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Environmentally induced programmed cell death in leaf protoplasts of Aponogeton madagascariensis

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An Erratum to this article was published on 15 December 2012

Abstract

Within plant systems, two main forms of programmed cell death (PCD) exist: developmentally regulated and environmentally induced. The lace plant (Aponogeton madagascariensis) naturally undergoes developmentally regulated PCD to form perforations between longitudinal and transverse veins over its leaf surface. Developmental PCD in the lace plant has been well characterized; however, environmental PCD has never before been studied in this plant species. The results presented here portray heat shock (HS) treatment at 55°C for 20 min as a promising inducer of environmental PCD within lace plant protoplasts originally isolated from non-PCD areas of the plant. HS treatment produces cells displaying many characteristics of developmental PCD, including blebbing of the plasma membrane, increased number of hydrolytic vesicles and transvacuolar strands, nuclear condensation, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive nuclei, as well as increased Brownian motion within the vacuole. Results presented here for the first time provide evidence of chloroplasts in the vacuole of living protoplasts undergoing environmentally induced PCD. Findings suggest that the mitochondria play a critical role in the cell death process. Changes in mitochondrial dynamics were visualized in HS-treated cells, including loss of mitochondrial mobility, reduction in ΔΨm, as well as the proximal association with chloroplasts. The role of the mitochondrial permeability transition pore (PTP) was examined by pre-treatment with the PTP agonist cyclosporine A. Overall, HS is depicted as a reliable method to induce PCD within lace plant protoplasts, and proves to be a reliable technique to enable comparisons between environmentally induced and developmentally regulated PCD within one species of plant.

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Abbreviations

CMXRos:

Chloromethyl-X-rosamine

CyD:

Cyclophilin D

CsA:

Cyclosporin A

FDA:

Fluorescein diacetate

FITC:

Fluorescent fluorescein isothiocyanate

GA:

Glutaraldehyde

HS:

Heat shock

ΔΨm :

Membrane potential

PTP:

Permeability transition pore

PCD:

Programmed cell death

PI:

Propidium iodide

SAM:

Shoot apical meristem

TUNEL:

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling

TRITC:

Tetramethyl rhodamine iso-thiocyanate

TEM:

Transmission electron microscopy

VDAC:

Voltage-dependent anion channel

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Acknowledgments

The authors thank Dr. M. E. Kane (University of Florida, USA) for providing the original sterile lace plant cultures, Jaime Wertman (Dalhousie University, Canada) for critical review of this article, Bruno J. Roy (Dalhousie University, Canada) for assistance in formatting supplementary videos, Dr. Nancy Dengler (University of Toronto, Canada) and Dr. Wouter G. van Doorn (Wageningen University, The Netherlands) for help throughout the manuscript, and Dr. Jennifer Sheen (Mass General Hospital, Molecular Biology, Boston, MA, USA) for help regarding protoplast isolation. The authors also greatly acknowledge the Canadian Foundation for Innovation (CFI) for the Leaders Opportunity Fund, the Natural Sciences and Engineering Research Council (NSERC) for discovery and equipment grants for A.G., and Dalhousie University for partial doctoral funding for C.L.

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  1. Department of Biology, Dalhousie University, 1355 Oxford Street, Halifax, NS, B3H 4J1, Canada

    Christina E. N. Lord & Arunika H. L. A. N. Gunawardena

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  1. Christina E. N. Lord
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  2. Arunika H. L. A. N. Gunawardena
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Correspondence to Arunika H. L. A. N. Gunawardena.

Additional information

An erratum to this article can be found online at http://dx.doi.org/10.1007/s00425-012-1825-5.

Electronic supplementary material

Below is the link to the electronic supplementary material.

425_2010_1304_MOESM1_ESM.mov

Online Resource 1 Increased brownian motion of small unknown objects in the vacuole of a protoplast HS at 55°C for 20 min. Scale bar = 15μm (MOV 21,505 kb)

425_2010_1304_MOESM2_ESM.mov

Online Resource 2 Protoplasts HS at 55°C for 20 min and allowed to rest for 1 h at room temperature. Note chloroplasts actively entering the vacuole of a cell, followed by slight brownian motion within the vacuole. Chloroplasts are presumably brought into the vacuole to be degraded. Chloroplast material floating freely in cell culture in cellular debris left from enzymatic isolation. Scale bar = 10μm (MOV 11,164 kb)

425_2010_1304_MOESM3_ESM.mov

Online Resource 3a Control protoplast depicting CMXRos stained mitochondria (red) and chlorophyll autofluorescence (green). Note actively streaming mitochondria in control protoplast. Also note the abundance of chloroplasts within the cell. Scale bar = 10μm (MOV 1,318 kb)

425_2010_1304_MOESM4_ESM.mov

Online Resource 3b Protoplast HS at 55°C for 20 min followed by a 30 min rest period at room temperature. CMXRos stained mitochondria (red) and chlorophyll autofluorescence (green). Note there is no actively streaming mitochondria within the HS challenged cell, when compared to the control sample (Online resource 3a). Also note the decrease in chloroplast abundance within the cell. Scale bar = 10μm (MOV 1,136 kb)

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Lord, C.E.N., Gunawardena, A.H.L.A.N. Environmentally induced programmed cell death in leaf protoplasts of Aponogeton madagascariensis . Planta 233, 407–421 (2011). https://doi.org/10.1007/s00425-010-1304-9

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