Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress
Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress
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DOI：
10.1111/tpj.14286
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发表时间：
2019-06-01
期刊：
影响因子：
7.2
通讯作者：
Miller, Gad
中科院分区：
文献类型：
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作者：
Luria, Gilad;Rutley, Nicholas;Miller, Gad
开花植物的有性繁殖取决于受精过程中雄配子体的适应性。由于花粉发育对极端高温和低温高度敏感，因此评估花粉活力的可靠方法对于提高生殖热应激 (HS) 耐受性的研究非常重要。在这里，我们描述了一种使用活性氧（ROS）探针二氯二氢荧光素二乙酸酯（即 H(2)DCFDA 染色）结合流式细胞术快速评估花粉活力的方法。在使用流式细胞术分析从拟南芥和番茄花中收获的成熟花粉时，我们发现花粉双峰分布为“低ROS”和“高ROS”亚群。荧光激活细胞分选后的花粉萌发测定表明，高 ROS 花粉的萌发频率比低 ROS 花粉高 35 倍，这支持了这样一种模型：即使在水合后，花粉的很大一部分仍处于低代谢或休眠状态。 DCF 荧光响应氧化应激或抗氧化治疗的剂量依赖性变化显示了使用流式细胞术量化大型花粉群体内 ROS 动态的能力。 HS 处理（35 摄氏度）增加了 ROS 水平，这与花粉萌发率减少了 60% 类似。这些结果证明了使用基于流式细胞术的方法来研究花粉应激反应期间代谢变化的潜力。
Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H(2)DCFDA-staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into 'low-ROS' and 'high-ROS' subpopulations. Pollen germination assays following fluorescence-activated cell sorting revealed that the high-ROS pollen germinated with a frequency that was 35-fold higher than the low-ROS pollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose-dependent alterations in DCF-fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35 degrees C) increased ROS levels, which correlated with a similar to 60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry-based approaches to investigate metabolic changes during stress responses in pollen.