JXB Advance Access originally published online on February 27, 2009
Journal of Experimental Botany 2009 60(3):815-828; doi:10.1093/jxb/ern330
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© 2009 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Different ways to die: cell death modes of the unicellular chlorophyte Dunaliella viridis exposed to various environmental stresses are mediated by the caspase-like activity DEVDase
1Department of Ecology, Faculty of Sciences, University of Málaga, Bvd. Louis Pasteur s/n, E-29071 Málaga, Spain
2Department of Renal Diseases and Hypertension, School of Medicine, University of Colorado Health Sciences Center, 4200 E. 9th Av. Denver, CO 80262, USA
3Department of Pathology, School of Medicine, University of Colorado Health Sciences Center, 4200 E. 9th Av. Denver, CO 80262, USA
* To whom correspondence should be addressed: E-mail: segovia{at}uma.es
Programmed cell death is necessary for homeostasis in multicellular organisms and it is also widely recognized to occur in unicellular organisms. However, the mechanisms through which it occurs in unicells, and the enzymes involved within the final response is still the subject of heated debate. It is shown here that exposure of the unicellular microalga Dunaliella viridis to several environmental stresses, induced different cell death morphotypes, depending on the stimulus received. Senescent cells demonstrated classical and unambiguous apoptotic-like characteristics such as chromatin condensation, DNA fragmentation, intact organelles, and blebbing of the cell membrane. Acute heat shock caused general swelling and altered plasma membrane, but the presence of chromatin clusters and DNA strand breaks suggested a necrotic-like event. UV irradiated cells presented changes typical for necrosis, together with apoptotic characteristics resembling an intermediate cell-death phenotype termed aponecrosis-like. Cells subjected to hyperosmotic shock revealed chromatin spotting without DNA fragmentation, and extensive cytoplasmic swelling and vacuolization, comparable to a paraptotic-like cell death phenotype. Nitrogen-starved cells showed pyknosis, blebbing, and cytoplasmic consumption, indicating a similarity to autophagic/vacuolar-like cell death. The caspase-like activity DEVDase was measured by using the fluorescent substrate Ac-DEVD-AMC and antibodies against the human caspase-3 active enzyme cross-reacted with bands, the intensity of which paralleled the activity. All the environmental stresses tested produced a substantial increase in both DEVDase activity and protein levels. The irreversible caspase-3 inhibitor Z-DEVD-FMK completely inhibited the enzymatic activity whereas serine and aspartyl proteases inhibitors did not. These results show that cell death in D. viridis does not conform to a single pattern and that environmental stimuli may produce different types of cell death depending on the type and intensity of the stimulus, all of which help to understand the cell death-dependent and cell death-independent functions of caspase-like proteins. Hence, these data support the theory that alternative, non-apoptotic programmed cell death (PCDs), exist either in parallel or in an independent manner with apoptosis and were already present in single-celled organisms that evolved some 1.2-1.6 billion years ago.
Key words: Aponecrosis-like, apoptosis-like, autophagic/vacuolar cell death, caspase-like, cell death, DEVDase activity, Dunaliella, environmental stress, microalgae, necrosis-like, paraptosis-like, phytoplankton, TUNEL
Received 4 July 2008; Revised 20 November 2008 Accepted 24 November 2008