Abstract
Aponogeton madagascariensis produces perforations over its leaf surface via programmed cell death (PCD). PCD begins between longitudinal and transverse veins at the center of spaces regarded as areoles, and continues outward, stopping several cells from these veins. The gradient of PCD that exists within a single areole of leaves in an early stage of development was used as a model to investigate cellular dynamics during PCD. Mitochondria have interactions with a family of proteases known as caspases, and the actin cytoskeleton during metazoan PCD; less is known regarding these interactions during plant PCD. This study employed the actin stain Alexa Fluor 488 phalloidin, the actin depolymerizer Latrunculin B (Lat B), a synthetic caspase peptide substrate and corresponding specific inhibitors, as well as the mitochondrial pore inhibitor cyclosporine A (CsA) to analyze the role of these cellular constituents during PCD. Results depicted that YVADase (caspase-1) activity is higher during the very early stages of perforation formation, followed by the bundling and subsequent breakdown of actin. Actin depolymerization using Lat B caused no change in YVADase activity. In vivo inhibition of YVADase activity prevented PCD and actin breakdown, therefore substantiating actin as a likely substrate for caspase-like proteases (CLPs). The mitochondrial pore inhibitor CsA significantly decreased YVADase activity, and prevented both PCD and actin breakdown; therefore suggesting the mitochondria as a possible trigger for CLPs during PCD in the lace plant. To our knowledge, this is the first in vivo study using either caspase-1 inhibitor (Ac-YVAD-CMK) or CsA, following which the actin cytoskeleton was examined. Overall, our findings suggest the mitochondria as a possible upstream activator of YVADase activity and implicate these proteases as potential initiators of actin breakdown during perforation formation via PCD in the lace plant.
Highlights
Programmed Cell Death (PCD) in Plants Programmed cell death (PCD) is an active process resulting in the death of cells within an organism and is pervasive throughout eukaryotes
The mitochondria [8],[9], caspases [10],[11] (Cysteine ASPartate-specific proteASES), and the actin cytoskeleton [12] have been implicated in animal PCD, though less is known regarding the dynamics of these cellular components during PCD in plants
Actin breakdown in the center of areoles was extensive in perforation formation leaves (Figure 2C, H and M) and continued to progress to the perforation border throughout leaf development, from perforation expansion (Figure 2D, I and N) to mature leaves (Figure 2E, J and O)
Summary
Programmed Cell Death (PCD) in Plants Programmed cell death (PCD) is an active process resulting in the death of cells within an organism and is pervasive throughout eukaryotes. Developmentally regulated PCD occurs throughout the life cycle [1],[2]. Mitochondria have been implicated in plant PCD [2],[14,15,16,17,18,19] where the release of IMS proteins, including cyt-c, has been detected in a number of systems [15],[20,21,22,23]. Work completed by Balk and Leaver (2003) [23] suggests that cyt-c release in plant systems may not be responsible for PCD activation. The mitochondrial permeability transition pore inhibitor cyclosporine A (CsA) has been shown to prevent PCD in a variety of animal [11],[24] and plant [18],[20],[25] systems
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