A method for analyzing programmed cell death in xylem development by flow cytometry

Abstract

Programmed cell death (PCD) is a genetically regulated developmental process leading to the death of specific types of plant cells, which plays important roles in plant development and growth such as wood formation. However, an efficient method needs to be established to study PCD in woody plants. Flow cytometry is widely utilized to evaluate apoptosis in mammalian cells, while it is rarely used to detect PCD in plants, especially in woody plants. Here, we reported that the xylem cell protoplasts from poplar stem were stained with a combination of fluorescein annexin V-FITC and propidium iodide (PI) and then sorted by flow cytometry. As expected, living cells (annexin V-FITC negative/PI negative), early PCD cells (annexin V-FITC positive/PI negative), and late PCD cells (annexin V-FITC positive/PI positive) could be finely distinguished through this method and then subjected for quantitative analysis. The expression of cell-type- and developmental stages-specific marker genes was consistent with the cell morphological observation. Therefore, the newly developed fluorescence-activated cell sorting (FACS) method can be used to study PCD in woody plants, which will be beneficial for studying the molecular mechanisms of wood formation.

Morpho‐histology of co‐occurrence of somatic embryos, shoots, and inflorescences within a callus of Limonium ‘Misty Blue’

“…During shoot bud differentiation, after periclinal cell division, the precambial cells form xylem mother cells (Du et al, 2023) that are subjected to polyphenol accumulation, cell wall thickening, lignin deposition, and PCD to develop primary vascular elements in the inner portion of the callus (Du and Groover, 2010; He et al, 2022). The role of PCD during the process of xylem differentiation has been reported by several authors (Pyo et al, 2007; Bollhöner et al, 2012; Escamez and Tuominen, 2014; Liu et al, 2023). The dying of neighboring cells accomplished successful somatic embryo and shoot bud development.…”