ABSTRACT

In plants, macroautophagy/autophagy has mainly been associated with stress-related processes but how it impacts normal physiological and developmental processes remains largely unexplored. Pollen germination is the critical first step toward fertilization in flowering plants. It is metabolically demanding and relies on high levels of cytoplasmic reorganization activities to support a dramatic morphological transformation that underlies the development of a pollen tube as the conduit to deliver sperm for fertilization. The role of autophagy in this process remains unclear. Here we provide evidence that pollen germination is accompanied by elevated autophagic activity and successful pollen tube emergence depends on autophagy-mediated cytoplasmic deletion. Genetic and cytological experiments demonstrate that inhibition of autophagy prevents pollen germination while induces the persistence of a layer of undegraded cytoplasm at the germination aperture. Together, these results unveil a novel compartmentalized autophagy. Furthermore, high-throughput comparative lipidomic analyses show that suppressed autophagy-induced inhibition of pollen germination is accompanied by altered profiles of stored and signaling lipids. Proteomic analyses reveal that autophagy likely exert its role in pollen germination via downstream mitochondria-related pathways. These findings reveal a critical role for autophagy in initiating pollen germination and provide evidences for compartmental cytoplasmic deletion being crucial for male fertility.

Abbreviations: 3-MA: 3-methyladenine; ATG: autophagy-related gene; Cer: ceramide; CL: cardiolipin; Con A: concanamycin A; DAG: diradylglycerol; GO: gene ontology; HAG: hour after germination; LC-MS: liquid chromatography-mass spectrometry; MAG: min after germination; MDC: monodansylcadaverine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; PLD: phospholipase D; PtdIns3K: phosphatidylinositol 3-kinase; RT-qPCR: quantitative real-time reverse transcription PCR; TAG: triradylglycerol; TEM: transmission electron microscopy; TMT: tandem mass tagging.

摘要

在植物中，巨自噬/自噬主要与应激相关过程有关，但它如何影响正常的生理和发育过程在很大程度上仍未得到探索。花粉萌发是开花植物受精的关键第一步。它在代谢方面要求很高，并依赖于高水平的细胞质重组活动来支持戏剧性的形态转变，这是花粉管作为输送精子进行受精的管道发展的基础。自噬在这一过程中的作用尚不清楚。在这里，我们提供的证据表明，花粉萌发伴随着自噬活性的升高，并且成功的花粉管出现取决于自噬介导的细胞质缺失。遗传和细胞学实验表明，抑制自噬可防止花粉萌发，同时诱导萌发孔处未降解的细胞质层持续存在。总之，这些结果揭示了一种新的分隔自噬。此外，高通量比较脂质组学分析表明，抑制自噬诱导的花粉萌发抑制伴随着储存和信号脂质的改变。蛋白质组学分析表明，自噬可能通过下游线粒体相关途径在花粉萌发中发挥作用。这些发现揭示了自噬在启动花粉萌发中的关键作用，并为细胞质区室缺失对雄性生育能力至关重要提供了证据。

缩写：3-MA：3-甲基腺嘌呤；ATG：自噬相关基因；Cer：神经酰胺；CL：心磷脂；Con A：刀豆霉素 A；DAG：双二醇甘油；GO：基因本体；HAG：发芽后小时；LC-MS：液相色谱-质谱法；MAG：发芽后分钟；MDC：单丹酰尸胺；PE：磷脂酰乙醇胺；PI：磷脂酰肌醇；PLD：磷脂酶 D；PtdIns3K：磷脂酰肌醇 3-激酶；RT-qPCR：定量实时逆转录PCR；标签：甘油三酯；TEM：透射电子显微镜；TMT：串联质量标记。