Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress

Abstract

Summary 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. H2DCFDA‐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.

Heat stress during flowering in cereals – effects and adaptation strategies

“…Although, there are no studies in this direction involving pistils per se , a plausible hypothesis would be that the above‐mentioned compounds will have a similar alleviation potential in pistils exposed to heat stress (Box 2). Support for this hypothesis, can be drawn from the fact that both pollen and pistil accumulate increased levels of ROS under heat stress, leading to damage in membranes and other organelles (Djanaguiraman et al , ,b; Luria et al , ). Hence, extending or developing similar techniques such as flow cytometry (Luria et al , ) to quantify ROS levels with higher throughput in stigmas/ovaries will help to identify germplasm with enhanced reproductive organs viability under heat stress.…”

“…A significantly positive relationship between pollen germination and spikelet fertility/pod set has been documented in sorghum ( R 2 = 0.79–0.91; Singh et al , ; Chiluwal et al , ), peanut ( Arachis hypogaea L.; R 2 = 0.94; Prasad et al , ), rice ( R 2 = 0.52–0.77; Rang et al , , Shi et al , ) and soybean ( R 2 = 0.52–0.84; Djanaguiraman et al , ). Recent breakthrough achieved by coupling ROS probe with flow cytometry allowed the authors to quantify changes in ROS under heat stress (35°C for 30 min), resulting in a 60% reduction in pollen germination potential in pollen population from different plant species (Luria et al , ). Hence, this recent development provides a unique opportunity to explore genetic diversity in pollen viability from different species, large mapping populations including wild accessions to enhance the male reproductive organ tolerance to severe heat stress conditions.…”

Regulation of High-Temperature Stress Response by Small RNAs

“…The extreme heat exposure damages lipid membranes, proteins, and nucleic acids, etc. Being sessile organisms, plants are especially exposed to extreme temperature fluctuations and heat stress (Luria et al 2019;Mazzeo et al 2018;Mittler et al 2012;Ohama et al 2017;Scharf et al 2012). To mitigate the negative effects, heat stress response (HSR) pathways are initiated.…”

Dynamic imaging in pollen morphology

“…Such indicators include thiazolyl blue tetrazolium bromide 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or 2,3,5-triphenyl tetrazolium chloride (TTC), which stain living pollens dark purple-black or orange (7). A signi cant disadvantage of these histochemical methods is that since they are cytotoxic, it is not possible to perform further studies requiring a living cell after their use (21). As technology advances, microscopic digital imaging techniques are also being used to various studies related to pollens (6,13,22,28).…”