https://orcid.org/0000-0001-8392-5006
![Sample our Bioscience journals, sign in here to start your access, Latest two full volumes FREE to you for 14 days]({"type":"image" , "src" : "/sda/5925/TOC-Subject-Sample-2021-Bioscience.jpg"})
Abstract
Reports of programmed cell death (PCD) across the taxonomic spectrum of photosynthetic unicellular organisms raise questions concerning its ecological and evolutionary roles. However, prior to ecological studies or evolutionary interpretations, it is essential to document phenotypic changes associated with PCD at the single-cell level, since death-related responses vary between taxa and within a single taxon depending on environmental stimuli. Here, we report responses to rapidly changing light, temperature and fluctuations in macronutrients in the model selenastracean green microalga Ankistrodesmus densus (Chlorophyta, Chlorophyceae, Sphaeropleales). We used stringent, but environmentally appropriate, conditions of prolonged darkness, nitrogen starvation (4 days), heat (1 h at 44°C) and cold shock (3 h at 2 ± 2°C). PCD phenotypes were examined by ultrastructural changes, phosphatidylserine (PS) externalization and DNA degradation. Flow cytometric Annexin V FITC analyses revealed that darkness and nitrogen-deprived cultures had significantly higher proportions of cells with PS externalization compared with controls (p < 0.05). Heat and cold treatments did not affect PS externalization (p = 0.44 and p = 0.99, respectively). Transmission electron microscopy (TEM) of light-deprived cells demonstrated, among other ultrastructural changes, marked cytoplasmic vacuolization suggesting a subtype of PCD known as vacuolar cell death. Nitrogen-starved cells had less vacuolization but presented more typical ultrastructural markers of PCD such as chromatin condensation and marginalization. In contrast, the more severe heat and cold shock treatments resulted in necrotic-like features. These findings suggest that prolonged darkness and nitrogen starvation induce PCD in a small (8.4 3.5 and 7.42 2.6%, respectively) but significant (p < 0.05) fraction of the A. densus population. Documenting these different death-related phenotypes depending on different environmental inducers is essential for interpreting ecological studies. Furthermore, our data support the hypothesis that autophagic/vacuolar cell death (VCD), which is central to organism homeostasis in plants (Streptophyta), occurs in Chlorophyta. VCD probably arose long before the evolution of multicellularity in plants.
HIGHLIGHTS
Darkness and nitrogen deprivation induce different programmed cell death markers in Ankistrodesmus densus;
Plant vacuolar-like cell death occurs in Chlorophyta;
There is crossover between the vacuolar and apoptosis-like death morphotypes.
Acknowledgements
We thank Prof. Stuart Sym from the School of Animal, Plant and Environmental Sciences and Prof. Theresa Coetzer from the School of Pathology both at the University of Witwatersrand, Johannesburg, South Africa, for kindly providing their laboratory facilities and some materials and reagents. We also thank Karen van Niekerk for assistance with the flow cytometry experiments. We are also grateful to Prof. Armando Vieira, Prof. Odete Rocha, Prof. James Jeffrey Morris and Dr. Thaís Garcia for valuable comments and suggestions.
Author contributions
MMBF, ILB & PMD: conceived the original ideas, designed the study; MMBF: Culture experiments, transmission electron microscopy, flow cytometry, data analysis, drafting and editing manuscript; ILB: Main supervisor, data analysis, drafting and editing manuscript; PMD: Co-supervisor, data analysis, drafting and editing manuscript; NEA: culture experiments; AJ: Transmission electron microscopy; HS: flow cytometric analysis.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Supplementary information
The following supplementary material is accessible via the Supplementary Content tab on the article’s online page at https://doi.org/10.1080/09670262.2021.1938240
Supplementary fig. S1. Growth curve of Ankistrodesmus densus under standard growth conditions (see text for more details). The black arrow indicates the day when cells were harvested for the assays of PCD induction.
Supplementary fig. S2. Experimental design for the experiments performed.
Supplementary fig. S3. The relative abundance of each characteristic marker under exposure to standard conditions (control), darkness, nitrogen deprivation, heat and cold shock. Raw data is included in table S6. chm = chromatin; chl = chloroplast; mit = mitochondria; prot = protoplasm.
Supplementary fig. S4. Additional TEM micrographs showing (a) a structure that could be highly disorganized thylakoids (white arrows). The black arrow indicates the electron-dense material inside the vacuoles. (b) A mitochondrion with ‘balloon’-shaped membrane protrusion (black arrow).
Supplementary fig. S5. Agarose gel electrophoresis (1% agarose, 45 min, 80V) of genomic DNA extracted from cultures under control standard conditions, darkness, nitrogen deprivation, heat and cold shock. No fragmentation could be observed.
Supplementary table S1. Shapiro–Wilk test for normality assessment.
Supplementary table S2. Levene’s homogeneity for assessment of homogeneity of variances.
Supplementary table S3. Dunnet’s multiple-comparison post-test fitting the ANOVA with FITC as the outcome variable.
Supplementary table S4. Shapiro–Wilk test for the assessment of normality of the residuals in the ANOVA model with FITC+ and FITC+ PI+ as the outcome variables.
Supplementary table S5. Kruskal–Wallis test for the comparison of means with PI as the outcome variable.
Supplementary table S6. The frequency and relative abundance for each marker under the different treatments. N = number of cells analyzed per treatment; ƒ positive = presence of the marker; ƒ negative = absence of the marker; rel. abundance positive (%) = ƒ positive divided by N; rel. abundance negative (%) = ƒ negative divided by N.
Supplementary table S7. Dunnet’s multiple-comparison post-test fitting the ANOVA with PI as the outcome variable.