Journal
PLANT JOURNAL
Volume 98, Issue 5, Pages 942-952Publisher
WILEY
DOI: 10.1111/tpj.14286
Keywords
pollen; flow cytometry; reactive oxygen species; dormancy; viability; fluorescence-activated cell sorting; Arabidopsis thaliana; Solanum lycopersicum; heat stress; oxidative stress; technical advance
Categories
Funding
- BARD [IS-4652-13]
- NSF [IOS 1656774]
- UNR Hatch [NEV00384]
- [BSF-2016605]
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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.
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