组蛋白乙酰化是生物体内重要的染色质重塑机制之一，参与调控植物生长发育的各个阶段。NuA4（Nucleosome Acetyltransferase of Histone H4）是酵母体内必需且唯一的乙酰转移酶复合物，影响酵母的正常生长发育。在物种进化过程中，动物的乙酰转移酶复合物NuA4/TIP60（NuA4/Tat-interactive protein, 60 kilodalton）的组成发生改变，由酵母SWR1（SWI2/SNF2-Related 1 chromatin remodeling complex）复合物与NuA4复合物的亚基经过保守进化后融合形成。在拟南芥中，存在绝大部分酵母NuA4复合物亚基的同源蛋白，并已证实各亚基的功能，但不存在关键组分EAF5（Esa1-associated factor 5）的同源蛋白。目前在已有的拟南芥NuA4复合物的质谱研究结果中均共纯化到一类BRD（Bromodomain）蛋白。这暗示了植物在进化过程中，可能演变出独特的组装构成。因此，进一步探究BRD蛋白是否参与拟南芥NuA4复合物的结构组成，有利于阐释其作用机制。

研究以模式植物拟南芥为研究对象，通过蛋白互作组学与系统进化分析方法解析BRD蛋白参与植物乙酰转移酶复合物NuA4的组装模式，并利用分子互作实验证实对组装模式的设想。另外，通过构建brd基因的多重突变体，解析突变体开花性状及其背后的分子机制，对BRD蛋白的功能进行初步解析。主要研究结果如下：

1. 整合多个拟南芥乙酰转移酶复合物NuA4/TIP60各亚基的亲和纯化串联质谱结果，以分析该复合物的组成结构及各亚基功能。结果表明，拟南芥中缺少酵母NuA4复合物亚基EAF5，但是存在一些BRD蛋白可与NuA4各亚基共纯化。通过系统进化树分析得出，BRD3/4/6/7/9属于一类特殊的拟南芥BRD蛋白分子，该类蛋白包含除了所有BRD蛋白共有的Bromodomain外，也拥有该分支特异性保守的N端序列。

2. 利用分子互作实验，从体内和体外分别证实BRD3/4/6/7/9蛋白与NuA4亚基SNS1（SnRK2 substrate 1）和EAF1（Esa1-associated factor 1）蛋白存在互作关系，桥接MRG1/2（Morf-Related Gene 1/2）-SNS1至NuA4的关键组分EAF1上，组成NuA4复合物。利用Alphafold3预测BRD3/4/6/7/9与拟南芥NuA4亚基SNS1互作的关键氨基酸位点，将这些氨基酸突变后，蛋白互作受到破坏。

3. 转录分析表明BRD3/4/6/7/9在拟南芥各组织中广泛表达。brds单突变体与双突变体brd3 brd6、brd3 brd9、brd6 brd7相较于野生型拟南芥开花时间没有明显差异。然而，brds多重突变体brd3 brd4 brd9、brd3 brd6 brd9、brd3 brd6 brd7 brd9、brd3 brd4 brd6 brd7 brd9表现出晚花表型。

4. brd3 brd4 brd6 brd7 brd9表现出类似sns1突变体的晚花性状，但没有eaf1a eaf1b突变体株型极小、叶片白化等性状，说明MRG-SNS1-BRD模块在NuA4复合物中可能是起着微调功能。转录表达确认BRD3/4/6/7/9与SNS1类似，通过调控开花关键基因FT、SOC1的表达，进而促进拟南芥在长日照下的开花。

综上所述，研究首次揭示了BRD3/4/6/7/9蛋白作为植物乙酰转移酶复合物NuA4的成员之一，初步确定其能够桥接MRG1/2-SNS1模块与EAF1，直接参与到植物乙酰转移酶复合物NuA4的组装。并且，研究也首次探明BRD3/4/6/7/9基因在拟南芥各组织中泛表达，其功能的缺失会影响拟南芥的开花时间，为进一步深度解析植物开花调控的复杂机制提供了新的视角，为丰富植物开花通路新机制的研究奠定了研究基础。

Histone acetylation is one of the crucial mechanisms of chromatin remodeling in vivo, which involved in regulating all stages of plant growth and development. NuA4 (Nucleosome Acetyltransferase of Histone H4) is an essential and unique acetyltransferase complex in yeast, affecting the normal growth and development of yeast. During the process of species evolution, the composition of the acetyltransferase complex NuA4/TIP60 (NuA4/Tat-interactive protein, 60 kilodalton) in animals has changed. It is formed by the fusion of the subunits of the yeast SWR1 (SWI2/SNF2-Related 1 chromatin remodeling complex) and the NuA4 complex after conserved evolution. In Arabidopsis thaliana, most of the homologous proteins of yeast NuA4 complex subunit exist, and the function of each subunit has been confirmed, but there is no homologous protein of the key component EAF5 (Esa1-associated factor 5). At present, in the existing mass spectrometry research results of the NuA4 complex of Arabidopsis thaliana, a type of BRD (Bromodomain) protein has been co-purified. This suggests that plants may have developed unique assemblies during evolution. Therefore, further research on the structure and composition of Arabidopsis NuA4 complex will help to elucidate its mechanism of function.

The study used the model plant Arabidopsis thaliana as the research object. In order to analyze the assembly mode of Bromodomain (BRD) protein, which involved in plant acetyltransferase complex NuA4, through protein interaction omics and phylogenetic analysis. The hypothesis of assembly mode was confirmed by molecular interaction experiments. In addition, the function of BRD protein was preliminarily analyzed. The main findings are as follows:

1. The affinity purification tandem mass spectrometry results of several Arabidopsis acetyltransferase complex NuA4/TIP60 subunits were integrated to analyze the composition and function of the subunits of the complex. The results showed that only the yeast NuA4 complex subunit EAF5 was missing in Arabidopsis thaliana, but some BRD proteins were co-purified with NuA4 subunits. According to phylogenetic tree analysis, BRD3/4/6/7/9 is a special group of Arabidopsis BRD proteins, which contain not only the conserved Bromodomain, but also the specific conserved N-terminal sequence.

2. Interactions between BRD3/4/6/7/9 and NuA4 subunit SNS1 (SnRK2 substrate 1) and EAF1 (Esa1-associated factor 1) were confirmed either in vivo or in vitro by molecular interaction experiments. Moreover, it can bridge MRG2 (Morf-Related Gene 2) -SNS1 sub-module to EAF1, a key component of NuA4, to form MRG2-SNS1-BRD3/4/6/7/9-EAF1 module. For another, Alphafold3 was used to predict the key sites of interaction between BRD3/4/6/7/9 and NuA4 subunit SNS1 of Arabidopsis thaliana, and the protein interaction relationship was destroyed after amino acid mutation of these sites.

3. BRD3/4/6/7/9 are widely express in various Arabidopsis tissues. There are no significant differences in flowering time between the brds single mutant and double nutant brd3 brd 6, brd3 brd9, brd6 brd7 compared with wild-type Arabidopsis thaliana. However, multiple mutant types of brd3 brd6 brd7 brd9 and brd3 brd4 brd6 brd7 brd9 exhibit a late-flowering phenotype.

4. brd3 brd4 brd6 brd7 brd9 exhibit late-flowering traits similar to those of the sns1 mutant, but do not have traits such as extremely small plant type and albino leaves of the eaf1a and eaf1b mutants. This indicates that the MRG-SNS1-BRD module may play a fine-tuning role in the NuA4 complex. Transcriptional expression confirmed that BRD3/4/6/7/9 is similar to SNS1. By regulating the expression of key flowering genes FT and SOC1, it promotes the flowering time of Arabidopsis thaliana under long-day conditions.

In summary, the study revealed that BRD3/4/6/7/9 protein, as a member of the plant acetyltransferase complex NuA4 for the first time. And they were preliminarily identified as a subunit to bridge MRG2-SNS1 module with EAF1, which directly involved in the assembly of the plant acetyltransferase complex NuA4. In addition, this study also proved that BRD3/4/6/7/9 are widely expressed in various tissues of Arabidopsis thaliana for the first time. The loss of their function will affect the flowering time. These provide a new perspective for further in-depth analysis of the complex mechanism of plant flowering regulation, and laying a research foundation for the study of new mechanisms of enriching plant flowering pathways.

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