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The Function of Autophagy in Lace Plant Programmed Cell Death.

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.

SUBMITTER: Dauphinee AN

PROVIDER: S-EPMC6817616 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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The Function of Autophagy in Lace Plant Programmed Cell Death.

Dauphinee Adrian N AN Denbigh Georgia L GL Rollini Alice A Fraser Meredith M Lacroix Christian R CR Gunawardena Arunika H L A N AHLAN

Frontiers in plant science 20191022

The lace plant (<i>Aponogeton madagascariensis</i>) 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 <i>in vivo</i> pharmacological experimentation. ATG8 immunostaini ...[more]

PMID: 31695708

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Project description:Aponogeton madagascariensis, commonly known as the lace plant, produces leaves that form perforations by programmed cell death (PCD). Leaf development is divided into several stages beginning with "pre-perforation" furled leaves enriched with red pigmentation from anthocyanins. The leaf blade is characterized by a series of grids known as areoles bounded by veins. As leaves develop into the "window stage", anthocyanins recede from the center of the areole towards the vasculature creating a gradient of pigmentation and cell death. Cells in the middle of the areole that lack anthocyanins undergo PCD (PCD cells), while cells that retain anthocyanins (non-PCD cells) maintain homeostasis and persist in the mature leaf. Autophagy has reported roles in survival or PCD promotion across different plant cell types. However, the direct involvement of autophagy in PCD and anthocyanin levels during lace plant leaf development has not been determined. Previous RNA sequencing analysis revealed the upregulation of autophagy-related gene Atg16 transcripts in pre-perforation and window stage leaves, but how Atg16 affects PCD in lace plant leaf development is unknown. In this study, we investigated the levels of Atg16 in lace plant PCD by treating whole plants with either an autophagy promoter rapamycin or inhibitors concanamycin A (ConA) or wortmannin. Following treatments, window and mature stage leaves were harvested and analyzed using microscopy, spectrophotometry, and western blotting. Western blotting showed significantly higher Atg16 levels in rapamycin-treated window leaves, coupled with lower anthocyanin levels. Wortmannin-treated leaves had significantly lower Atg16 protein and higher anthocyanin levels compared to the control. Mature leaves from rapamycin-treated plants generated significantly fewer perforations compared to control, while wortmannin had the opposite effect. However, ConA treatment did not significantly change Atg16 levels, nor the number of perforations compared to the control, but anthocyanin levels did increase significantly in window leaves. We propose autophagy plays a dual role in promoting cell survival in NPCD cells by maintaining optimal anthocyanin levels and mediating a timely cell death in PCD cells in developing lace plant leaves. How autophagy specifically affects anthocyanin levels remained unexplained.

Project description:BackgroundDevelopmentally regulated programmed cell death (PCD) is the controlled death of cells that occurs throughout the life cycle of both plants and animals. The lace plant (Aponogeton madagascariensis) forms perforations between longitudinal and transverse veins in spaces known as areoles, via developmental PCD; cell death begins in the center of these areoles and develops towards the margin, creating a gradient of PCD. This gradient was examined using both long- and short-term live cell imaging, in addition to histochemical staining, in order to establish the order of cellular events that occur during PCD.ResultsThe first visible change observed was the reduction in anthocyanin pigmentation, followed by initial chloroplast changes and the bundling of actin microfilaments. At this stage, an increased number of transvacuolar strands (TVS) was evident. Perhaps concurrently with this, increased numbers of vesicles, small mitochondrial aggregates, and perinuclear accumulation of both chloroplasts and mitochondria were observed. The invagination of the tonoplast membrane and the presence of vesicles, both containing organelle materials, suggested evidence for both micro- and macro-autophagy, respectively. Mitochondrial aggregates, as well as individual chloroplasts were subsequently seen undergoing Brownian motion in the vacuole. Following these changes, fragmentation of nuclear DNA, breakdown of actin microfilaments and early cell wall changes were detected. The vacuole then swelled, causing nuclear displacement towards the plasma membrane (PM) and tonoplast rupture followed closely, indicating mega-autophagy. Subsequent to tonoplast rupture, cessation of Brownian motion occurred, as well as the loss of mitochondrial membrane potential (ΔΨm), nuclear shrinkage and PM collapse. Timing from tonoplast rupture to PM collapse was approximately 20 minutes. The entire process from initial chlorophyll reduction to PM collapse took approximately 48 hours. Approximately six hours following PM collapse, cell wall disappearance began and was nearly complete within 24 hours.ConclusionResults showed that a consistent sequence of events occurred during the remodelling of lace plant leaves, which provides an excellent system to study developmental PCD in vivo. These findings can be used to compare and contrast with other developmental PCD examples in plants.

Project description:BackgroundProgrammed cell death (PCD) is the regulated death of cells within an organism. The lace plant (Aponogeton madagascariensis) produces perforations in its leaves through PCD. The leaves of the plant consist of a latticework of longitudinal and transverse veins enclosing areoles. PCD occurs in the cells at the center of these areoles and progresses outwards, stopping approximately five cells from the vasculature. The role of mitochondria during PCD has been recognized in animals; however, it has been less studied during PCD in plants.ResultsThe following paper elucidates the role of mitochondrial dynamics during developmentally regulated PCD in vivo in A. madagascariensis. A single areole within a window stage leaf (PCD is occurring) was divided into three areas based on the progression of PCD; cells that will not undergo PCD (NPCD), cells in early stages of PCD (EPCD), and cells in late stages of PCD (LPCD). Window stage leaves were stained with the mitochondrial dye MitoTracker Red CMXRos and examined. Mitochondrial dynamics were delineated into four categories (M1-M4) based on characteristics including distribution, motility, and membrane potential (??m). A TUNEL assay showed fragmented nDNA in a gradient over these mitochondrial stages. Chloroplasts and transvacuolar strands were also examined using live cell imaging. The possible importance of mitochondrial permeability transition pore (PTP) formation during PCD was indirectly examined via in vivo cyclosporine A (CsA) treatment. This treatment resulted in lace plant leaves with a significantly lower number of perforations compared to controls, and that displayed mitochondrial dynamics similar to that of non-PCD cells.ConclusionsResults depicted mitochondrial dynamics in vivo as PCD progresses within the lace plant, and highlight the correlation of this organelle with other organelles during developmental PCD. To the best of our knowledge, this is the first report of mitochondria and chloroplasts moving on transvacuolar strands to form a ring structure surrounding the nucleus during developmental PCD. Also, for the first time, we have shown the feasibility for the use of CsA in a whole plant system. Overall, our findings implicate the mitochondria as playing a critical and early role in developmentally regulated PCD in the lace plant.

Project description:BackgroundThe lace plant (Aponogeton madagascariensis) is an aquatic monocot that develops leaves with uniquely formed perforations through the use of a developmentally regulated process called programmed cell death (PCD). The process of perforation formation in lace plant leaves is subdivided into several developmental stages: pre-perforation, window, perforation formation, perforation expansion and mature. The first three emerging "imperforate leaves" do not form perforations, while all subsequent leaves form perforations via developmentally regulated PCD. PCD is active in cells called "PCD cells" that do not retain the antioxidant anthocyanin in spaces called areoles framed by the leaf veins of window stage leaves. Cells near the veins called "NPCD cells" retain a red pigmentation from anthocyanin and do not undergo PCD. While the cellular changes that occur during PCD are well studied, the gene expression patterns underlying these changes and driving PCD during leaf morphogenesis are mostly unknown. We sought to characterize differentially expressed genes (DEGs) that mediate lace plant leaf remodelling and PCD. This was achieved performing gene expression analysis using transcriptomics and comparing DEGs among different stages of leaf development, and between NPCD and PCD cells isolated by laser capture microdissection.ResultsTranscriptomes were sequenced from imperforate, pre-perforation, window, and mature leaf stages, as well as PCD and NPCD cells isolated from window stage leaves. Differential expression analysis of the data revealed distinct gene expression profiles: pre-perforation and window stage leaves were characterized by higher expression of genes involved in anthocyanin biosynthesis, plant proteases, expansins, and autophagy-related genes. Mature and imperforate leaves upregulated genes associated with chlorophyll development, photosynthesis, and negative regulators of PCD. PCD cells were found to have a higher expression of genes involved with ethylene biosynthesis, brassinosteroid biosynthesis, and hydrolase activity whereas NPCD cells possessed higher expression of auxin transport, auxin signalling, aspartyl proteases, cysteine protease, Bag5, and anthocyanin biosynthesis enzymes.ConclusionsRNA sequencing was used to generate a de novo transcriptome for A. madagascariensis leaves and revealed numerous DEGs potentially involved in PCD and leaf remodelling. The data generated from this investigation will be useful for future experiments on lace plant leaf development and PCD in planta.

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The Function of Autophagy in Lace Plant Programmed Cell Death.

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The Function of Autophagy in Lace Plant Programmed Cell Death.

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