Antarctic benthic diatoms after 10 months of dark exposure: consequences for photosynthesis and cellular integrity

Abstract

Antarctic algae are exposed to prolonged periods of extreme darkness due to polar night, and coverage by ice and snow can extend such dark conditions to up to 10 months. A major group of microalgae in benthic habitats of Antarctica are diatoms, which are key primary producers in these regions. However, the effects of extremely prolonged dark exposure on their photosynthesis, cellular ultrastructure, and cell integrity remain unknown. Here we show that five strains of Antarctic benthic diatoms exhibit an active photosynthetic apparatus despite 10 months of dark-exposure. This was shown by a steady effective quantum yield of photosystem II (Y[II]) upon light exposure for up to 2.5 months, suggesting that Antarctic diatoms do not rely on metabolically inactive resting cells to survive prolonged darkness. While limnic strains performed better than their marine counterparts, Y(II) recovery to values commonly observed in diatoms occurred after 4-5 months of light exposure in all strains, suggesting long recovering times. Dark exposure for 10 months dramatically reduced the chloroplast ultrastructure, thylakoid stacking, and led to a higher proportion of cells with compromised membranes than in light-adapted cells. However, photosynthetic oxygen production was readily measurable after darkness and strong photoinhibition only occurred at high light levels (>800 µmol photons m⁻² s⁻¹). Our data suggest that Antarctic benthic diatoms are well adapted to long dark periods. However, prolonged darkness for several months followed by only few months of light and another dark period may prevent them to regain their full photosynthetic potential due to long recovery times, which might compromise long-term population survival.

Full text

Antarctic benthic diatoms after
10 months of dark exposure:
consequences for
photosynthesis and
cellular integrity
Jacob Handy
1
, Desire
´e Juchem
2
, Qian Wang
1
,
Katherina Schimani
3
, Oliver Skibbe
3
, Jonas Zimmermann
3
,
Ulf Karsten
2
and Klaus Herburger
1
*
1
Cell Biology of Phototrophic Marine Organisms, Institute of Biological Sciences, University of
Rostock, Rostock, Germany,
2
Applied Ecology and Phycology, Institute of Biological Sciences,
University of Rostock, Rostock, Germany,
3
Botanischer Garten und Botanisches Museum Berlin, Freie
Universität Berlin, Berlin, Germany
Antarctic algae are exposed to prolonged periods of extreme darkness due to
polar night, and coverage by ice and snow can extend such dark conditions to up
to 10 months. A major group of microalgae in benthic habitats of Antarctica are
diatoms, which are key primary producers in these regions. However, the effects
of extremely prolonged dark exposure on their photosynthesis, cellular
ultrastructure, and cell integrityremainunknown.Hereweshowthatfive
strains of Antarctic benthic diatoms exhibit an active photosynthetic apparatus
despite 10 months of dark-exposure. This was shown by a steady effective
quantum yield of photosystem II (Y[II]) upon light exposure for up to 2.5
months, suggesting that Antarctic diatoms do not rely on metabolically
inactive resting cells to survive prolonged darkness. While limnic strains
performed better than their marine counterparts, Y(II) recovery to values
commonly observed in diatoms occurred after 4-5 months of light exposure in
all strains, suggesting long recovering times. Dark exposure for 10 months
dramatically reduced the chloroplast ultrastructure, thylakoid stacking, and led
to a higher proportion of cells with compromised membranes than in light-
adapted cells. However, photosynthetic oxygen production was readily
measurable after darkness and strong photoinhibition only occurred at high
light levels (>800 µmol photons m
-2
s
-1
). Our data suggest that Antarctic benthic
diatoms are well adapted to long dark periods. However, prolonged darkness for
several months followed by only few months of light and another dark period
may prevent them to regain their full photosynthetic potential due to long
recovery times, which might compromise long-term population survival.
KEYWORDS
Antarctica, dark adaptation, diatoms, photosynthesis, polar night, plastoglobules
Frontiers in Plant Science frontiersin.org01
OPEN ACCESS
EDITED BY
Benoit Schoefs,
Le Mans Universite
´, France
REVIEWED BY
Ansgar Gruber,
Academy of Sciences of the Czech Republic
(ASCR), Czechia
Torsten Jakob,
Leipzig University, Germany
*CORRESPONDENCE
Klaus Herburger
klaus.herburger@uni-rostock.de
RECEIVED 23 October 2023
ACCEPTED 08 March 2024
PUBLISHED 22 March 2024
CITATION
Handy J, Juchem D, Wang Q, Schimani K,
Skibbe O, Zimmermann J, Karsten U and
Herburger K (2024) Antarctic benthic
diatoms after 10 months of dark exposure:
consequences for photosynthesis
and cellular integrity.
Front. Plant Sci. 15:1326375.
doi: 10.3389/fpls.2024.1326375
COPYRIGHT
© 2024 Handy, Juchem, Wang, Schimani,
Skibbe, Zimmermann, Karsten and Herburger.
This is an open-access article distributed under
the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and that the
original publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or reproduction
is permitted which does not comply with
these terms.
TYPE Original Research
PUBLISHED 22 March 2024
DOI 10.3389/fpls.2024.1326375

1 Introduction
Studying organisms with adaptation capacities to extreme
environments is crucial, especially given the current climate change
scenarios that predict dramatic alterations of abiotic conditions in
evolutionary short periods in many environments worldwide (Turner
et al., 2020). This is particularly relevant in vulnerable regions like
Antarctica, where organisms must adapt to prolonged periods of
darkness or continuous light and cope with very low temperatures
(Cottier and Potter, 2020). Antarctica is characterized by extensive ice
and snow cover, and significant light v ariations, includin g long periods
of complete darkness. The lack of light during the three to 6 months of
polar night is a major challenge for primary producers in these regions
(McMinn et al., 2010;Cottier and Potter, 2020). Sea ice can extend the
dark period, reducing sunlight to only 2% of normal levels in some
areas for up to 10 months (Karsten et al., 2019a). Together with
overlyingsnow cover, 99.9%of the remaining surfaceirradiance arecut
out (Petrich and Eicken, 1998), frequently yielding light levels of less
than 5 mmol photons m
−2
s
−1
beneath the sea ice in summer (McMinn
et al., 1999). In parts of Antarctica, such as the Weddell Sea, large areas
of unbroken fast ice can persist for two or more seasons, resulting in
lack of lightfor several months (Peters and Thomas, 1996). Thus, polar
photosynthetic organisms must adapt to darkness/very low light
conditions for periods that can exceed the polar night by months,
but also cope with high light in summer, displaying flexibility in their
photosynthetic efficiency (Longhi et al., 2003;Gomez et al., 2009;
Zacher et al., 2009).
An important group of organisms in this context are diatoms
(Bacillariophyta); these microalgae dominate well-mixed water
columns across all oceans, benthic algal communities of shallow-
water soft bottoms and rock biofilms (Lacour et al., 2019). Diatoms
are responsible for 20-25% of global and up to 50% of marine primary
production, producing more oxygen than all rainforests combined
(Field et al., 1998). At depths of 30 meters, diatoms serve as the main
food source for benthic suspension/deposit feeders, thus occupying a
significant position in polar coastal food webs (Kowalke, 1998;Glud
et al., 2009). Moreover, as primary producers, benthic diatoms and
their planktic counterparts play a crucial role in global carbon dioxide
fixation, binding approximately 45% of marine CO
2
(Mann, 1999).
The physiological stateof polar diatoms surviving in darknessand their
underlying cellular/physiological processes remain largely unstudied.
This is even though diatom long-term dark survival might have been a
key adaptive trait to survive the Cretaceous mass extinction events,
where diatoms experienced a relatively slight generic loss compared to
other planktonic organisms such as coccolithophores (Ribeiro et al.,
2011). Thus, the implicationsof dark survival in diatomsmay therefore
be far reaching, and may be a contributing factor to the dominance of
diatoms within algal communities of polar regions and elsewhere.
Various mechanisms have been observed in a few diatoms to adapt to
the polar night (Reeves et al., 2011). This includes a reducedmetabolic
activity (Palmisano and Sullivan, 1982;Peters and Thomas, 1996),
maintenance of membrane integrity (Karsten et al., 2019a,b),
utilization of stored energy reserves like chrysolaminarin or lipids
(Karstenet al., 2012;Schaub et al.,2017;Juchem et al., 2023),formation
of resting stages (McQuoid and Hobson, 1996;Durbin, 1978),
phototaxis and/or switching to a mixotrophic lifestyle (Hellebust and
Lewin, 1977;Tuchman et al., 2006). A recent study on five Antarctic
benthic diatoms revealed their reliance on storage lipids during a 3-
month dark exposure experiment (Juchem et al., 2023). During this
time, a decline of photosynthetic performance was found for two
species, while all five displayed some chloroplast degradation.
However, the effects of extended dark exposure on photosynthesis,
cellular ultrastructure, and cell integrity in diatoms remain largely
unknown. This is a problem as it prevents us from understanding the
long-term population survival of these key photosynthetic organisms.
For example, whether they can easily restore their populations from a
long-lasting dark-adapted state relying on autophagy and/or lipolysis,
require input of viable cells from neighboring areas with less extreme
light conditions or from dormant resting spores (cysts).
Here, we explored the consequences of prolonged dark
adaptation (10 months) on the photo-physiological performance,
cellular structure, and survivability of five diatom strains isolated
from Antarctic benthic habitats. We hypothesized that when
exposed to light, these algae can rapidly regain photosynthetic
activity, despite prolonged dark exposure, which would suggest a
swift restoration of high metabolic activity.
2 Materials and methods
2.1 Algal origin and cultivation
Five benthic diatoms cultures established at the Botanic Garden
and Botanical Museum Berlin (Germany) were maintained in the in-
house algal culture collection at the University of Rostock. Algal
samples were collected during a field trip to Antarctica in 2020.
Diatom strains isolated from these samples were taxonomically
characterized as described elsewhere (Prelle et al., 2022;Juchem
et al., 2023). The four different sampling sites, three of which marine
and one limnic, are shown in Supplementary Figure S1 and described
in Supplementary Table S1. Information on the isolation and
establishment of unialgal cultures can be found in Juchem et al.
(2023). The diatoms were cultured in flasks containing sterile-
filtered Baltic Sea water enriched with Guillard’sf/2medium
(Guillard and Ryther, 1962)and0.6mMmetasilicate(Na
2
SiO
3
·5
H
2
O). Salinity was set to 33 S
A
for the marine cultures (Navicula
criophiliforma,Chamaepinnularia gerlachei,Melosira sp.) by adding
artificial sea salt (hw-Marinemix®professional, Wiegandt GmbH,
Germany) or to 1 S
A
for limnic cultures (Planothidium wetzelii
D300_015 + 025) by dilution with deionized water. Nutrients were
replenished by changing the mediaregularly. Algaewere maintained at
8° C and 15 mmol photons m
−2
s
−1
under a 16/8-hlight/dark cycleset in
a culture chamber. For light exposure following dark adaptation
experiments, algae were kept at constant 15 mmol photons m
−2
s
−1
at
5° C for up to 5 months.
2.2 Experimental design for dark
incubation and
photosynthesis measurements
Algal flasks were incubated under dark conditions at 5° C for 10
months in a culture chamber. To assess the photosynthetic
Handy et al. 10.3389/fpls.2024.1326375
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performance of dark-adapted diatoms, 150 µl of cell suspension was
transferred to 96-well microplates in a darkened room using a weak
red-light source (PPFD = ~1 µmol m
-2
s
-1
). This was done with two
independent replicates each consisting of 3 technical replicates. To
ensure comparable biomass in wells, cells were concentrated by
filtration. Plates were placed on a cooling block set to the dark
incubation temperature of 5° C or a cool metal shelf to prevent
temperature stress. Next, the effective quantum yield of
photosystem II (Y[II]) of algae was measured, which describes the
efficiency of energy transfer from the antenna to photosystem II and
is used as a proxy for the overall photosynthetic performance of
algae and plants (Herburger et al., 2015). The Y(II) was determined
with a pulse amplitude modulation (PAM) fluorimeter (PAM-2500,
Heinz Walz GmbH, Effeltrich, Germany). The PAM probe was
adjusted ~1 mm above wells containing algae and measurements
were performed through the well lid, avoiding disturbance of algae
or evaporation of media due to opening. The Y(II) was measured
every 15 min for the first 300 min after 10 months of dark
adaptation, followed by ~daily measurements for 2.5 months, and
after 4 and 5 months. As the Y(II) values did not differ significantly
in all five strains during the first 300 min after dark exposure, they
were averaged and expressed as day one of light exposure. Between
these measurements taking up to 5 months, algae were kept in a
culture chamber at 5° C and constant light (15 µmol photons m
-2
s
-
1
). We have chosen these relatively low light intensities to simulate a
continued light limitation as it can occur during, for example, snow/
ice coverage and to allow for comparability with an earlier study one
the five strains investigated (Juchem et al., 2023).
2.3 Photosynthetic oxygen production and
respiratory consumption
The photosynthetic oxygen production and respiratory
consumption was measured in algal cultures that have been dark-
adapted for 10 months and measured again in cultures after 2.5
months of light exposure (15 µmol photons m
-2
s
-1
,5°C).
Photosynthesis-irradiance (PI) curves were recorded at 11
increasing light levels (0~1600 mmol photons m
-2
s
-1
), each light
level was maintained for 30 min (n=4). Algae were enriched with 2
mM NaHCO
3
to avoid carbon deficiency and transferred to
measuring chambers placed on magnetic stirrers. Light was
generated by LEDs (LUXEON Rebel1 LXML-PWN1-0100,
neutral-white, Philips, Amsterdam, Netherlands). Chambers were
connected to an Oxy 4-mini meter (Presens Precision Sensing
GmbH, Regensburg, Germany), and data were recorded using the
software Presens OXY4v2_30. Temperatures in the chambers were
maintained at 5° C using a refrigerated circulator pump system.
More details for this experimental setup can be found elsewhere
(Prelle et al., 2019). After O
2
measurements, algae were transferred
onto Whatman GF/6 glass fiber filters, chlorophyll awas extracted
with 96% EtOH (v/v) and quantified spectrophotometrically
(Helcom, 2019). Oxygen values were expressed as µmol O
2
mg
-1
chlorophyll a h
-1
and PI curve data points fitted using the model of
Walsby (1997) to derive photosynthetic parameters. The risk of
overestimating diatom respiration due to the presence of bacteria in
cultures was considered low, because removing diatoms from
culture medium by sedimentation, followed by measuring O
2
consumption as described above produced only negligible
consumption values.
2.4 Cell integrity and cell number dark-
versus light-adapted
Cell integrity of 10 months dark-adapted samples was
compared with samples exposed to 15 µmol photons m
-2
s
-1
(5°
C) for 8 days and for 2.5 months by using SYTOX Green staining
(n=2), which yields green fluorescence after entering cells with
compromised membranes. Cells were harvested, stained with 0.5
µM SYTOX Green (Catalog no. S7020, Thermo Fisher Scientific,
Waltham, Massachusetts, USA) in culture medium for 5-10 min in
darkness. Stained and unstained cells were counted using an
epifluorescence microscope (BX-51, Olympus/Evident, Hamburg,
Germany) equipped with a GFP filter set. At least 400 unstained and
stained (compromised) cells were counted per group.
Algal growth (cell number) was determined in samples
preserved with 2.5% glutaraldehyde, which were taken after 6, 8
and 10 months of darkness. In a second experiments, cells were
taken at the end of the 10 months dark period, after 8 days of light
exposure and after 2.5 months of light exposure (15 µmol photons
m
-2
s
-1
, 5° C; n=6). Cells were counted in sterile channel slides
(Catalog no. 80601, ibidi, Gräfeling, Germany; 6 channels/slide:
channel volume 30 µl). At least 500 cells per sample were counted
and expressed as cell number per ml.
2.5 Cellular ultrastructure dark- versus
light-adapted
To assess ultrastructural changes occurring after prolonged
darkness, 10 months dark-adapted algal cells were compared with
samples used for Y(II) measurements, which were exposed to 15
µmol photons m
-2
s
-1
at 5° C for 2.5 months. Samples were prepared
for transmission electron microscopy (TEM) as described
previously (Herburger et al., 2015). Briefly, cells were fixed with
2.5% glutaraldehyde in 50 mM cacodylic acid buffer (pH 6.5) for 4 h
and postfixed in 1% OsO
4
in 50 mM cacodylic acid buffer for 22 h.
Fixed cells were dehydrated using a gradient of increasing ethanol,
embedded in modified low viscosity Spurr resin and sectioned using
a Reichert Ultracut S microtome. Ultrathin sections on grids were
counterstained with uranyl acetate, lead citrate and examined with a
transmission electron microscope (Zeiss EM902; 80 kV) equipped
with a 1x2k FT-CCD camera.
2.6 Quantifying lipid droplets in cells
The volume of lipid droplets was quantified using Nile red
staining (Aleman-Nava et al., 2016). Cells were stained in PBS
containing 0.05% nile red crystals (suspended in DMSO) for 10
min, followed by mounting in PBS. Nile red fluorescence was
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visualized using a Keyence BX-800 digital microscope equipped
with a TRITC filter set. Nile red fluorescence images were merged
with corresponding bright fieldimages.Thevolumeoflipid
droplets and cells was measured using Fiji (ImageJ; Schindelin
et al., 2012). Cell volumes were calculated with the assumption of
ellipsoid shapes or calculated using two hemispheres and a cylinder
(Melosira sp.) as done before (Juchem et al., 2023).
3 Results
3.1 Photosynthetic performance –dark-
versus light-adapted
Despite prolonged dark exposure for 10 months, all five strains
showed an appreciable photosynthetic performance during the first
day of light exposure (Figures 1,2). However, the Y(II) differed
among strains on day one (Figure 1). Limnic Planothidium wetzelii
(D300_015) consistently produced the highest Y(II) values (~0.5)
(Figure 1D). In contrast, marine N. criophiliforma,C. gerlachei and
Melosira sp. produced Y(II) values of ~0.3-0.4 (Figures 1A-C).
Limnic P. wetzelii (D300_025) showed intermediate Y(II) values
(Figure 1E). We then followed the Y(II) for 2.5 months, showing
that differences between strains persisted. The two limnic strains
showed the highest Y(II) (Figures 1D,E), while values of N.
criophiliforma,C. gerlachei and Melosira sp. remained lower
throughout the 2.5 months (0.3-0.4) (Figures 1A-C). The Y(II)
kinetics displayed overall constant Y(II) values in most strains
(Figure 1); only C. gerlachei showed a statistically significant Y(II)
decrease to ~70% after 2.5 months when compared to the values
measured on day one (Figure 1B). The Y(II) of other strains either
showed a minor increase (N. criophiliforma,Melosira sp.)
(Figures 1A,C) or decrease (P. wetzelii (D300_015 + 25))
(Figures 1D–F) over the 2.5 months of light exposure. We next
measured the Y(II) after 4 and 5 months of constant light adaptation
and found Y(II) values >0.5 in all five strains (Figure 1F), suggesting
A
B
D
E
F
C
FIGURE 1
Monitoring the effective quantum yield of PSII (Y[II]) over 2.5 months in five Antarctic benthic diatoms (A-E) at 15 µmol photons m
-2
s
-1
(n=6± SD: A,
C; n=9± SD: B,D-F). Samples were taken from cultures after 10 months of dark adaptation. Significances between day one and day 77 of light
exposure are indicated by letters and were determined using a student’s t test (p<0.05). (F) Y[II] after 4 and 5 months at 15 µmol photons m
-2
s
-1
.
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that the photosynthetic apparatus required several months to recover.
Y(II) measurements estimate indirectly, via chlorophyll fluorescence
properties, how efficiently light is utilized for photochemical processes
but providelittle information about direct photosynthetic parameters,
such as O
2
production rates. Even though the Y(II) values at the
beginning and end of the light adaptation phaseof 2.5 months did not
differ significantly in moststrains (Figure 1), it can be assumed that the
assimilation performance of light-adapted variants is significantly
higher than that of dark-adapted ones, for example, due to the
recovery of the reduced chloroplast ultrastructure under light
(see below).
We therefore compared the photosynthetic oxygen production
and respiratory consumption in response to increasing photon
fluence rates (PI curves) between dark adapted (10 months) and
light-adapted cells (2.5 months) (Figures 2A-E). In most strains, the
maximum net primary production (NPP
max
) of dark-adapted
strains was significantly lower than in light-adapted strains, but
never fell below ~60% of the light-adapted cultures (Figure 3A).
Differences in respiration between dark- and light-adapted strains
were less pronounced; while most strains tended to show lower
respiratory O
2
consumptioninthedark,thistrendwasnot
statistically significant (Figure 3A). PI curves suggested that light-
adapted strains lacked strong photoinhibition, even at high photon
fluence rates >1500 µmol photons m
-2
s
-1
, while O
2
production of
dark-adapted N. criophiliforma,C. gerlachei and Melosira sp.
(Figures 2A-C,3A) dropped to 20-30% of their NPP
max
values at
high light. Dark-adapted limnic strains showed less photoinhibition
than their marine counterparts. In all strains, both the light
A
BD
E
C
FIGURE 2
Photosynthetic oxygen production and respiratory consumption in response to increasing PPFD up to ~1600 mmol photons m
−2
s
−1
(PI curves, n=4
± SD). (A-E) five Antarctic benthic diatoms exposed to 10 months of darkness (DA), followed by light adaptation at 15 µmol photons m
-2
s
-1
for 2.5
months (LA). Data points were fitted according to Walsby (1997).
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compensation point (I
c
) and the I
k
point, the ladder expressing the
initial value of light-saturated photosynthesis, were higher in light-
adapted strains, which was statistically significant in Melosira sp.
and P. wetzelii (D300_015 + 025) (Figure 3B). The avalue, which
describes the PI curve slope at limiting photon fluence rates, differed
among dark and light adapted strains, with the exception of P.
wetzelii (D300_015) (Figure 3C). Overall, these PI curve-derived
values suggest a low-light adaptation for all strains tested, indicated
by low I
c
and I
k
values and high avalues.
3.2 Membrane integrity, algal growth,
and ultrastructure
To assess whether membrane integrity contributed to long-term
dark survival, Sytox Green staining was applied. The percentage of
cells with compromised membranes differed strongly between
strains and amounted to ~30-40% in the two limnic strains (P.
wetzelii (D300_015 + 25)) but was much lower in the three marine
strains (2~15%) (Figure 4A). When exposing cultures to light for 8
days, the percentage of compromised cells decreased, and a further
decrease was found in strains adapted to light for 2.5 months, where
the number of compromised cells was <10% in all
strains (Figure 4A).
Counting cells after 6, 8 and 10 months of dark exposure
indicated a slight increase by up to ~4% from month 6 to month
10 (Figure 4B), however, this was not statistically significant
(p<0.05). The cell numbers after dark exposure differed strongly
among treatments, ranging from 1200 to 4300 cells ml
–1
(Figure 4C). Light exposure for 8 days increased the cell number
significantly (p<0.05) in all strains, up to doubling the number in
cultures of marine Melosira sp. and in both limnic P. wetzelii
cultures. Light exposure for 2.5 months produced cell numbers of
>130.000 cells in P. wetzelii (D300_015) cultures (Figure 4C).
TEM was used to examine the cellular ultrastructure of 10
months dark-adapted diatoms and compared to cells adapted to
light for 2.5 months after dark exposure. Here, a marine (N.
criophiliforma)andlimnic(P. wetzelii (D300_015)) strain is
A
BC
FIGURE 3
Comparison of five photosynthetic parameters derived from fitted PI curves (Walsby, 1997)infive Antarctic benthic diatoms (n=4 ± SD). Dark-
adapted (10 months; DA) and light-adapted (2.5 months; LA) strains were compared. (A) Maximum net primary production (NPP
max
) and respiration.
(B) Light compensation (Ic) and Ik point, expressing the initial value of light-saturated photosynthesis. (C) avalue, PI curve slope at limiting PPFD.
Significantly different means between DA and LA groups within each strain are indicated by small letters (underlined for respiration values in (A)).
Comparison was performed by a student’s t test (p<0.05) or one-way ANOVA followed by Tukey’spost hoc test (p<0.05).
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exemplified. Both strains exhibited an appreciable Y(II) after dark
exposure (Figures 1A,D), even though both dark-adapted strains
possessed a significantly reduced and less organized chloroplast
ultrastructure (Figures 5A,C). The thylakoids were scattered, hardly
organized into grana, and numerous plastoglobules were visible
(Figures 5A,C). Moreover, multivesicular bodies (MVBs) were
found in P. wetzelii (D300_015) (Figure 5C). In light-adapted
strains, the chloroplast ultrastructure appeared regenerated as
evident by pronounced thylakoid stacks (grana), especially in P.
wetzelii (D300_015) (Figure 5D), indicating a good structural
condition of the chloroplasts. This is well in line with the
improved photosynthetic performance in light-adapted strains
(Figures 2A,E). Moreover, a significantly lower plastoglobule
density was evident in light-adapted strains. P. wetzelii
A
B
C
FIGURE 4
Membrane integrity and growth rates in five Antarctic benthic diatoms. (A) Cells stained with SYTOX Green (i.e. compromised membranes) as
percentage of total cells counted after dark adaptation for 10 months (DA) and after light adaption (15 µmol photons m
-2
s
-1
) for 8 days (LA 8 days)
and 2.5 months (LA 2.5 months). At least 400 cells were counted per sample (n=2 ± SD). (B) Cell number in cultures during dark adaptation, counted
after 6, 8 and 10 months. (C) Cell number in cultures after dark adaptation for 10 months (DA) and after light adaption (15 µmol photons m
-2
s
-1
) for
8 days (LA 8 d) and 2.5 months (LA 2.5 m.). The increase of cells during light exposure is shown as a percentage above data points n=6 ± SD).
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(D300_015) contained multivesicular bodies and clearly identifiable
cell wall pores (Figure 5C).
Quantifying the total lipid content detectable by nile red after 10
months of dark incubation revealed that cells only contained few
small lipid droplets (Figure 6). Overall, <1% of the cell volume was
occupied by lipid droplets (Figures 6A,C,E,G,I,K) and many cells
showed complete lipid depletion (Figures 6B,D,F,H,J).
4 Discussion
Here we showed that both limnic and marine Antarctic diatoms
from benthic habitats sustain prolonged dark exposure for 10
months and show an appreciable photosynthetic performance as
soon as light becomes available. This might be key for their survival
as phototrophic organisms in polar regions, where they face
extreme light fluctuations. A recent review (Pavlov et al., 2019)
summarized the complex underwater light conditions in the well-
studied Kongsfjorden in the Arctic, which is used here as a
representative example due to limited Antarctic data. Factors like
seasonal changes, clouds, sea ice, and water properties affect light
penetration. Run-off, glacial meltwater, and phytoplankton blooms
add complexity. Similar conditions likely exist in Potter Cove and
other Antarctic Peninsula bays (Hoffmann et al., 2019).
Additionally, glacier retreat and global change introduce more
particles, reducing available light (Hoffmann et al., 2019). Thus,
studying the photophysiological responses of diatoms to extreme
light conditions is timely and crucial to understand their reactions
to changing environments.
4.1 Dark exposure has long-lasting effects
on photosynthesis
A recent study assessed the photosynthetic performance of the
same diatom strains investigated in the present work and found Y
(II) values of ~0.6 for light-adapted strains (Juchem et al., 2023).
Earlier studies on other Antarctic benthic diatoms reported similar
values (0.6-0.7; Longhi et al., 2003;Wulff et al., 2008). While Juchem
et al. (2023) found no decrease of Y(II) after 3 months of darkness,
we showed that 10 months of dark exposure reduced these values in
all strains and no strain exceeded a Y(II) of >0.6, suggesting that
prolonged lack of light impairs the physiological condition of
diatoms. Moreover, a reduced Y(II) remained for at least 2.5
months at light after 10 months of darkness. Recovery was only
possible after 4-5 months of cultivation under light, restoring Y(II)
FIGURE 5
Transmission electron micrographs of (A, B) Navicula criophiliforma and (C, D) Planothidium wetzelii (D300_015) after (A, C) 10 months of dark
exposure, followed by (B, D) 2.5 months at light. (A) Chloroplast with disorganized thylakoid (Ty) membranes, numerous plastoglobulus (PG) and
electron-dense structures (EB); the cell wall (CW) is adjacent to the chloroplast. (B) Chloroplast with few plastoglobules, clearly visible thylakoid
stacks and thylakoid-free spaces (TyF). (C) Chloroplast with numerous plastoglobules, disorganized thylakoids and multivesicular bodies (MVB); the
cell walls exhibit pores. (D) Numerous plastoglobules but with thylakoids organized in stacks. Scale bars: 1 mm.
Handy et al. 10.3389/fpls.2024.1326375
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values to >0.5 in all five strains (Figure 1F). Interestingly, the
photosynthetic oxygen production did not fully reflect Y(II)
measurements. In all investigated strains, NPP
max
values where
relatively low after 10 months of darkness, however, a strong
increase of O
2
production was measurable after 2.5 months of
light exposure. In contrast, the Y(II) hardly changed during these
2.5 months of light exposure or even declined in some strains. It
possible that the recovery after dark exposure strongly improves the
carboxylation capacity, while the light harvesting efficiency –
measured as Y(II) –increases to a lesser extent or requires more
than 2.5 months to recover. This is suggested by PI curve
parameters, where in all 5 strains, NPP
max
increases >1.5-fold
during recovery under light for 2.5 months (Figure 3A); in
contrast, the avalue, which reflects the photosynthetic efficiency
at the light-limiting range, increases <1.5-fold or even declines in
some strains (Figure 3C).
Decreasing NPP
max
in Antarctic benthic diatoms after ~2
months of dark exposure was found before and explained by a
degradation of antennae complexes in the photosynthetic reaction
centers (Wulff et al., 2008), which likely coincides with a decline in
chlorophyll aconcentration (Juchem et al., 2023) and reduction of
light-harvesting complexes and photosystems (Kennedy et al.,
2019). Correspondingly, in all five strains, we found a
significantly lower chlorophyll acontent in dark adapted cells
when compared with cells exposed to light for up to 2.5 months
(Supplementary Figure S2). N. criophiliforma (~7 ng chlorophyll a
per cell; Supplementary Figure S2), Melosira sp. (~4.5 ng
chlorophyll aper cell; Supplementary Figure S2)andbothP.
wetzelii strains (D300_015 + 25; 3.3 and 4.1 ng chlorophyll aper
cell) had similar chlorophyll acontents per cell after 2.5 months
light exposure following a 10 months dark period as reported by
Juchem et al. (2023) for cells from light-adapted cultures. In
contrast to Juchem et al. (2023), we found a ~7 times higher
chlorophyll acontent per cell in C. gerlachei (~3 ng chlorophyll a
per cell). This content is well within the range of values measured in
the other four strains (see Supplementary Figure S2 and Juchem
et al., 2023). Moreover, considering that C. gerlachei showed an
overall similar photophysiological behavior to the four other strains
(e.g., Figure 3) and all five strains were isolated from similar
habitats, similar chlorophyll acontents can be expected. Thus, we
suggest that the differences in C. gerlachei’s chlorophyll acontent
between the present study and Juchem et al. (2023) might be due to
an underestimation in the earlier study. Importantly, a drop in
chlorophyll acontent during dark adaption for up to 10 months was
consistently found in all strains tested (Supplementary Figure S2).
While the chlorophyll acontent strongly decreased during long-
term dark adaption, sampling in months 6-10 of the 10 months
dark exposure period indicated a minor trend for increasing cell
numbers (3-5% from month 8 to 10), which, however, was not
statistically significant.
4.2 Prominent changes of chloroplast
ultrastructure after dark and light adaption
The chloroplast ultrastructure showed clear signs of
degradation, most prominently a strongly reduced and
disorganized thylakoid structure and high abundance of
plastoglobules. These are osmophilic lipid bodies that often occur
in association with stressed chloroplasts (van Wijk and Kessler,
FIGURE 6
Quantification of cellular lipid deposition. Lipid droplets are shown as
bright red fluorescence signal and these images were merged with
bright field images. (A, C, E, G, I) Cells containing lipid droplets, (B,D,F,
H, J) cells without lipid droplets. (K) Amount of lipid droplets in cells (in
% of cell volume), averaging between 0 and 1% in N. criophiliforma,C.
gerlachei and P. wetzelii (D300_025). Melosira sp. and P. wetzelii
(D300_015) showed the highest proportion of cells with complete lipid
droplet depletion. Significant difference between means (n=30 ± SD)
were determined via one-way ANOVA (p<0.01, **). Scale bar = 10 µm.
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2017). Plastoglobules contain thylakoid building blocks and several
proteins that are involved in lipid metabolism or the structural
integrity of plastoglobules, such as fibrillins, which might help
stabilizing the globules and preventing their coalescence (Rottet
et al., 2015). As found for land plants, both darkness or continuous
light for 7 days can down- and upregulate certain fibrillins,
suggesting that the formation dynamics and maintenance of
plastoglobules is strongly linked to the light conditions (Ytterberg
et al., 2006). As plastoglobules appear to actively participate in
thylakoid biogenesis to senescence, it is possible that Antarctic
diatoms “store”most of their thylakoids as plastoglobules during
long-term darkness; as soon as light becomes available, they can be
used as building blocks to restore the grana system, allowing to
regain an appreciable photosynthetic performance. Degradation of
the chloroplast seems to be a key mechanism in benthic diatoms to
survive the polar night; dark exposure condenses the chloroplast in
Antarctic diatoms (Wulff et al., 2008), and reduces the chloroplast
lengths by up to 50% in Arctic diatoms (Karsten et al., 2012,2019b),
but recovery is possible after some hours in light. Even though
strongly reduced, after 10 months of darkness, thylakoids are still
visible in plastoglobules-rich chloroplasts, helping to explain why
appreciable photosynthetic oxygen production is measurable
immediately upon light exposure.
Another possibility is that chloroplast lipids are converted to
plastoglobules and ultimately transferred to the cytoplasm,
degraded by autophagy to mobilize energy reserves and allow
cells to remain metabolically active during prolonged darkness.
Such mechanism was found in green algal model system
Micrasterias (Schwarz et al., 2017). However, cells adapted to
darkness for 10 months showed a high abundance of
plastoglobules (Figure 5), suggesting that plastoglobules do not
serve as a major energy resource. Measurable photosynthesis in
diatoms investigated in the present study suggest that none of them
relied on metabolically inactive resting cells to survive prolonged
darkness. Similar results were found for the Antarctic diatom
Thalassiosira tumidu, which survived for 9 months in darkness in
the vegetative state and regained an appreciable growth rate when
exposed to light again (Peters and Thomas, 1996). Considering the
dynamic degradation processes in chloroplasts during darkness, it is
remarkable that diatoms can maintain an appreciable
photosynthetic capacity in the absence of light for 10 months. On
the other hand, full recovery of the photosynthesis proxy Y(II) took
several months, suggesting that diatom populations facing long-
term dark exposure can never regain their full photosynthetic
potential after darkness and when exposed to relatively low light
(15 µmol photons m
-2
s
-1
) for less than 4-5 months. Nevertheless, all
strains produced appreciable cell numbers during 2.5 months under
such light conditions (see. Figure 4C), which might be explained by
theirlowlightrequirementsasPIcurveparameterssuggest;
however, it is possible that higher irradiances (e.g. 50 µmol
photons m
-2
s
-1
;Peters and Thomas, 1996) would allow for a
quicker recovery of the Y(II).
Long-term dark survival can be enhanced significantly by the
formation of resting cells (cysts; McMinn and Martin, 2013),
allowing cells to survive with minimal effort on cellular energy.
When buried in benthic sediments, such cells enable Melosira sp. to
survive for 20 years (Sicko-Goad et al., 1986) and certain other
marine diatom genera (e.g., Chaetoceros,Skeletonema) can even
survive for up to 100 years as resting cells and germinate as soon as
brought back to light (Lundholm et al., 2011). The latter taxa exhibit
a planktonic life-style, and Smetacek (1985) discussed the mass
sinking of such diatom cells after a bloom event as a transition from
a growing phase to a resting stage in the life history of these protists.
Mass sinking into the dark region of the water column is considered
as ecologically important since these diatom cells often retain
viability under cold conditions, which is particularly true in
coastal regions (Smetacek, 1985). Such deep-water resting stages
are considered as seeding population when freshly recycled
nutrients are available (Smetacek, 1985). This makes diatom
resting cells a potential source of phytoplankton to their overlying
waters as soon as disturbance releases them from dormant state and
exposes them to light (Lewis et al., 1999).
4.3 Membrane integrity differs between
marine and limnic strains
Few studies investigated the cell and membrane integrity of
polar benthic diatoms. In a dark exposure experiment, Arctic
Surirella cf. minuta cultures exhibited ~20% dead cells after 1
month incubation (Karsten et al., 2019a). In contrast, after 5
months of dark exposure, >95% of Arctic Navicula directa cells
were still alive as indicated by their intact membrane systems
(Karsten et al., 2019b). This suggests that some diatom cells can
cope better with long-term darkness than others, which we also
confirmed after 10 months of dark-exposure of Antarctic strains.
Interestingly, both limnic strains [P. wetzelii (D300_015 + 25)]
exhibited a higher proportion of cells with compromised
membranes (~1/3) than their marine counterparts, in some of
which ~95% of cells were not compromised (Figure 4A).
However, dark exposure for 3 months led to a higher proportion
of compromised cells in these strains, spanning ~20-60% (Juchem
et al., 2023). It is possible that these marine diatoms undergo a
lengthy adaption process to darkness, allowing them to establish a
higher membrane integrity over time. In contrast, the limnic strains
appear to have a lower cell lifespan (more cells with compromised
membranes) yet higher cell proliferation rate as indicated by higher
growth rates after a dark period (Figure 4C). This might suggest
different strategies to maintain appreciable cell numbers during
dark and light periods, i.e. improving membrane integrity during
darkness or allowing for higher proliferation rates in the light.
When transferred to light after 10 months of darkness, the cell
number recovered relatively slowly during a period of 8 days and
was higher after 2.5 months, suggesting that cell division processes
require time to recover under light. Overall, these observations are
in line with earlier studies suggesting that diatoms cultured in
darkness for several months do not grow; however, cells entered
proliferation immediately after light exposure (Miquel, 1892).
Lewin (1953) found that in the presence of glucose, several
Navicula spp. show “unlimited”growth in the dark, at least for 2
months. While these studies added carbon sources to culture media
to support heterotrophic growth, we did not supplement glucose or
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other compounds for heterotrophic growth, also preventing
bacterial growth in non-axenic cultures. This suggests that the 5
Antarctic diatom strains tested can sustain cellular survival in the
vegetative state, while showing relatively high respiration as
indicated by a respiration rate of ~15-20 µmol O
2
mg
-1
chlorophyll ah
-1
(Figure 3A), which was measured immediately
after the 10 months dark-period. Assuming a respiratory quotient
of 0.8 (Lewin et al., 1955) and constant respiration rates, this
would result in a respiratory CO
2
production of up to ~16
µmol CO
2
mg
-1
chlorophyll a h
-1
and thus a carbon loss of ~192
µg C mg
-1
chlorophyll a h
-1
, resulting in a total carbon loss of ~1400
Cmg
-1
chlorophyll ain 10 months. Such carbon loss might be even
higher, because diatom cultures exposed to darkness for 3 months
showed a higher respiratory O
2
consumption (up to ~36 µmol O
2
consumption per mg chlorophyll aand hour; Juchem et al., 2023).
In part, this might be explained by co-occurring heterotrophic
bacteria in cultures, however, we consider this risk relatively low as
removing diatom cells from cultures showed low O
2
consumption,
assuming that co-occurring bacteria do not strongly stick to
diatoms and remained in cultures. P. wetzelii (D300_015 + 025)
showed relatively high respiration rates, which coincided with a
high proportion of dead cells in cultures (~33%; Figure 4A) and
cells might have used organic matter released from dead cells as a
substrate to sustain a relatively high heterotrophic metabolism. In
general, high respiration levels during prolonged darkness of polar
night are not uncommon (Berge et al., 2015) and our data confirm
that this can also be true for Antarctic diatoms. Antarctic diatoms
can use their lipid storage pools during long-term darkness. During
3 months of darkness, marine N. criophiliforma,C. gerlachei, and
Melosira sp. used up ~90% of their lipids, while the content
decreased by less than 40% in limnic P. wetzelii (D300_015 +
025) (Juchem et al., 2023). As confirmed by nile red staining, the
cellular lipid pool is depleted after 10 months of dark inception,
where only <1% of the cellular volume remained occupied by lipid
droplets in all 5 strains. Drawing energy from the lipid pool via
autophagy or lipophagy during darkness was also suggested for the
polar diatom Fragilariopsis cylindrus (Joli et al., 2023). Another
source for energy in benthic diatoms can be anaerobic respiration
using intercellular storage pools of NO
3-
to reduce them to NH
4
(Kamp et al., 2011). However, depletion of NO
3-
occurs very
rapidly, suggesting that the energy provided by dissimilatory
NO3- reduction might be sufficient for entering a resting stage
but cannot directly fuel long-term dark survival (Kamp et al., 2011).
In conclusion, we found that long-term dark-exposure strongly
reduced the photosynthetic performance and chloroplast
ultrastructure in all five Antarctic benthic diatoms. Nevertheless,
algae showed appreciable photosynthesis rates as soon as darkness
ceased, while full recovery of photosynthesis required several
months under light. Future studies may explore the molecular
mechanism leading to the observed long-term down-regulation of
photosynthesis. In an ecological context, it will be interesting to
explore whether prolonged darkness of several months, for example
due to polar night and ongoing ice coverage, followed by a short
light periods (2-4 months) and another dark period, such as the
next polar night, would allow for long-term population survival or
whether such light periods are too short to fully restore
photosynthesis, accumulate sufficient assimilates, and sustain the
next period(s) of darkness.
Data availability statement
The original contributions presented in the study are included
in the article/Supplementary Material. Further inquiries can be
directed to the corresponding author.
Author contributions
JH: Writing –review & editing, Writing –original draft,
Visualization, Validation, Investigation, Formal analysis,
Conceptualization. DJ: Resources, Writing –review & editing,
Validation. QW: Visualization, Investigation, Formal analysis,
Writing –review & editing, Validation. KS: Writing –review &
editing, Validation, Resources, Investigation. OS: Writing –review
& editing, Validation, Resources, Investigation. JZ: Writing –review
& editing, Validation, Resources, Funding acquisition. UK: Writing
–review & editing, Validation, Resources, Project administration,
Methodology, Funding acquisition, Conceptualization. KH: Writing
–review & editing, Writing –original draft, Visualization, Validation,
Supervision, Resources, Project administration, Methodology,
Investigation, Funding acquisition, Formal analysis, Data
curation, Conceptualization.
Funding
The author(s) declare that financial support was received for the
research, authorship, and/or publication of this article. This study
was funded by the DFG under the reference HE 9354/3-1 (project
no. 520755331) and within the framework of the SPP 1158
Antarktisforschung by the DFG under grant numbers ZI 1628/2-1
and KA899/38-1.
Conflict of interest
The authors declare the research was conducted in the absence
of any commercial or financial relationships that could be construed
as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations,
or those of the publisher, the editors and the reviewers. Any product
that may be evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the publisher.
Handy et al. 10.3389/fpls.2024.1326375
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Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fpls.2024.1326375/
full#supplementary-material
SUPPLEMENTARY FIGURE 1
Isolation sites of diatoms in Potter Cove. (A) Map of Antarctica. (B) Overview
King George Island). (C) Potter cove with 3 marine (APC06, APC14, APC41)
and 1 limnic (APC18) diatom isolation sites. Maps were obtained via
Google Maps.
SUPPLEMENTARY FIGURE 2
Chlorophyll content in 5 Antarctic diatom strains after 6 and 10 months of dark
adaptation (DA) and 8 daysand 2.5 months of light adaption (LA). Comparison of mean
values was done using one-way ANOVA followed by Tukey’spost hoc test (p<0.05)
and different small letters indicate significantly different values for each diatom strain.
SUPPLEMENTARY TABLE 1
List of diatom samples collected at Potter Cove, King George Island
(Antarctica) with information about the 5 strains and the sampling site.
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