The Function of Autophagy in Lace Plant Programmed Cell Death.

Adrian N Dauphinee,Christian R Lacroix,Alice Rollini,Arunika H L A N Gunawardena,Meredith Fraser,Georgia L Denbigh

doi:10.3389/fpls.2019.01198

Abstract

The lace plant (Aponogeton madagascariensis) is an aquatic monocot that utilizes programmed cell death (PCD) to form perforations throughout its mature leaves as part of normal development. The lace plant is an emerging model system representing a unique form of developmental PCD. The role of autophagy in lace plant PCD was investigated using live cell imaging, transmission electron microscopy (TEM), immunolocalization, and in vivo pharmacological experimentation. ATG8 immunostaining and acridine orange staining revealed that autophagy occurs in both healthy and dying cells. Autophagosome-like vesicles were also found in healthy and dying cells through ultrastructural analysis with TEM. Following autophagy modulation, there was a noticeable increase in vesicles and vacuolar aggregates. A novel cell death assay utilizing lace plant leaves revealed that autophagy enhancement with rapamycin significantly decreased cell death rates compared to the control, whereas inhibition of autophagosome formation with wortmannin or blocking the degradation of cargoes with concanamycin A had an opposite effect. Although autophagy modulation significantly affected cell death rates in cells that are destined to die, neither the promotion nor inhibition of autophagy in whole plants had a significant effect on the number of perforations formed in lace plant leaves. Our data indicate that autophagy predominantly contributes to cell survival, and we found no clear evidence for its direct involvement in the induction of developmental PCD during perforation formation in lace plant leaves.

Highlights

Autophagy is a major catabolic pathway critical for the survival of eukaryotes as it enables cells to maintain homeostasis under stressful conditions such as nutrient deprivation or starvation (Klionsky et al, 2016)
It has been proposed that there are three classes of autophagy in plants: i) microautophagy which involves the direct passing of contents into a lytic vacuole; ii) macroautophagy is coordinated by evolutionarily conserved AuTophaGyrelated (ATG) proteins and involves either the bulk or selective sequestration of cytoplasmic cargoes into double-membrane vesicles known as autophagosomes, which are delivered to a lytic compartment for degradation; and iii) mega-autophagy, defined by cellular degradation following
To assess whether autophagy occurs during lace plant programmed cell death (PCD), immunostaining of Non-programmed cell death (NPCD) and LPCD cells in fixed window stage leaves was carried out with ATG8 and DyLight 488 antibodies and a negative control with the α-ATG8 preimmune serum (Figure 3A)

Summary

Introduction

Autophagy is a major catabolic pathway critical for the survival of eukaryotes as it enables cells to maintain homeostasis under stressful conditions such as nutrient deprivation or starvation (Klionsky et al, 2016). Autophagy and Lace Plant PCD the release of hydrolases from the vacuole after tonoplast rupture (Van Doorn and Papini, 2013; Marshall and Vierstra, 2018). Of these classes, macroautophagy, hereafter autophagy, is the only well-characterized form of autophagy in plants (Batoko et al, 2017) and is the focus of this study. Because of the significant involvement of autophagy in a wide range of developmental processes and stress responses, there has been a substantial effort to identify chemicals that modulate autophagic flux (Figure 1). Autophagy can be inhibited indirectly toward the end of autophagic flux by halting the breakdown of autophagic bodies via raising the vacuolar pH through the specific inhibition of vacuolar ATPases with concanamycin A (Huss et al, 2002)

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