DOI: 10.5061/dryad.rn8pk0pb4
title: Cell size, chlorophyll fluorescence and cadmium bioaccumulation between wild-type and mutant strains of Chlamydomonas reinhardtii upon exposure to cadmium

description: Heavy metal contamination presents a constant threat to biological
 systems. Simultaneously, heavy metals have become one of the major
 contaminants in the aquatic ecosystem. In this regard, the investigation
 of heavy metal-tolerance genes in algae is relevant. Chlamydomonas
 reinhardtii is a unicellular green alga, and an excellent model organism
 used in heavy metal studies. In C. reinhardtii, a novel gene designated as
 Cia7, was hypothesized to play a role in heavy metal homeostasis due to
 CIA7’s conserved cysteine-residue motif. This study compared two strains
 of C. reinhardtii, cc4425, the wild-type possessing the functional CIA7
 protein and cc5013, the mutant strain possessing the disrupted cia7- gene.
 The hypothesis was that the expression of Cia7 contributes to an increased
 cadmium (Cd)-tolerance in C. reinhardtii. The Cd-tolerance would be
 described by physiological markers of microalgae health, and by
 intracellular accumulation of the metal. Methods: The objectives of this
 study were (1) to compare chlorophyll fluorescence and cell size in cc4425
 and cc5013 exposed to Cd2+, and (2) to compare Cd2+ bioaccumulation in
 cc4425 and cc5013 strains in different growth media. Flow cytometry, and
 inductively coupled plasma optical emission spectrometry (ICP-OES)
 analysis were performed. Results: There was no significant statistical
 difference in Cd2+ bioaccumulation between the two strains, cc4425 and
 cc5013, regardless of growth media. However, a statistically significant
 difference in Cd2+ bioaccumulation (p&lt;0.0001) was determined
 between the media (with acetate and without acetate). The cia7- mutant,
 cc5013 was found to be more susceptible to a Cd2+-induced decrease in
 chlorophyll fluorescence and had a reduced cell size compared to cc4425,
 the wild-type strain. Conclusions: These observed differences between the
 strains suggest that CIA7’s biological activity could play a direct or
 indirect role in increasing Cd tolerance in C. reinhardtii.
description: Cell
 growth: Liquid cultures were initiated in 50 mL of TAP medium by using two
 loopfuls (4 mm spherical scraping) of cells for inoculation. Culture
 flasks were placed in an orbital shaker (VWR DS2-500-1) at 130 rpm under
 normal fluorescent room lighting until cultures reached optical density
 (OD650) of 0.800 +/- 0.050, measured using a Spectronic Genesys 8
 spectrophotometer. This OD650 of 0.800 has been calibrated to be equal to
 1.0-3.0 x 10<sup>6</sup> cells/mL which is log phase in C.
 reinhardtii. The data were generated from a 2 x 2 x 2 factorial experiment
 in randomized complete block design.  The factorial arrangement of
 treatments was the result of two levels of strain
 (<i>cc5013</i>, <i>cc4425</i>), two levels of Cd
 concentration (0 µM, 10 µM), and two levels of media (1/4X TAP, Min). In
 this experiment there were 3 blocks. 
 Flow
 cytometry: Three replicates (in this experiment blocks are synonymous to
 replicates) with three trials were performed per concentration and per
 strain. Chlorophyll intensity and cell size were taken using a Cytek DxP8
 FACSCalibur Flow Cytometer acquiring 10,000 events. Chlorophyll intensity
 was determined by utilizing laser BluFL3 with a wavelength filter 695/40
 nm. Cell size was determined as a function of the forward scatter signal
 (FSC). Cell cultures <i>cc4425</i> and
 <i>cc5013</i> were treated with 0, 5, and 10 µM CdCl2 at
 midlog phase (OD650 = 0.400). Samples were analyzed at 24-hour time points
 for a duration of 96 hours via flow cytometry.<br> <br> The
 data were generated from a 2 x 3 x 4 factorial experiment in randomized
 complete block design. The factorial arrangement of treatments was the
 result of two levels of strain (<i>cc5013</i>,
 <i>cc4425</i>), three levels of Cd concentration (0 µM, 5 µM,
 and 10 µM), and four levels of time (1, 2, 3, and 4 days).
 ICP-OES: The treatment period was
 4 days, after which cells reached the logarithmic phase of growth. On the
 4th day, cells were equilibrated at OD650 = 0.800 (an absorbance at log
 phase) and harvested by centrifugation at 3,000 rpm for 10 minutes. The
 pellets were washed twice with millipore H2O, then resuspended, followed
 by centrifugation at 3,000 rpm for 5 minutes to rinse pellets of
 extracellular metals. Washed pellets were desiccated at 100°C, and either
 frozen at -80°C or used for downstream analysis. Dry pellets were
 acidified with 5 mL of HNO3, incubated for 20 minutes, and digested in a
 MARS 6 microwave following the machine’s internal protocol for plant
 material. Following digestion, samples were diluted 1:10, and analyzed for
 cadmium, by ICP-OES (Agilent Varian 720-ES). The data were generated from
 a 2 x 2 x 2 factorial experiment in randomized complete block design. The
 factorial arrangement of treatments was the result of two levels of strain
 (cc5013, cc4425), two levels of media (¼X TAP and Min), and two levels of
 Cd concentration (0 ppb [0 μM] and 1830 ppb [10 μM]).
description: Cell
 growth: The data were generated from a 2 x 2 x 2 factorial experiment in
 randomized complete block design.  The factorial arrangement of treatments
 was the result of two levels of strain (<i>cc5013</i>,
 <i>cc4425</i>), two levels of Cd concentration (0 µM, 10 µM),
 and two levels of media (1/4X TAP, Min). In this experiment there were 3
 blocks. Flow
 cytometry: Three replicates (in this experiment blocks are synonymous to
 replicates) with three trials were performed per concentration and per
 strain. The data were generated from a 2 x 3 x 4 factorial experiment in
 randomized complete block design. The factorial arrangement of treatments
 was the result of two levels of strain (<i>cc5013</i>,
 <i>cc4425</i>), three levels of Cd concentration (0 µM, 5 µM,
 and 10 µM), and four levels of time (1, 2, 3, and 4 days).
 ICP-OES: The data were generated
 from a 2 x 2 x 2 factorial experiment in randomized complete block design.
 The factorial arrangement of treatments was the result of two levels of
 strain (cc5013, cc4425), two levels of media (¼X TAP and Min), and two
 levels of Cd concentration (0 ppb [0 μM] and 1830 ppb [10 μM]).