Functional Characterization and Phenotyping of Protoplasts on a Microfluidics-Based Flow Cytometry

Abstract

A better understanding of the phenotypic heterogeneity of protoplasts requires a comprehensive analysis of the morphological and metabolic characteristics of many individual cells. In this study, we developed a microfluidic flow cytometry with fluorescence sensor for functional characterization and phenotyping of protoplasts to allow an unbiased assessment of the influence of environmental factors at the single cell level. First, based on the measurement of intracellular homeostasis of reactive oxygen species (ROS) with a DCFH-DA dye, the effects of various external stress factors such as H2O2, temperature, ultraviolet (UV) light, and cadmium ions on intracellular ROS accumulation in Arabidopsis mesophyll protoplasts were quantitatively investigated. Second, a faster and stronger oxidative burst was observed in Petunia protoplasts isolated from white petals than in those isolated from purple petals, demonstrating the photoprotective role of anthocyanins. Third, using mutants with different endogenous auxin, we demonstrated the beneficial effect of auxin during the process of primary cell wall regeneration. Moreover, UV-B irradiation has a similar accelerating effect by increasing the intracellular auxin level, as shown by double fluorescence channels. In summary, our work has revealed previously underappreciated phenotypic variability within a protoplast population and demonstrated the advantages of a microfluidic flow cytometry for assessing the in vivo dynamics of plant metabolic and physiological indices at the single-cell level.

Effect of Mercuric Ion on the Oxidant Response and Calcium Ion in the Protoplasts of Arabidopsis Thaliana

“…The fluorescence probe that is generally used to detect intracellular ROS is DCFH-DA. In addition, DCFH is oxidized by ROS to produce the fluorescent substance DCF and the fluctuation of ROS can be investigated by detecting the fluorescence intensity of DCF (Dai et al, 2022). In this study, DCFH-DA was used to label ROS in Arabidopsis protoplast cells.…”

Microfluidics identify moso bamboo and henon bamboo by leaf protoplast subpopulations with single‐cell analysis

“…The plant origins, efficacies, and traditional uses of these Article Related Abbreviations: CVM, Cramer Von Mises; DC-iDEP, direct-current insulator-based dielectrophoresis; DEP, dielectrophoresis; EK, electrokinetic; EKMr, electrokinetic mobility to dielectrophoresis mobility ratio; EP, electrophoresis; FITC, fluorescein isothiocyanate; FSC, forward scatter; IR, infrared. herbal medicines differ [3]. They have not been officially recognized due to a lack of adequate or accepted research methodology for their authentication [4].…”

“…Motivated by economic profits, high‐quality herbal medicines may be adulterated with lower‐quality herbals having a less expensive price to defraud the consumer [1, 2]. The plant origins, efficacies, and traditional uses of these herbal medicines differ [3]. They have not been officially recognized due to a lack of adequate or accepted research methodology for their authentication [4].…”