菊花（Chrysanthemum morifolium）是我国十大传统名花之一，被誉为花中四君子之一，拥有悠久的栽培历史。菊花不仅具有深厚的文化意义，还具备显著的经济和审美价值。花色是菊花重要的观赏性状之一，主要由类黄酮、类胡萝卜素和叶绿素等色素呈现。其中类黄酮是最主要的花色素类型，其主要成分花青苷赋予花瓣粉、红和紫等不同颜色。花青苷作为植物中广泛存在的次生代谢物质，目前对其研究相对较为深入，其在植物体内的合成受相关结构基因和上游调节基因的调控，并被外界环境因素影响。温度是影响花青苷生物合成的主要环境因子，在拟南芥和苹果等植物的研究中发现，高温通常导致花青苷合成减少，而低温则促进花青苷积累。

菊花多在秋季冷凉季开花，其自然花期的平均温度约为10℃左右，大部分红色系菊花在该环境下着色良好，但适度低温促进菊花花瓣中花青苷积累和花色形成的分子机制尚不清楚。在本研究中，我们以‘南农粉翠’菊花为研究材料，对菊花开花阶段进行了24℃和10℃温度处理。主要研究内容及结论如下：

10℃低温下菊花花瓣中花青苷可被诱导积累。RT-qPCR结果表明，花青苷合成通路的主要结构基因CmCHS、CmCHI、CmDFR、CmANS、CmF3H和CmUFGT在10℃低温下均上调表达，暗示存在上游调节机制参与低温下菊花花青苷生物合成结构基因的表达调控。R2R3-MYB转录因子在调控植物花青苷生物合成结构基因表达中具有核心作用。我们对菊花中已经报道参与调控花青苷生物合成的R2R3-MYB转录因子响应低温情况进行了分析，发现CmMYB6和CmMYB73被10℃低温处理上调表达，且CmMYB73被诱导的程度更高，其在10℃低温下的表达约为常温对照的5倍，因此我们选择CmMYB73作为进一步研究的对象。

CmMYB73的氨基酸序列含有保守EAR转录抑制基序，是一个可能的转录抑制因子，但其在菊花花青苷生物合成中的功能尚不明确。为此，我们构建了CmMYB73基因的超表达和干扰表达载体，对‘南农粉翠’进行遗传转化，成功获得了相应的转基因株系。相比于野生型（Wild type, WT）对照，CmMYB73超表达株系花瓣中花青苷含量显著降低，而干扰株系则呈现出相反的表型，说明CmMYB73负调控菊花花瓣中花青苷生物合成。通过酵母单杂交（Yeast One-Hybrid, Y1H）实验和双荧光素酶报告基因实验（Dual-Luciferase Reporter Assay, Dual-Luc）发现CmMYB73通过直接结合花青苷生物合成结构基因CmCHS和CmCHI的启动子抑制其表达，并间接抑制结构基因CmANS的表达，说明CmMYB73通过转录抑制花青苷合成结构基因CmCHS、CmCHI和CmANS的表达负调控菊花花瓣中花青苷生物合成。

Western-blot实验结果表明，相比于24℃对照组，CmMYB73蛋白在10℃低温处理下加速降解，暗示CmMYB73在蛋白水平的降解是低温下菊花花瓣中花青苷积累的原因之一。为了明确CmMYB73在低温下降解的具体机制，以CmMYB73为诱饵蛋白进行了酵母双杂交筛库，筛选与CmMYB73相互作用的蛋白。鉴定到一个U-box型E3泛素连接酶CmPUB15。通过酵母双杂交（Yeast Two-Hybrid, Y2H）实验、蛋白Pull-down实验（Protein Pull-down Assay）和双分子荧光素酶互补实验（Bimolecular Luciferase Complementation, BiFC）确认了CmMYB73与CmPUB15之间的相互作用。RT-qPCR结果表明，CmPUB15基因表达在10℃低温处理下相比于24℃对照组显著上调。蛋白降解实验结果表明，CmPUB15在10℃低温处理下的稳定性高于24℃对照组。基于‘南农粉翠’的遗传转化实验表明，CmPUB15能够正向调控菊花花瓣中的花青苷积累。

体外泛素化实验结果表明，在E1（泛素激活酶）、E2（泛素结合酶）和ATP的存在下，CmPUB15蛋白能够介导CmMYB73蛋白的泛素化修饰。同时，植物体内验证结果表明，在CmPUB15蛋白的存在下，CmMYB73蛋白的泛素化水平显著提高。基于CmPUB15转基因菊花植株的半体内蛋白降解实验表明，相比于WT对照，CmMYB73蛋白在CmPUB15过量表达背景下的降解速率显著增加，而在沉默CmPUB15的背景下，CmMYB73蛋白的降解速率显著减缓，而加入26S蛋白酶体抑制剂MG132后，CmMYB73蛋白在各个背景下的降解均被显著抑制，说明CmPUB15介导的CmMYB73的泛素化修饰导致了CmMYB73蛋白通过26S 蛋白酶体途径降解。对‘南农粉翠’的瞬时转化实验结果表明，在OE-CmMYB73的背景下瞬时干扰CmPUB15的表达会进一步抑制花瓣中的花青苷积累，下游结构基因CmCHS、CmCHI、CmANS基因的表达相比于OE-CmMYB73株系中进一步下降，说明干扰CmPUB15的表达可以增强CmMYB73对菊花花青苷下游结构基因CmCHS、CmCHI和CmANS的抑制作用，进一步抑制菊花的花青素的积累。

综上，本研究初步揭示了菊花CmPUB15-CmMYB73分子模块调控低温下花瓣花青苷合成的分子机制。在常温下，CmPUB15的表达和CmMYB73蛋白的泛素化修饰水平较低，CmMYB73蛋白更为稳定，从而抑制其下游花青苷生物合成结构基因CmCHS、CmCHI、CmANS的表达，导致菊花花瓣中较低的花青苷积累水平。在10℃低温条件下，CmPUB15的表达被诱导，积累的CmPUB15蛋白与CmMYB73蛋白相互作用，并对其进行泛素化修饰，导致CmMYB73蛋白经由26S蛋白酶体途径降解，从而解除其对下游CmCHS、CmCHI、CmANS的转录抑制，促进菊花花瓣中的花青苷积累。此外，菊花花青苷生物合成的正调控转录因子CmMYB6在转录水平响应10℃低温，从而促进花瓣中花青苷在低温下的积累。这些发现加深了对低温下菊花花青苷积累的分子调控机制的理解，并为菊花周年生产以及花色分子改良提供了理论基础和优异靶基因。

Chrysanthemum is one of the ten traditional famous flowers in China, honored as one of 'the Four Gentlemen' among Flowers, with a long history of cultivation. Chrysanthemum not only carries profound cultural significance but also possesses significant economic and aesthetic values. Flower color, primarily exhibited by pigments such as flavonoids, carotenoids, and chlorophyll, is a crucial ornamental trait of chrysanthemum. Among these, flavonoids are the primary type of floral pigment, with anthocyanins as their main component, imparting hues like pink, red, and purple to the petals. Anthocyanins, as widely occurring secondary metabolites in plants, have been relatively extensively studied. Their synthesis within plants is regulated by related structural genes and upstream regulatory genes and influenced by external environmental factors. Temperature is a major environmental factor affecting anthocyanin biosynthesis. Studies in plants such as arabidopsis thaliana and apples have found that high temperatures generally lead to reduced anthocyanin synthesis, while low temperatures promote anthocyanin accumulation.

Chrysanthemums predominantly bloom during the cool autumn season, with an average temperature of approximately 10°C during their natural flowering period. Most red-colored chrysanthemum varieties exhibit good coloration under these conditions, yet the molecular mechanism underlying the promotion of anthocyanin accumulation and flower color formation in chrysanthemum petals at moderate low temperatures remains unclear. In this study, we used the chrysanthemum cultivar 'Nannong Fencui' as the research material and subjected it to temperature treatments of 24°C and 10°C during the flowering stage. The main research contents and conclusions are as follows:

Anthocyanins in chrysanthemum petals can be induced to accumulate at 10°C. RT-qPCR results indicated that the major structural genes involved in anthocyanin biosynthesis, including CmCHS, CmCHI, CmDFR, CmANS, CmF3H, and CmUFGT, were upregulated under low temperature at 10°C, suggesting the involvement of upstream regulatory mechanisms in the expression regulation of these structural genes during anthocyanin biosynthesis in chrysanthemum under low temperature. R2R3-MYB transcription factors play a central role in regulating the expression of structural genes involved in plant anthocyanin biosynthesis. We analyzed the response of R2R3-MYB transcription factors in chrysanthemum, which have been reported to regulate anthocyanin biosynthesis, to low temperature and found that CmMYB6 and CmMYB73 were upregulated by low temperature treatment at 10°C, with CmMYB73 being induced to a higher level, exhibiting an expression level approximately five times that of the control at room temperature. Therefore, we selected CmMYB73 for further study.

 2. The amino acid sequence of CmMYB73 contains a conserved EAR transcription repressor motif, suggesting it may be a transcriptional repressor, but its function in chrysanthemum anthocyanin biosynthesis is unclear. To address this, we constructed overexpression and interference expression vectors for the CmMYB73 gene and genetically transformed 'Nannong Fencui', successfully obtaining corresponding transgenic lines. Compared to the wild-type (WT) control, the anthocyanin content in the petals of CmMYB73 overexpression lines was significantly reduced, while the interference lines exhibited the opposite phenotype, indicating that CmMYB73 negatively regulates anthocyanin biosynthesis in chrysanthemum petals. Yeast one-hybrid (Y1H) experiments and dual-luciferase reporter assays (Dual-Luc) revealed that CmMYB73 inhibits the expression of structural genes CmCHS and CmCHI by directly binding to their promoters and indirectly inhibits the expression of CmANS, suggesting that CmMYB73 negatively regulates anthocyanin biosynthesis in chrysanthemum petals by transcriptionally inhibiting the expression of CmCHS, CmCHI, and CmANS.

3. Western-blot results showed that CmMYB73 protein degraded more rapidly under low temperature treatment at 10°C compared to the 24°C control, suggesting that the degradation of CmMYB73 at the protein level is one of the reasons for anthocyanin accumulation in chrysanthemum petals under low temperature. To elucidate the specific mechanism of CmMYB73 degradation under low temperature, we conducted yeast two-hybrid screening using CmMYB73 as the bait protein to identify proteins that interact with CmMYB73. We identified a U-box E3 ubiquitin ligase, CmPUB15. The interaction between CmMYB73 and CmPUB15 was confirmed through yeast two-hybrid (Y2H) experiments, protein pull-down assays, and bimolecular luciferase complementation (BiFC) experiments. RT-qPCR results showed that CmPUB15 gene expression was significantly upregulated under low temperature treatment at 10°C compared to the 24°C control. Protein degradation experiments demonstrated that CmPUB15 was more stable under low temperature treatment at 10°C than at 24°C. Genetic transformation experiments based on 'Nannong Fencui' showed that CmPUB15 positively regulates anthocyanin accumulation in chrysanthemum petals.

4. In vitro ubiquitination experiments showed that CmPUB15 protein can mediate the ubiquitination of CmMYB73 protein in the presence of E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and ATP. Meanwhile, in vivo validation results indicated that the ubiquitination level of CmMYB73 protein was significantly increased in the presence of CmPUB15 protein. Semi-in vivo protein degradation experiments based on CmPUB15 transgenic chrysanthemum plants showed that the degradation rate of CmMYB73 protein increased significantly in the context of CmPUB15 overexpression compared to the WT control, while the degradation rate of CmMYB73 protein slowed significantly in the context of CmPUB15 silencing. The degradation of CmMYB73 protein was significantly inhibited in all contexts after the addition of the 26S proteasome inhibitor MG132, suggesting that ubiquitination of CmMYB73 mediated by CmPUB15 leads to degradation of CmMYB73 protein via the 26S proteasome pathway. Transient transformation experiments in 'Nannong Fencui' showed that transient interference of CmPUB15 expression in the background of OE-CmMYB73 further inhibited anthocyanin accumulation in the petals, and the expression of downstream structural genes CmCHS, CmCHI, and CmANS further decreased compared to OE-CmMYB73 lines, indicating that interference of CmPUB15 expression enhances the inhibitory effect of CmMYB73 on the downstream structural genes CmCHS, CmCHI, and CmANS of chrysanthemum anthocyanin biosynthesis, further inhibiting the accumulation of anthocyanins in chrysanthemum.

In summary, this study initially revealed the molecular mechanism by which the CmPUB15-CmMYB73 molecular module regulates anthocyanin synthesis in chrysanthemum petals under low temperature. At room temperature, the expression of CmPUB15 and the ubiquitination modification level of CmMYB73 protein are low, resulting in a more stable CmMYB73 protein that inhibits the expression of its downstream structural genes CmCHS, CmCHI, and CmANS involved in anthocyanin biosynthesis, leading to low levels of anthocyanin accumulation in chrysanthemum petals. Under low temperature conditions at 10°C, the expression of CmPUB15 is induced, and the accumulated CmPUB15 protein interacts with CmMYB73 protein and ubiquitinates it, leading to the degradation of CmMYB73 protein via the 26S proteasome pathway, thereby relieving its transcriptional inhibition on downstream CmCHS, CmCHI, and CmANS and promoting anthocyanin accumulation in chrysanthemum petals. Additionally, CmMYB6, a positive regulatory transcription factor for chrysanthemum anthocyanin biosynthesis, responds to low temperature at 10°C at the transcriptional level, thereby promoting anthocyanin accumulation in petals under low temperature. These findings deepen our understanding of the molecular regulatory mechanisms underlying anthocyanin accumulation in chrysanthemum under low temperature and provide a theoretical basis and excellent target genes for the year-round production of chrysanthemum and molecular improvement of flower color.

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