The Mina and Everard Goodman, Faculty of Life Sciences, Bar Ilan University, Ramat Gan, 5290002, Israel.
Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV, USA.
Plant J. 2019 Jun;98(5):942-952. doi: 10.1111/tpj.14286. Epub 2019 Mar 18.
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 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°C) increased ROS levels, which correlated with a ~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.
有性生殖依赖于植物在受精过程中雄性配子体的适合度。由于花粉的发育对高温和低温极端温度非常敏感，因此评估花粉活力的可靠方法对于研究提高生殖热应激（HS）耐受性非常重要。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 DCFDA 染色）结合流式细胞术快速评估花粉活力的方法。通过流式细胞术分析从小麦和番茄花朵中收获的成熟花粉，我们发现花粉呈双峰分布，分为“低 ROS”和“高 ROS”亚群。荧光激活细胞分选后的花粉萌发实验表明，高 ROS 花粉的萌发频率比低 ROS 花粉高 35 倍，支持这样一种模型，即即使在水合作用后，花的花粉仍有很大一部分处于低代谢或休眠状态。通过氧化应激或抗氧化剂处理对 DCF-荧光的剂量依赖性改变，显示了流式细胞术定量分析大花粉群体中 ROS 动力学的能力。HS 处理（35°C）增加了 ROS 水平，这与花粉萌发率降低约 60%相关。这些结果表明，使用基于流式细胞术的方法研究花粉在应激反应过程中的代谢变化具有潜力。
