Developmentally Regulated and Environmentally Induced Programmed Cell Death (PCD) in the Lace Plant (Aponogeton madagascariensis)

Abstract

Programmed cell death (PCD) is pervasive in eukaryotes, playing a fundamental role in development. PCD in animals has been studied in detail, partly due to Caenorhabditis elegans, a worm whose anatomy allowed for the investigation of exactly 131 cells that die via PCD. Elucidating this complex pathway in this simple worm laid the foundation for further insights into mammalian PCD. Overall, less is known regarding PCD in plants, where cell death is broadly separated into developmentally regulated and environmentally induced. The lace plant (Aponogeton madagascariensis) undergoes developmentally regulated PCD to form perforations between longitudinal and transverse veins over its leaf surface. The optimization of protoplast isolation and induced cell death via heat shock (HS) in the lace plant is detailed here. Following HS, protoplasts displayed characteristics of PCD including: Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) positive nuclei, increases in vesicles as well as Brownian motion, and plasma membrane blebbing. Additionally, mitochondrial dynamics were investigated, and a role for the mitochondrial permeability transition pore (MPTP) was indirectly established via cyclosporine A (CsA) experimentation. The main focus of this dissertation was to elucidate cellular dynamics during developmentally regulated PCD in the lace plant, which is visibly discernable during the window stage of leaf development. A single areole within a window stage leaf was further divided into three areas based on the progression of PCD; non-PCD (NPCD) cells, early PCD (EPCD) cells, and late PCD (LPCD) cells. Using this gradient, mitochondria were delineated into four stages based on distribution, motility, and membrane potential. Additionally, it was determined that the MPTP also played a role in developmental lace plant PCD, as inhibition of the pore with CsA not only reduced caspase-like proteases (CLPs) but also stopped perforation formation. Furthermore, the actin cytoskeleton was also investigated, with evidence suggesting it as a possible target for CLPs. The novel use of lace plant leaves for long-term live cell imaging allowed for the establishment of a timeline of cellular events that occur during developmental PCD. Major conclusions of this dissertation reveal various similarities between environmental induced and developmentally regulated PCD in this one plant species.