang Sun & Shengjun Dong

范文轩、陈建华、冯俊欣、杨金娣、孙永强、董盛军

Key Laboratory for Silviculture of Liaoning Province, Shenyang Agricultural University, Shenyang, 110866, Liaoning Province, China

辽宁省森林培育重点实验室，沈阳农业大学，沈阳，110866，辽宁省，中国

Wenxuan Fan, Jianhua Chen, Junxin Feng, Jindi Yang, Yongqiang Sun & Shengjun Dong

范文轩、陈建华、冯俊欣、杨金娣、孙永强、董胜军

Bureau of Forestry and Grassland of Kazuo County, Kazuo, Chaoyang, 122300, Liaoning Province, China

中国辽宁省朝阳市喀左县林业和草原局，邮编122300

Yuncheng Zhang

运城张

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Contributions

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All authors have read and agreed to the published version of the manuscript. S.D., W.F. and J.C. conceived and designed the work. W.F., Y.Z., J.Y. and J.F. collected the data. W.F. analyzed and interpreted the data. W.F. drafted the manuscript. S.D., J.C. and Y.S. revised the manuscript.

所有作者均已阅读并同意手稿的发表版本。S.D.、W.F. 和 J.C. 构思并设计了这项工作。W.F.、Y.Z.、J.Y. 和 J.F. 收集了数据。W.F. 分析并解释了数据。W.F. 起草了手稿。S.D.、J.C. 和 Y.S. 修订了手稿。

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陈建华

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Shengjun Dong

董圣俊

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Fan, W., Zhang, Y., Chen, J.

范, W., 张, Y., 陈, J.