组蛋白修饰H3K9ac与转录因子AREB1协同调控毛果杨响应干旱胁迫的分子机制研究

Drought severely affects plant growth and forest productivity. Plants respond and adapt to drought stress through coordinated changes at transcriptional level of drought responsive gene networks. This requires complex transcriptional regulation and a change in chromatin accessibility. However, the molecular connections among these factors and with drought response are not known. Here, we found that the transcriptional activity of PtrNACs was highly associated with H3K9ac alteration through integrative analysis of chromatin immunoprecipitation with sequencing (ChIP-seq) and RNA-seq data in Populus trichocarpa subjected to soil-water depletion. We analyzed promoters of PtrNACs, and identified transcription factor (TF) PtrAREB1s that may be involved in regulating transcription of PtrNACs. Moreover, we identified a drought inducible histone acetyltransferase (HAT) gene, PtrGCN5-1, encoding a protein which is the catalytic subunit of several multiprotein HAT complexes, and a drought inducible adaptor protein gene PtrADA2b which is integral parts of GCN5-containing complexes. We also identified a drought inducible alternative PtrADA2b splice variant, PtrADA2b-α, which may be involved in an alternative splicing regulatory mechanism linking histone acetylation and TFs in plant response to drought stress. In this proposed work, we will investigate crosstalk between PtrGCN5-1, PtrADA2b, PtrADA2b-α and PtrAREB1s in regulation of H3K9ac on PtrNACs to activate their transcription, and analyze the roles of PtrGCN5-1, PtrADA2b, PtrADA2b-α, PtrAREB1s and PtrNACs in P. trichocarpa response to drought stress. We will also identify the direct targets of PtrNACs and find out their metabolic pathway in response to drought stress. Learning crosstalk between TFs and H3K9ac in response to drought stress will allow us to reveal the mechanisms of the combined action of TFs and histone modifications in regulation of drought responsive genes and also have broad utility in improving drought tolerance of tree species and forest productivity.

植物通过整个抗旱基因网络快速、协调的变化适应干旱胁迫，其中转录因子和表观遗传修饰发挥着关键调控作用。然而，在此过程中表观遗传修饰及其与转录因子相互协调机制还不清楚。本项目前期的研究发现PtrNACs基因表达与H3K9ac水平密切相关，并且转录因子PtrAREB1s可能调控PtrNACs。同时，鉴定出了干旱诱导的组蛋白乙酰化酶复合体基因PtrGCN5-1、PtrADA2b及一个新的可变剪切产物PtrADA2b-α。在此基础上，本项目将研究组蛋白乙酰化酶复合体与PtrAREB1s调控PtrNACs基因H3K9乙酰化激活其表达响应干旱胁迫的分子机制，以及这些基因在毛果杨应答干旱胁迫中的功能，并鉴定PtrNACs调控的靶基因及分析其调节的抗旱代谢途径。该项目的实施将揭示组蛋白修饰H3K9ac与转录因子AREB1协同调控毛果杨响应干旱胁迫的分子机制，为培育高抗林木树种进行遗传改良提供理论基础。

多年生的林木在漫长的生命周期中，会遇到不断变化的各种外界环境条件。其中，干旱是主要环境限制因子之一。转录因子和组蛋白修饰在调节植物适应干旱胁迫中发挥关键作用，但是二者之间如何相互协调控制耐旱基因表达的机制尚不清楚。.在前期的研究中，我们从毛果杨木质部中鉴定出PtrNAC006等干旱胁迫响应基因。本研究中，我们发现过量表达PtrNACs基因显著增强转基因植株的耐旱性。这些基因在干旱条件下受到诱导并且H3K9ac标记增多，基因启动子中含有ABRE顺式作用元件，该元件能够被AREB1转录因子所特异性的识别并结合。因此，我们推测AREB1转录因子和组蛋白乙酰化修饰对干旱响应基因具有协同调控作用。进一步研究发现，AREB1转录因子通过特异性识别并结合PtrNACs启动子上的ABRE元件，激活PtrNACs基因表达，并且AREB1与组蛋白乙酰化酶复合体GCN5-ADA2b相互作用形成蛋白三聚体。利用原生质体瞬时转化系统，证明了组蛋白乙酰化酶复合体可以通过增加H3K9ac、RNA聚合酶II的富集水平，增强AREB1对干旱响应基因的转录调控。利用PtrGCN5-1、PtrAREB1-2的RNAi和PtrADA2b-3的CRISPR突变体植株进行ChIP-PCR及RT-qPCR分析，结果显示转基因毛果杨及突变体植株干旱胁迫后，H3K9ac、RNA聚合酶II的富集水平和PtrNAC基因的转录水平均显著下降，并且转基因植株与突变体耐旱性降低。综合分析以上结果，转录因子AREB1与GCN5-ADA2b互作将组蛋白乙酰化酶复合体招募到PtrNACs启动子上，引起H3K9c及RNA聚合酶II富集，激活这些基因表达，从而使杨树适应干旱胁迫。.综上所述，该研究揭示了组蛋白修饰与转录因子协同调控林木次生生长适应干旱环境的新机制，为从表观遗传角度进行林木分子育种开拓了新思路，具有重要的理论意义和潜在的应用价值。

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