拟南芥种子萌发中EIN2的生物学功能与机制研究

  种子萌发是植物生命周期中至关重要的一个过程。它标志着种子的休眠状态结束，转而开始进行生长和发育。在种子萌发的过程中，种子脱离休眠状态，开始吸收水分，进而激活一系列生理和生化过程，包括DNA复制、细胞分裂、蛋白质合成等，最终导致胚轴伸长和胚根的突出。在这个过程中，植物激素脱落酸（abscisic acid，ABA）起着重要作用。种子内ABA主要通过受体以及其介导的信号通路实现对种子萌发的抑制。随着水分吸收和光照刺激，种子内高水平的ABA开始经历分解代谢而下降，ABA信号通路去激活，随之种子开始萌发。环境中多种不利因素对ABA代谢的干扰或ABA信号通路的促进均可能激活ABA响应，从而抑制种子的萌发。相反地，植物激素乙烯（ethylene，ET）则被发现在种子萌发中起促进作用，与ABA存在明显的拮抗调控关系。乙烯主要通过受体（包括ETR1）、CTR1、EIN2、EBFs以及核心转录因子EIN3/EIL1等组成一个近似线性的经典信号通路实现对植物生长、发育进行调控。然而，乙烯信号通路如何在种子萌发过程中参与拮抗ABA的作用尚不清楚。

  本研究首先通过种子萌发表型分析，发现乙烯不敏感突变体etr1与ein2的萌发明显晚于另一个乙烯不敏感的双重突变体ein3 eil1及野生型，暗示了乙烯对种子萌发的促进作用依赖于受体ETR1和正调因子EIN2而不依赖于核心转录因子EIN3/EIL1。进一步，在验证乙烯是否通过拮抗ABA信号来促进种子萌发的研究中发现：（1）乙烯拮抗ABA信号的作用依赖于ETR1和EIN2而不依赖于EIN3/EIL1；（2）EIN2/CEND抑制ABA信号不依赖于EIN3/EIL1；（3）EIN2抑制ABA信号依赖于ABI3和ABI5。这些结果均支持乙烯拮抗ABA存在一条不依赖于EIN3/EIL1的非经典作用途径。本研究进一步发现，EIN2可以与一个正调控ABA信号且具潜在组蛋白乙酰化酶活功能的蛋白HLS1直接相互作用。遗传表型分析发现，HLS1位于EIN2的遗传学下游，且对于EIN2抑制ABA信号的功能的发挥是必需的。染色质免疫共沉淀（ChIP）实验发现，HLS1介导的ABI3和ABI5染色质组蛋白乙酰化调控受EIN2的影响。最终，体外蛋白互作实验表明了EIN2能够直接干扰HLS1与其底物组蛋白的结合。这些结果揭示了，EIN2在乙烯拮抗ABA信号的作用中通过干扰HLS1调控的ABI3和ABI5染色质组蛋白乙酰化来促进种子的萌发，而并非经典途径中通过对转录因子EIN3/EIL1活性的调控。

  同时，本研究在种子萌发表型实验中，发现经冷吸胀处理后的etr1、ein2、ein3 eil1与Col-0在萌发上无显著差异。加之已有研究表明冷吸胀的处理会诱导ABA的糖基化代谢发生。因此，这暗示了在乙烯促进种子萌发过程中可能还存在着对ABA糖基化代谢的调控。通过ABA糖基化代谢基因（UGT71B6/7/8）在种子萌发过程中的表达模式分析发现，ein2与ein3 eil1的UGT71B6/7/8基因表达水平变化不足以解释其在种子萌发表型上的差异。而随后的蛋白互作研究发现EIN2与ABA糖基化代谢酶UGT71B6存在直接相互作用，并且体外ABA糖基化的酶活实验结果发现EIN2/CEND可以显著增强UGT71B6重组蛋白的ABA糖基化代谢酶活性。这些结果表明，乙烯可能部分通过EIN2促进ABA的糖基化代谢来促进种子的萌发。

  本研究从多个层次解析了乙烯信号通路在种子萌发过程中参与拮抗ABA的作用机制，为深入阐明植物激素交互调控的分子机制提供了重要线索。同时，本研究也展示了EIN2功能的多样性和复杂性。这些发现对于我们更好地理解植物生长和发育的调控机制具有重要的意义，为提高农作物产量和改善种子耐储藏与萌发齐性的质量提供了新的理论基础和技术支撑。

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