International Journal of
Molecular Sciences
Article
Metabolic Adjustment of High Intertidal Alga Pelvetia
canaliculata to the Tidal Cycle Includes Oscillations of Soluble
Carbohydrates, Phlorotannins, and Citric Acid Content
Renata Islamova 1 , Nikolay Yanshin 1 , Elizaveta Zamyatkina 1 , Ekaterina Gulk 1 , Ekaterina Zuy 1 , Susan Billig 2 ,
Claudia Birkemeyer 2,3 and Elena Tarakhovskaya 1,4, *
1
2
3
4
*
Citation: Islamova, R.; Yanshin, N.;
Zamyatkina, E.; Gulk, E.; Zuy, E.;
Billig, S.; Birkemeyer, C.;
Tarakhovskaya, E. Metabolic
Adjustment of High Intertidal Alga
Pelvetia canaliculata to the Tidal Cycle
Includes Oscillations of Soluble
Carbohydrates, Phlorotannins, and
Department of Plant Physiology and Biochemistry, Faculty of Biology, Saint Petersburg State University,
Universitetskaya Nab., 7/9, 199034 Saint Petersburg, Russia; renata.tag.isl@gmail.com (R.I.)
Faculty of Chemistry and Mineralogy, Leipzig University, 04103 Leipzig, Germany;
birkemeyer@chemie.uni-leipzig.de (C.B.)
German Center for Integrative Biodiversity Research (iDiv) Halle-Leipzig-Jena, 04103 Leipzig, Germany
Vavilov Institute of General Genetics, Saint Petersburg Branch, Russian Academy of Science, Universitetskaya
Nab., 7/9, 199034 Saint Petersburg, Russia
Correspondence: e.tarahovskaya@spbu.ru or elena.tarakhovskaya@gmail.com
Abstract: The brown alga Pelvetia canaliculata is one of the species successfully adapted to intertidal
conditions. Inhabiting the high intertidal zone, Pelvetia spends most of its life exposed to air, where
it is subjected to desiccation, light, and temperature stresses. However, the physiological and
biochemical mechanisms allowing this alga to tolerate such extreme conditions are still largely
unknown. The objective of our study is to compare the biochemical composition of Pelvetia during
the different phases of the tidal cycle. To our knowledge, this study is the first attempt to draft
a detailed biochemical network underneath the complex physiological processes, conferring the
successful survival of this organism in the harsh conditions of the high intertidal zone of the polar
seas. We considered the tide-induced changes in relative water content, stress markers, titratable
acidity, pigment, and phlorotannin content, as well as the low molecular weight metabolite profiles
(GC-MS-based approach) in Pelvetia thalli. Thallus desiccation was not accompanied by considerable
increase in reactive oxygen species content. Metabolic adjustment of P. canaliculata to emersion
included accumulation of soluble carbohydrates, various phenolic compounds, including intracellular
phlorotannins, and fatty acids. Changes in titratable acidity accompanied by the oscillations of citric
acid content imply that some processes related to the crassulacean acid metabolism (CAM) may be
involved in Pelvetia adaptation to the tidal cycle.
Citric Acid Content. Int. J. Mol. Sci.
2023, 24, 10626. https://doi.org/
10.3390/ijms241310626
Keywords: Pelvetia; brown algae; desiccation; tidal cycle; phlorotannins; metabolomics; citric acid;
salicylic acid; titratable acidity; CAM-photosynthesis
Academic Editor: Se-Kwon Kim
Received: 31 May 2023
Revised: 17 June 2023
Accepted: 22 June 2023
Published: 25 June 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
The intertidal zone is a part of a shoreline lying between the high water and low water
marks and thus covered with water at high tide and exposed to the air at low tide. The
relative duration of underwater and waterless periods varies for the different subzones
(low, mid, or high intertidal), and also depends on the tidal amplitude, which is maximal
during spring tides and minimal during neap tides. Typically, the intertidal zones of the
seas host rich and diverse ecosystems, though such a specific habitat may be considered
among the most stressful ones due to the recurring changes of environmental conditions.
Thus, seaweeds inhabiting this zone are constantly exposed to fluctuations of humidity,
light intensity, temperature, salinity, nutrient availability, etc. [1,2].
The brown alga Pelvetia canaliculata is one of the species that has successfully adapted
to the intertidal conditions. This miniature fucoid is abundant on the shores of the Arctic
Int. J. Mol. Sci. 2023, 24, 10626. https://doi.org/10.3390/ijms241310626
https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2023, 24, 10626
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and North Atlantic seas, growing as a narrow belt on the wave-exposed rocks of the high
intertidal zone. Pelvetia thalli lack airbladders and look like dichotomously branching
bushlets of 2–15 cm high, firmly attached to the substratum with a small basal disc. The
mature thalli can bear multiple receptacles on the tips of the branchlets. The color of the
Pelvetia thalli varies from olive-yellow (wet) to dark brown (dried) [3]. An interesting
ecophysiological feature of P. canaliculata is its intimate association with the endophytic
ascomycete fungus Stigmidium ascophylli. Living in the form of such association called
mycophycobiosis gives Pelvetia some lichen features and is supposed to contribute to the
high tolerance of this organism [4,5].
Inhabiting the high intertidal zone, Pelvetia systematically faces extreme differences
in environmental conditions. This part of the shore is covered with seawater for no more
than 8 h per day during the spring tides and may remain dry for several days during
the periods of the neap tides, thus Pelvetia spends most of its life exposed to air, where
beyond desiccation, it is also subjected to high light and temperature stress in the daytime and sharp drops of temperature in the night. Adaptive responses of P. canaliculata
to such a complicated environment are expected to require systematic rearrangements of
all key physiological processes according to the phase of the tidal cycle. However, the
physiological and biochemical mechanisms allowing this alga to tolerate such conditions
are still largely unknown, as very few studies of intertidal seaweeds focus on Pelvetia [6,7].
These works and the studies of the mid and low intertidal fucoids (Fucus vesiculosus,
F. serratus, Ascophyllum nodosum) suggest that fluctuations of physiological parameters and
biochemical composition induced by the tidal cycle include changes in photosynthetic
performance and pigment content [7–9], nutrient uptake [10], reactive oxygen species
metabolism [7,11], osmolyte accumulation [1], and secondary metabolism [12]. Moreover,
intertidal fucoids were shown to use an adaptive strategy similar to CAM-photosynthesis,
storing CO2 in the form of organic acids during submersion and then consuming it in
the Calvin cycle during the first hours of air exposure [13,14]. Phlorotannins, the dominating secondary metabolites of brown algae, also contribute to resilience of intertidal
fucoids [15,16]. These phenolic compounds have considerable UV-protective and antioxidant capacities, and thus may provide chemical defense against high light and temperature
stress during the low tide [12]. All these data imply that the physiological adjustment
of P. canaliculata to the variation of environmental conditions imposed by the tidal cycle is accompanied with dramatic changes of cell metabolism, which remain obscure at
the moment.
Here, we used a GC-MS-based metabolomics approach to explore the peculiarities
of Pelvetia biochemistry in the context of the unique tolerance to recurring desiccation
demonstrated by this species. This method allows simultaneous analysis of hundreds
of low-molecular weight primary and secondary metabolites, which makes it an ideal
method for comprehensive characterization of such potentially complicated processes.
Metabolomic studies of brown macrophytes are still rare. The most detailed investigations
were carried out on Ectocarpus siliculosus [17,18] and F. vesiculosus [19,20], whereas most
other metabolite profiling studies focus on dominating metabolites and compounds having
a potential applied relevance [21–23]. In our study, we discuss and relate the results of
metabolite profiling of Pelvetia to data on relative water content, stress markers, titratable
acidity, pigment, and phlorotannin content.
Therefore, the objective of the current study is to compare the biochemical composition
of the thalli of P. canaliculata during the different phases of the tidal cycle. To our knowledge,
this study is the first attempt to draft a detailed biochemical network underneath the
complex physiological processes conferring the successful survival of this organism in the
harsh conditions of the high intertidal zone of the polar seas.
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2. Results
ffff
2.1. Water Content and Titratable Acidity in the Algal Thalli at Different Tidal Phases
Relative water content (RWC) in the thalli of P. canaliculata changed dramatically
during the tidal cycle (Figure 1). When under water (HW and ET phases), the thalli had
RWC of ~100%, but after the emersion, this parameter decreased by more than 3.5 times
and then was stable during the whole period of air exposure (LW and RT phases). For
the details of the local tidal regime and the sampling timepoints, please see Section 4.1 of
Materials and Methods.
Figure 1. Dynamics of relative water content in the thalli of Pelvetia canaliculata during the tidal
cycle. HW—high water; ET—ebb tide; LW—low water; RT—rising tide. Means are given with ±SD
ff
tttt letters indicate significant
ff
ff different
ff differences (p < 0.05, Student’s t-test).
(n = 12–20);
The changes in RWC of algal thalli were accompanied by the oscillations of titratable
acidity (Figure 2). This parameter was maximal (~144 µmol −−H+ g−1 DW) during the
underwater phases (HW and ET) and decreased significantly (p < 0.05) at the waterless
phases (LW and RT).
Figure 2. Dynamics of titratable acidity of the thalli of Pelvetia canaliculata during the tidal cycle.
HW—high water; ET—ebb tide; LW—low water; RT—rising tide. Means are given with ±SD
ff
tttt
ff
ff different
ff differences (p < 0.05, Student’s t-test).
(n = 10–12);
letters indicate significant
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ffff
2.2. Hydrogen Peroxide and Malondialdehyde Content in the Algal Thalli at Different Tidal Phases
Hydrogen peroxide accumulated in the cells of P. canaliculata to its maximum
− −
(0.19 µmol
g−1 DW) soon after submersion (HW phase), then tended to decrease (though
not significantly) till the ET phase and dropped abruptly after emersion of the thalli (LW
phase) (Figure 3). This reduction in H2 O2 content was accompanied by the increase in
− −1
malondialdehyde (MDA) level, which was the highest (74 nmol g−
DW) at the LW phase
(Figure 4). In progression of the air exposure, there were no considerable changes in H2 O2
and MDA content in Pelvetia cells (Figures 3 and 4). After submersion, the MDA amount
gradually decreased, reaching minimal values during the ET phase, when the alga spent
~4–4.5 h under water (Figure 4).
Figure 3. Dynamics of hydrogen peroxide content in the thalli of Pelvetia canaliculata during the tidal
cycle. HW—high water; ET—ebb tide; LW—low water; RT—rising tide. Means are given with ±SD
(n = 8–10); different
letters
(p < 0.05, Student’s t-test).
ffff
tttt indicate significant differences
ffff
Figure 4. Dynamics of malondialdehyde (MDA) content in the thalli of Pelvetia canaliculata during
the tidal cycle. HW—high water; ET—ebb tide; LW—low water; RT—rising tide. Means are given
ffff
tttt
ffff
with ±SD (n = 6); different
letters
indicate significant differences
(p < 0.05, Student’s t-test).
2.3. Pigment Content in the Algal Thalli at Different Tidal Phases
The most considerable changes in pigment content in the thalli of P. canaliculata during
the tidal cycle related to the amounts of chlorophyll a and the product of its degradation,
pheophytin a (Figure 5). Chlorophyll a content was maximal (0.8–0.9 mg g−1 DW) during
ff
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−
the underwater phases (HW and ET) and dropped almost two-fold during the waterless
phases (LW and RT). In contrast, the amount of pheophytin was the lowest (1.8% of
summarized contents chlorophyll a and pheophytin a) at the end of the underwater phases
(ET) and increased dramatically in progression of the air exposure.
Figure 5. Dynamics of photosynthetic pigments content in the thalli of Pelvetia canaliculata during the
tidal cycle. HW—high water; ET—ebb tide; LW—low water; RT—rising tide. Means are given with
±SD (n = 6); different letters indicate significant differences (p < 0.05, Student’s t-test).
Int. J. Mol. Sci. 2023, 24, 10626
ff
tt
ff
6 of 21
Chlorophyll c contributed to 13% of the total chlorophyll amount during the HW
phase and up to 23% during the LW phase. Generally, the tide-induced oscillations of the
content of this additional chlorophyll were not as clear as those of chlorophyll a, though
chlorophyll c also tended to accumulate in the submerged thalli, reaching its maximum at
the ET phase (Figure 5).−The total amount of carotenoids in the cells of P. canaliculata varied
from 0.43 to 0.64 mg g−1 DW, gradually decreasing during the underwater phases (HW
and ET) and increasing during the waterless phases (LW and RT) (Figure 5).
ff
2.4. Phlorotannin Content in the Algal Thalli at Different Tidal Phases
ff
Total phlorotannin content in the thalli of P. canaliculata at different
tidal phases varied
ff
from ~4.5 to 7.4% DW (Figure 6). Notably, different tide-induced behavior was shown
for the two subcellular pools of these phenolic metabolites: intracellular phlorotannins,
located in physodes, and phlorotannins associated with the cell wall (CW). The amount of
intracellular phlorotannins was highest in the air-exposed thalli (LW and RT phases) and
sharply decreased after ~2–2.5 h of immersion (HW phase). The dynamics of CW-bound
phlorotannin content was just the opposite: these molecules accumulated in the Pelvetia
thalli under water (up to 15.6% of total phlorotannins) and had the lowest content at the
LW phase, after 3.5–4 h of air exposure (Figure 6).
Figure 6. Dynamics of intracellular and cell wall (CW)-bound phlorotannin content in the thalli of
Pelvetia canaliculata during the tidal cycle. HW—high water; ET—ebb tide; LW—low water; RT—
rising tide. Means are given with ±SD (n = 8–12); different letters indicate significant differences
(p < 0.05, Student’s t-test).
ff
tt
Int. J. Mol. Sci. 2023, 24, 10626
ff
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ff
2.5. Metabolic Profiles in the Algal Thalli at Different Tidal Phases
GC-MS-based metabolite profiling of P. canaliculata revealed 132 compounds identified by retention indices and spectral similarity (excluding 110 unknowns), represented
α
by 66 carbohydrates (sugars, polyols, and sugar acids),
17 amino acids including three
tt ones (5-hydroxypipecolic acid, α-aminoadipic acid, ornithine), 11 ornon-proteinogenic
ganic acids, 9 fatty acids and their derivatives, 9 phenolic compounds (phloroglucinol
and its dimers, homogentisic acid, salicylic acid etc.), and a miscellaneous group of
metabolites including fucosterol, tocopherols, ascorbic acid, squalene, urea, etc. (Table S1).
The most abundant compounds in the metabolite profiles were mannitol, volemitol, citric acid, fucosterol, threonic acid, and several polyols and sugars, which could not be
ff
identified unambiguously.
The main changes of metabolite profiles of the P. canaliculata thalli during different
tidal phases were revealed by a partial least squares discriminant analysis (PLSDA), where
the first three components explain ~60% of the variance (Figure 7). The first component
divides all the samples into two distinct groups, separating underwater phases (HW and
ET) from waterless phases (LW and RT). Analysis of the component loadings showed
that the metabolites contributing significantly to component 1 are mostly carbohydrates
(fructose, myo-inositol, threonic acid), amino acids (threonine, glutamine, proline, etc.),
phenolic compounds (phloroglucinol, 5-hydroxypipecolic, and homogentisic acids) and, to
a lesser extent, organic acids (citric and isocitric acids) and components of lipid metabolism
(cholesterol, glycerol-myristate). Component 2 allows separating HW and ET phases.
ff The
most significant constituents of this component are photosynthetic products, different
sugars and polyols (glucose, sucrose, dulcitol), ffthe amino acid phenylalanine, and several
compounds related to lipid metabolism. The difference between the metabolite profiles
of Pelvetia thalli during LW and RT phases was only revealed at the level of component 3
(Figure 7). Among the compounds showing high loadings for this component are salicylic
tt
acid, sugar acids (gluconic and galacturonic), several organic acids (malic, succinic), fatty
acids, and squalene.
Figure 7. Sample scores for the first three components derived from PLSDA of the relative metabolite
concentrations in the thalli of Pelvetia canaliculata during the tidal cycle. HW—high water; ET—ebb
tide; LW—low water; RT—rising tide.
The specific ‘metabolite signatures’ of Pelvetia passing through different
tidal phases
ff
are illustrated by a heatmap (Figure 8). During the HW phase, cells featured relatively
low contents of carbohydrates, compounds related to lipid metabolism and phenolics,
but accumulated free amino acids (except for phenylalanine) and several organic acids of
TCA cycle (citric, isocitric, cis-aconitic, malic). Among phenolic compounds, the notable
exception is homogentisic acid, which showed the highest relative content at this tidal
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phase. In progression of the underwater period (ET phase), the changes in the carbohydrate
profiles occurred: several sugars and polyols were up-regulated (glucose, sucrose, polyol
RI2858), whereas the content of the other compounds (inositols, dulcitol, threonic acid)
decreased (Figure 8).
Figure 8. A heatmap of significantly (p < 0.05) changing key metabolites detected in the thalli of
Pelvetia canaliculata during the tidal cycle. Mean area values of five samples are presented on a log10
scale. Arrows mean direction of the tidal current.
The emersion and transition to the air-exposed conditions lead to dramatic rearrangements of the Pelvetia metabolome. The most prominent features of metabolite profiles
related to the LW phase are an accumulation of low molecular weight carbohydrates, phenolic compounds (including tocopherols), and dicarboxylic acids in Pelvetia cells, and a
decrease in relative amounts of free amino acids and citric and isocitric acids (Figures 8–10).
In addition, homogentisic acid behavior is inconsistent with the other phenolic metabolites,
demonstrating a significant (p < 0.05) decrease in relative content after the emersion of the
algal thalli (Figures 8 and 10).
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Figure 9. Presumable consumption pathways of TCA cycle metabolites in the cells of Pelvetia
ff different tidal phases. Blue lines indicate the pathways activated in the water
canaliculata during
(high water, HW and ebb tide, ET), red lines indicate the pathways activated in the air (low water, LW
and rising tide, RT). Direct reactions are presented as straight lines, and reactions involving several
steps are presented as dashed lines. Metabolites which were not determined are labeled in grey. Bars
represent the means ± SD. Arbitrary units are normalized peak areas of extracted ion chromatograms.
Figure 10. Scheme of the salicylic acid and tocopherols biosynthesis. Direct reactions are presented as
straight lines and reactions involving several steps are presented as dashed lines. HW—high water,
ET—ebb tide, LW—low water, RT—rising tide. Bars represent the means ± SD. Arbitrary units are
normalized peak areas of extracted ion chromatograms.
tt
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During the RT phase, after 7–8 h of air exposure, fatty acids and squalene were considerably up-regulated together with several sugars (glucose, trehalose, sucrose). Meanwhile
the contents of free amino acids and most organic acids (in particular, the tricarboxylic
ones) kept decreasing, reaching the minimum values at this tidal phase (Figures 8 and 9).
While most of phenolic compounds maintained their relatively high level throughout both
waterless tidal phases (LW and RT), the content of salicylic acid, after a sharp 10-fold
increase during the first hours of air exposure, dropped down with progressing desiccation
(Figures 8 and 10). The only carbohydrate demonstrating a clear down-regulation at the RT
phase was gluconic acid (Figure 8).
3. Discussion
In this study, we addressed, to our knowledge for the first time, the metabolic adjustment of the high intertidal brown alga P. canaliculata to the recurrent changes of environmental conditions during the tidal cycle. For all intertidal organisms derived from the sea, such
as algae, the most crucial event is the emersion during the waterless period of the cycle [1].
For Pelvetia, these periods of desiccation last for at least 7–8 h, even during the spring
tides and up to four days during the neap tides. From our data, we can see the dramatic
changes in RWC in the Pelvetia cells (Figure 1). Notably, more than a three-fold decrease in
RWC already occurs 3–3.5 h after emersion and then this parameter is stable for the next
several hours in the air. The rehydration is also very fast, and, as known from the literature
data, the complete recovery of key physiological processes took no more than 2 h, even
after more prolonged and severe desiccation [24]. For most terrestrial plants, a decrease of
RWC below 60% results in permanent wilting [25,26]; thus RWC dynamics in Pelvetia with
regular drops to 28% (as in our study) or lower values (e.g., in the period of neap tides)
resembles that of the poikilohydrous photosynthetic organisms, such as the resurrection
plants or lichens [27,28]. Indeed, such extreme desiccation tolerance may arise from the
mycophycobiosis state of P. canaliculata and ubiquitous presence of the endophyte fungus
in its thallus [4]. This hypothesis is supported by another example of intimate association
between a brown alga and a fungus: Petroderma maculiforme (Ishigeales) lichenized by the
ascomycete Wahlenbergiella tavaresiae inhabits the high intertidal zone (the same biotope
as P. canaliculata), whereas the free-living individuals of P. maculiforme typically grow in
the low intertidal or even subtidal zone [29–31]. It is suggested that symbiosis with W.
tavaresiae gave the alga higher resistance to desiccation and wave action and thus promoted
the colonization of a new habitat [32].
The poikilohydrous nature of P. canaliculata has an important consequence from the
perspective of the methodology used to study this organism. Considerable changes of
thallus moisture make any measurements of its physiological or biochemical parameters
calculated on the fresh weight (FW) basis inadequate, as it is frequently conducted for other
algae or vascular plants. In our study, we calculated all the parameters per dry weight,
while FW-based calculations were used in several earlier studies of intertidal seaweeds
(e.g., [7,9]), which hampers data comparison.
The other specific feature of Pelvetia, which should be taken into account while discussing its biochemical composition, is the ubiquitous presence of the hyphae of the
endophyte fungus in its thalli [4,5]. Though the total biomass of the fungus is several orders
of magnitude lower than that of the alga, some especially abundant fungal metabolites may
be detected by GC-MS analysis and contribute to the results. In this study, we did not find
any metabolites in Pelvetia sample that were specific for fungi and not reported for algae.
Considering the repeating of waterless periods in the life of Pelvetia and its rapid full
recovery after rehydration, we wondered whether air exposure is a true physiological stress
for this organism, resulting in significant damage to cellular constituents. As we took the
LW and RT samples when these tidal phases occurred in the daytime in warm and sunny
weather, desiccation was accompanied by light and temperature impacts. Altogether these
factors may lead to disbalance in reactive oxygen species (ROS) metabolism, oxidative
stress, and loss of integrity of cell macromolecules and membranes. As hydrogen peroxide
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and MDA contents are widely accepted biochemical markers of oxidative stress and level
of lipid peroxidation [33,34], we monitored these parameters in Pelvetia cells throughout
the tidal cycle (Figures 3 and 4). Both H2 O2 and MDA content data measured in our
study were within the range of the values reported for different macroalgae (including
other populations of P. canaliculata) and aquatic vascular plants [7,35–39]. According to
the literature data, in seaweeds, different abiotic stresses are accompanied by considerable
changes in ROS production and MDA level. Thus, H2 O2 production enhanced more than
thrice in the thalli of F. evanescens, F. distichus, and Scytosiphon lomentaria after desiccation
stress [9,40] and in Ectocarpus siliculosus after heavy metal stress [41]. MDA content was
shown to increase by one and a half time in F. vesiculosus thalli exposed to enhanced
temperature [39] and in Macrocystis pyrifera treated with high temperature and UV-light [42].
In the current study, there was a slight increase (by ~25%) of MDA level in Pelvetia cells
after the emersion, but it did not grow further, either in progression of the waterless
period, nor after rehydration (Figure 4), and thus can hardly be interpreted as a serious
threat to cell membranes’ integrity. As for H2 O2 content dynamics, surprisingly, Pelvetia
cells accumulated more of this ROS when underwater, compared to the waterless period
(Figure 3), and again the amplitude of these oscillations was not large enough to consider
them as clearly stress-related. One of the main sources of H2 O2 in the cells of plants and
algae are photosynthetic processes [34], and rehydration of Pelvetia thalli is accompanied
by a considerable increase in photosynthesis rate [6,24], which may account for the rise of
H2 O2 production in the submersed thalli.
The only clear evidence of damage to functional molecules in Pelvetia cells during the
waterless tidal phases came from the analysis of the pigment content (Figure 5). The amount
of chlorophyll a decreased almost two-fold after the emersion, and, moreover, the content
of pheophytin increased dramatically to the end of the waterless period. Pheophytin is one
of the first products of chlorophyll degradation, and its accumulation refers to either cell
senescence or damage of the light-harvesting apparatus [43]. Relative pheophytin content
was shown to increase in different algae and vascular plants subjected to osmotic, high
light, and heavy metal stresses [44–46]. Notably, soon after immersion, pheophytin content
in Pelvetia cells declines to a level not exceeding 5% of the sum of chlorophyll + pheophytin,
thus showing the high sustainability of the pigment metabolism.
As a whole, analysis of the stress markers dynamics during the tidal cycle showed that
7–8 h long emersion even in combination with warm and sunny weather may be considered
as a relatively mild stress, if any at all, for the White Sea populations of P. canaliculata.
Apparently, the metabolic protection mechanisms in this alga (and, presumably, also in
its fungus symbiont) are well tuned to preclude the dramatic changes of ROS level, lipid
peroxidation intensity, or irreversible damage of photolabile molecules. Thus, it might be
more correct to consider waterless periods of Pelvetia life not as a permanent stress, but
as a specific metabolism state, characterized by appropriate biochemical rearrangements
conferring the successful survival.
The results of PLSDA with evident separation of the HW+ET samples from the LW+RT
samples at the level of the first component (Figure 7), clearly indicate that during the tidal
cycle, Pelvetia passes through two distinctive metabolism states depending on the current
environment: water or air. According to the literature data, very soon after the submersion
and rehydration, P. canaliculata begins to actively photosynthesize [6,24], which corresponds
well with our data showing the increase in the chlorophyll content between the RT and HW
phases (Figure 5). As we cannot see a considerable accumulation of the early photosynthetic products, such as glucose, mannitol, and other low molecular weight carbohydrates
in Pelvetia cells at the HW phase (Figure 8), we may suppose that these compounds are
immediately consumed for the biosynthesis reactions. The spectrum of the synthesized
products may include laminaran (specific brown algal storage polysaccharide) and the
other structural and functional molecules, such as amino acids, proteins, and cell wall constituents. Relatively high amounts of free amino acids (glutamic acid, glutamine, aspartic
acid, threonine, etc.) imply active efflux of ketoacids from the TCA cycle to form different
Int. J. Mol. Sci. 2023, 24, 10626
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amino acids and then proteins (Figures 8 and 9). The most stable intermediates of TCA cycle
(citric, isocitric, malic acids) are also present in relatively high concentrations indicating the
active cell respiration. The drop of the intracellular phlorotannin content accompanied by
increase in the CW-bound phenolics may be the result of the intensification of the thallus
growth and CW formation at the underwater tidal phases. During biosynthesis of brown
algal cell walls, intracellular phlorotannins are secreted into the forming apoplast where
they cross-link with the alginate molecules, thus conferring the wall rigidification [38,47].
Apparently, at the end of the underwater period (ET phase), the photosynthetic machinery of Pelvetia gets optimized: the cells contain a minimal amount of pheophytin,
the content of chlorophyll c increases, and contents of H2 O2 and MDA tend to decrease
(Figures 3–5). Accumulation of glucose, together with the metabolically linked sugars
(sucrose, mannose), may indicate the stabilization of the intracellular laminaran pool
and slow its further replenishment (Figure 8). Interesting features of the metabolite
profiles of the ET samples are a sharp increase in phenylalanine content and gradual
accumulation of tocopherols (Figures 8 and 10). These changes find their logical explanation in the subsequent events, connected with the emersion of Pelvetia thalli. Our data
show that adaptation of P. canaliculata to air exposure includes extensive metabolic rearrangements (Figures 8–10). Notably, one of the most affected compounds, transiently
up-regulated (fold change > 10) at the beginning of the waterless period, was salicylic acid
(Figures 8 and 10). Salicylic acid is a biologically active compound of phenolic nature,
known as a signal molecule, involved in the induction of diverse adaptive reactions to
abiotic and biotic stresses in both vascular plants and algae [48–50]. Thus, in the red
macrophyte Neoporphyra haitanensis expression level of the genes related to the salicylate
biosynthesis and content of endogenous salicylic acid increased considerably after exposure
to high temperature, high light, desiccation, and ultraviolet irradiation [49,51]. Moreover,
pretreatment with salicylic acid was shown to improve thermotolerance of the brown and
red seaweeds due to activation of the enzymes contributing to antioxidative defense and
stabilizing the content of reactive oxygen species in cells [52,53]. Wang et al. [51] showed
that in the cells of N. haitanensis, biosynthesis rate and content of salicylic acid increased
sharply in an hour after beginning the exposure to high temperature, desiccation, and
high light, but returned to the initial level after 6 h of treatment. This coincides with our
data: in P. canaliculate, salicylic acid content grows dramatically in the beginning of the
waterless period (LW phase), but then drops down in progression of the air exposure (RT
phase) (Figures 8 and 10). Thus, we may suggest, that the spike of this phytohormone is a
signal activating protective mechanisms conferring Pelvetia resilience during the waterless
part of its life. To our knowledge, in our study, the endogenous content of salicylic acid in
P. canaliculata was analyzed for the first time. The values measured in the underwater
samples (12–16 nmol g−1 DW) are relatively high and comparable to the highest values
reported for the other macroalgae, including the intertidal ones [54,55]. It is possible that
the enhanced level of salicylate biosynthesis is one of the biochemical adaptations of Pelvetia to the high intertidal habitat. Salicylic acid can be synthesized via two independent
pathways, operated by isochorismate synthase or phenylalanine ammonia lyase (PAL) as
the key enzymes. Both these routes are supposed to occur in the vascular plants and algae,
though their relative contributions to the level of this hormone in the cells vary considerably in different species [51,56,57]. The key intermediate of PAL-dependent biosynthesis
of salicylic acid is phenylalanine, and in P. canaliculata, accumulation of this amino acid
precedes the spike of salicylate during the tidal cycle (Figures 8 and 10). Thus, we suggest
that in Pelvetia salicylic acid is mainly formed via the PAL-dependent pathway.
Besides the salicylate precursor, during the underwater period, Pelvetia cells also
accumulated the metabolically linked precursors of tocopherols, such as tyrosine and homogentisic acid (Figure 10). A considerable down-regulation of these metabolites at the
LW phase coincided with the increase in tocopherols content. Due to their potent antioxidant capacities, tocopherols may contribute to the prevention of the oxidative stress in
the air-exposed Pelvetia thalli. The other phenolic compounds, both low molecular weight
Int. J. Mol. Sci. 2023, 24, 10626
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(phloroglucinol, ditertbutylphenol, etc.) and polymeric (intracellular phlorotannins), also
accumulated in Pelvetia cells during the waterless tidal phases (Figures 6 and 8). These
metabolites are known for their UV-protective and radical-scavenging activities [16,58].
The increase in amounts of tocopherols and phenolic compounds was earlier shown in F.
vesiculosus thalli exposed to enhanced temperature [59]. Notably, only the intracellular fraction of phlorotannins, but not the CW-bound one, increased in the air-exposed Pelvetia cells
(Figure 6). This corresponds well to the results of our previous studies, where we showed
that these two subcellular pools of phlorotannins differ in their molecular composition,
and that free radical scavenging activity of the CW-bound phlorotannins of P. canaliculata
is substantially lower compared to that of the intracellular phlorotannins [60,61]. Generally, among studied fucoid algae, P. canaliculata is distinguished by its unusually complex
phlorotannin profile, which was suggested to reflect both adaptation of this species to high
intertidal conditions and interaction with the endophyte fungus [60,62].
The adaptive responses of plants and algae to desiccation typically include the upregulation of osmolytes, such as soluble sugars and polyols. In Pelvetia, we could also see
the accumulation of diverse low-molecular weight carbohydrates (~2–9-fold for a broad
range of identified and unknown sugars and polyols) during the waterless tidal phases.
Interestingly, one of the most affected carbohydrate derivatives was gluconic acid, showing
a spike at the LW phase (Figure 8). The biological function of this metabolite in plants and
algae is still very poorly studied, though there are several reports of its up-regulation in
response to different stresses as well as to exogenous salicylic acid treatment in vascular
plants [63–66]. We suggest that the spike of gluconic acid in Pelvetia cells may indicate a
transient switch of glucose catabolism from glycolysis to the oxidative pentose phosphate
pathway (PPP), which was shown to activate under oxidative stress conditions and to
contribute to stabilization of the cell redox status [67,68]. Gluconic acid can be produced
due to glucose oxidation by NADP+ glucose dehydrogenase in the gluconate shunt, an
alternative route for the entrance of glucose into the PPP, which was shown to function in
plants and algae [69]. According to our data, increase in gluconate content coincided with
the transient drop of glucose level (Figure 8).
The biochemical rearrangements in the emersed Pelvetia thalli included decrease in
free amino acid content and gradual accumulation of lipid exchange metabolites. Such
events may be explained by the changes in the TCA cycle configuration, the more that
relative amounts of the organic acids also changed dramatically after emersion (Figure 8). It
is known that intermediates of the TCA cycle such as citric, α-ketoglutaric, and oxaloacetic
acids can act as “switch valves” for the efflux of metabolites from the cycle for various
biosynthetic processes [70]. Apparently, in the submerged thalli, there was no considerable
efflux of citric acid from the cycle; meanwhile, α-ketoglutarate and oxaloacetate were
largely consumed for amino acid biosynthesis. On the contrary, after the emersion, citric
acid might start to leave the cycle providing material for the synthesis of phlorotannins,
squalene, and fatty acids, resulting in a two-fold decrease in total citrate content (Figure 9).
Both fatty acids and squalene are membrane constituents, so they may be used to maintain
cell membranes’ integrity during desiccation of the thalli. The spikes of succinate and
fumarate at the LW phase may be the result of limited flow of tricarboxylic acids and
general inhibition of cell respiration occurring during the waterless period [24,71]. One
more organic acid found in Pelvetia extracts (citramalate) co-behaved with succinate and
fumarate (Figure 8). This metabolite, previously only described in microorganisms, was
recently found in several plants, brown algae, and filamentous fungi [72–74]. In apples,
citramalate is supposed to be synthesized from pyruvate and acetyl-CoA by citramalate
synthase and to contribute to isoleucine and 2-methylbutanoate and propanoate ester
biosynthesis [75]. The biochemical role of this compound in brown algae is still to be
investigated. As citramalate may be a fungal metabolite, in Pelvetia it may also be produced
by the other member of mycophycobiosis, S. ascophylli.
Citric acid is one of the predominant metabolites of brown algae with cellular concentrations several orders of magnitude higher than the other organic acids (Table S1; [18,20]).
Int. J. Mol. Sci. 2023, 24, 10626
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Apparently, this compound, standing on the crossroads of several metabolic pathways,
is largely involved in the metabolic adjustment of Pelvetia to extreme conditions. One
of its putative functions may be the carbon storage and recycling in the processes similar to CAM-photosynthesis. According to the literature data, another intertidal alga,
F. vesiculosus, can photosynthesize for some time even in CO2 -free air, which suggests the
presence of an organic carbon store in the cells [13,14]. Moreover, Fucus and Pelvetia not
only maintain photosynthesis during the first hours of emersion, but even demonstrate a
transient increase in CO2 fixation rate, despite the lower inorganic carbon concentration
in the air compared to seawater [24,71,76]. As measurements of tissue titratable acidity
during the periods differing in inorganic carbon and water availability (day vs. night,
for terrestrial CAM plants) were shown to be a highly sensitive method for detecting
even low-level CAM activity [77], we carried out such analysis and revealed considerable
changes of H+ content in emersed and submersed Pelvetia thalli (Figure 2). The amplitude
of these oscillations is comparable to the values reported for some terrestrial CAM plants
(e.g., Pilea peperomioides, Werauhia sanguinolenta) [77], and citric acid is the only metabolite
present in Pelvetia cells in the amount high enough to account for such acidity changes. Stoichiometrically, each mol of citrate accumulated in algal cells during the underwater period
should be balanced by 3 mol H+ during titration, and indeed the ratio of the amplitudes
of citrate and H+ oscillations, measured in our study, is very close to 1:3 (exactly, 1:2.97)
(Figures 1 and 9). Though the principal organic acid involved in typical CAM-photosynthesis
is malate, considerable diurnal oscillations of citrate level were shown for many terrestrial
CAM plants (reviewed in: [78]). E.g., during the salinity-induced C3 –CAM shift in Mesembryanthemum crystallinum, a nocturnal accumulation of citrate preceded that of malate,
and the CAM-dependent increase in citric acid content was ~100 times higher than that
of malic acid [79]. Currently, the biochemical and ecophysiological significance of these
CAM-associated citrate oscillations have been studied very little. Lüttge [78] suggested
that using citric acid for carbon storage in plant cells may be reasonable and even favorable
if it is synthesized from acetyl-CoA obtained not from glycolysis (as it would mean the loss
of CO2 due to pyruvate decarboxylation), but from other sources, such as β-oxidation of
fatty acids. Such a process implies oscillations of fatty acid levels in counter-phase to those
of citrate, and this is exactly what we found in the tide-dependent dynamics of Pelvetia
metabolite profiles (Figures 8 and 9). The “open”, non-cyclic, configuration of TCA cycle
may provide export of citrate to the cytosol and/or vacuole, and its decarboxylation (via
isocitrate) may occur then due to activity of cytosolic NADP-isocitrate dehydrogenase [79].
Thus, we suggest that besides providing organic carbon for biosynthetic reactions, citric
acid may also serve as a CO2 store and, thus, contribute to maintaining the photosynthetic
activity in the emersed Pelvetia thalli. To our knowledge, our study provided the first data
implying that citrate-based CAM-like processes may occur in brown algae. This issue
definitely warrants further investigation.
4. Materials and Methods
4.1. Algal Material Collection
Thalli of Pelvetia canaliculata (L.) Dcne and Thur. were collected at the rocky shores
of Srednii island in the Keret Archipelago (Kandalaksha Bay, White Sea) in July–August
2021–2022. This location features semi-diurnal tides with typical ranges of 1.0 m at neap
tides and 1.8 m at spring tides. The material collection was carried out at four timepoints
corresponding to different tidal phases (Table 1). To minimize the effects of varying
environmental conditions, the sampling was made only in the daytime, in quiet and sunny
weather and the air temperature was in the range of 20–25 ◦ C. Most samples were taken
during the spring tides. As at the neap tidal cycles Pelvetia cannot be regularly submerged,
the thalli were not collected during this period.
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Table 1. Sampling timepoints and condition of the thalli of Pelvetia canaliculata during the typical
tidal cycle in the period of spring tides at the rocky shores of Srednii island.
Timepoint Label
High water (HW)
Ebb tide (ET)
Low water (LW)
Rising tide (RT)
Timepoint Description
The maximum water level, no considerable tidal current
2–2.5 h after the water level started to decrease, tidal current
is flowing seaward
The minimum water level, no considerable tidal current
3.5–4 h after the water level started to increase, tidal current
is flowing inland
Condition of Pelvetia thalli
in the Moment of Sampling
Totally submersed
Still under water, ready to emerge
In the air (exposure time: 3.5–4 h)
Still in the air (exposure time: 7–8 h),
ready to be flooded
Algae collected during high water or ebb tide were delivered to the laboratory in
seawater. Algae collected during low water or rising tide were transported in dry condition.
All fixations and analyses were started no later than 0.5 h after the sampling.
4.2. Fresh Weight, Dry Weight, and Relative Water Content (RWC) Determination
RWC was calculated according to [80]. For the determination of fresh weight,
12–20 samples, taken from different individual thalli during each tidal phase, were blotted with filter paper (for HW and ET samples) and weighed. For determination of dry
weight, the samples were weighed again after drying at 70 ◦ C to the constant weight.
For determination of the saturated weight (SW), Pelvetia thalli were kept in water for
3 days and then blotted with filter paper and weighed. After that, RWC was calculated as
( FW − DW )×100%
[80].
SW − DW
Because of high variation of moisture between the samples taken at different tidal
phases, all measured biochemical parameters were calculated on the DW basis.
4.3. Hydrogen Peroxide Analysis
Measurement of H2 O2 content in thalli of P. canaliculata was carried out using the
ferrous–xylenol orange assay (FOX) [81,82] with modifications. From 50 to 150 mg FW
of algal material was homogenized in cold 0.2 M perchloric acid and the extracts were
centrifuged (10,000× g, 5 min, 4 ◦ C). The supernatants were neutralized with KOH and
then treated with ascorbate oxidase (0.25 mL of sample, 0.75 mL of 0.1 M potassium
phosphate buffer pH 5.6, 1 U of ascorbate oxidase, Sigma-Aldrich, Taufkirchen, Germany,
A0157) for 10 min. Then each extract was divided into two aliquots, one of which was
treated with catalase (Sigma-Aldrich, Taufkirchen, Germany, C9322). Equal volumes of
FOX reagent (0.2 mM xylenol orange, 200 mM sorbitol, 50 mM H2 SO4 , 0.5 mM (NH4 )2 SO3 ,
and 0.5 mM FeSO4 ) was added to both solutions. After 30 min, the extinction of the
solutions was measured at 560 nm using a SPEKOL 1300 spectrophotometer (Analytik Jena
AG, Jena, Germany). H2 O2 concentration in the reaction mixture was calculated as the
difference in absorbance between catalase-free and catalase-treated aliquots according to
the calibration curve.
4.4. Malondialdehyde Analysis
Malondialdehyde (MDA) content was determined based on the protocol of Velikova
et al. [83]. Samples of algal material (40 mg FW) were homogenized in cold 5% trichloroacetic
acid (TCA), and the extracts were centrifuged (10,000× g, 5 min, 4 ◦ C). The supernatants
were transferred into 5 mL polypropylene tubes and supplemented with a 3-fold volume
of 0.5% thiobarbituric acid (TBA, Sigma-Aldrich, Taufkirchen, Germany, T5500) in 20%
TCA. The reaction mixture was incubated at 95 ◦ C for 30 min and then cooled on ice. The
extinction of the solutions was measured at 532 and 600 nm using a SPEKOL 1300 spectrophotometer (Analytik Jena AG, Jena, Germany). The content of TBA-reactive substances
was calculated according to the published equations and expressed as MDA equivalent [83].
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4.5. Pigment Analysis
Content of photosynthetic pigments in the thalli of P. canaliculata was measured
spectrophotometrically. Fragments of algal thalli (5–10 mg FW) were ground using a
mortar and pestle in aqueous acetone (90% for chlorophylls a and c and pheophytin a or
80% for total carotenoids) with a small amount of MgCO3 . Several rounds of extraction
were completed with additional acetone until the extracts were colorless. The pigment
content was calculated according to the published equations [84–86] from data obtained
with a SPEKOL 1300 spectrophotometer (Analytik Jena, Jena, Germany).
4.6. Determination of Total Phlorotannin Content
Extraction of intracellular and CW-bound phlorotannins was carried out based on the
standard protocol of Koivikko et al. [87,88] with modifications described in [60]. Briefly,
20 mg of fresh algal material was poured with 70% aqueous acetone, homogenized, and left
soaking in 1 mL aqueous acetone for one hour to extract intracellular phenolics. Then, the
extract was centrifuged (5000× g, 10 min), the supernatant was transferred to another tube,
and the pellet was re-extracted with another portion of aqueous acetone. The supernatants
of four extraction rounds were combined.
The CW-bound phlorotannin fraction was extracted from the precipitate of the remaining algal material after the extraction of intracellular phlorotannins. The precipitate
was resuspended in 0.5 mL of 1 M aqueous NaOH solution (80 ◦ C) and then incubated
for 2.5 h at room temperature with continuous shaking (750 rpm). After centrifugation
(5000× g, 10 min,), the supernatant was transferred to another tube. The alkaline extraction
was repeated three times. The combined supernatants were neutralized with concentrated
HCl to pH 6.8–7.0.
A modification of the Folin–Ciocalteu micro-method was used to measure the total
phenolic content in the extracts [89]. The reaction mixture containing 0.3 mL of sample
(diluted as needed), 0.3 mL of Folin reagent and 2.4 mL of 5% (w/v) Na2 CO3 , was incubated
for 20 min at 45 ◦ C, and then the absorbance was measured at 750 nm using a SPEKOL
1300 spectrophotometer. Phloroglucinol (Sigma-Aldrich, Taufkirchen, Germany, 79330) was
used as the standard.
4.7. Determination of Titratable Acidity
Samples of Pelvetia thalli (1–1.5 g FW) were fixed with boiling distilled water and
homogenized. The homogenates were centrifuged (5000× g, 10 min), the supernatants
were collected, and the pellets were re-extracted with another portion of boiling water. The
supernatants of three extraction rounds were combined and cooled to 20 ◦ C. Content of H+
in the extracts was determined by titration with 5 mM NaOH to pH 7.0.
4.8. Metabolite Profiling
Before starting the sample preparation, feasibility and appropriate sample amount
were assessed in pilot experiments with ascending amounts of algal material. Fragments of
algal thalli (10–20 mg FW, depending on the tidal phase) were poured with cold methanol
(−25 ◦ C), quickly ground in a pre-cooled mortar and left soaking in 1 mL of cold methanol
for extraction. Aliquots of 500 µL of methanol extracts were transferred to clean 1.5 mL
polypropylene Eppendorf tubes (VWR, Dresden, Germany) and vacuum-dried in the
CentriVap vacuum concentrator system (Labconco, Kansas City, MO, USA) for subsequent analysis.
Metabolite profiling analyses were carried out according to [90]. Briefly, vacuum-dried
extracts were incubated by shaking in methoxyamine hydrochloride (Alfa Aesar by Thermo
Fisher Scientific, Kandel, Germany) solution in pyridine (Sigma-Aldrich, Taufkirchen,
Germany) and N,O-bis(trimethylsilyl)-trifluoroacetamide (Macherey-Nagel GmbH and Co
KG, Düren, Germany). After derivatization, samples were transferred to glass vial microinserts and subjected to GC-MS analysis on an Agilent 6890 gas chromatograph coupled to
an Agilent 5973N quadrupole mass selective detector (Agilent Technologies, Böblingen,
Int. J. Mol. Sci. 2023, 24, 10626
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Germany) with standard electron impact ionization (70 eV). Separation was accomplished
on a DB-5MS Ultra Inert column (Agilent, Waldbronn, Germany; 30 m × 0.25 mm ID and
0.25 µm film) at 0.9 mL/min carrier gas flow (He 5.0 Alphagaz, Air Liquide, Germany)
after splitless injection at 250 ◦ C. Within each sequence, a mixture of alkanes (C10–C32)
in hexane was measured for the calculation of Kovats retention indices (RI). A mix of
authentic reference standards containing 21 amino acids, 23 sugars and polyols, 19 organic
acids, phloroglucinol, etc., was co-spiked to confirm the identity of expected compounds.
Peak deconvolution was accomplished using AMDIS 2.66. The retention indices were
automatically calculated using an AMDIS calibration file containing the batch retention
times of each alkane. GMD (Golm metabolome database, GMD_20100614_VAR5_ALK,
24 September 2010, [91]) and NIST14 (National Institute of Standards and Technology,
Gaithersburg, MD, USA) were used for identification of the peaks based on spectra comparison. Where applicable, absolute quantitation was performed by standard addition using
five calibration levels.
4.9. Data Analysis
Experiments were carried out with 5 to 20 biological replicates (taken from different
individuals). Quantitation of metabolites in GC-MS analysis was performed by peak integration of the corresponding extracted ion chromatograms (m/z ± 0.5) for representative
intense signals at specific retention times (RT) using Xcalibur 3.0. Excel 2013 (Microsoft, Redmond, WA, USA) and MetaboAnalyst 5.0 Web application (http://www.metaboanalyst.ca,
accessed on 23 May 2023) were used for data processing and normalization procedures, creation of figures, and heatmap construction [92]. The metabolomic data processing included
peak area normalization to the median of all areas within the corresponding chromatogram,
generalized logarithm transformation, and range data scaling (mean-centered and divided
by the range of each variable). The normalized metabolomics data were analyzed by a partial least square discriminant analysis (PLSDA). Where appropriate, data were confirmed
for normality using the Kolmogorov–Smirnov test and for homogeneity of variance using
the Levene test. Student’s t-test was used to confirm the significant differences between the
means. All values are expressed as means and standard deviations.
5. Conclusions
During the underwater period, various growth-maintaining biosynthetic processes
activate in the cells of P. canaliculata, which manifest as accumulation of chlorophyll, amino
acids, and CW-bound phlorotannins. Emersion resulted in a dramatic decrease in RWC
in Pelvetia thalli, though such severe desiccation did not lead to considerable increase
in hydrogen peroxide and malondialdehyde content in the cells. Metabolic adjustment
of P. canaliculata to emersion included accumulation of soluble carbohydrates, various
phenolic compounds, including intracellular phlorotannins, and fatty acids. Apparently,
the dominating metabolite of Pelvetia, citric acid, has a key role in switching the algal
metabolism between the underwater and waterless conditions. Changes in titratable acidity
underlain by the oscillations of citric acid content imply that some specific CAM-like
processes may be involved in Pelvetia adaptation to the tidal cycle.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/ijms241310626/s1.
Author Contributions: Conceptualization, E.T.; data curation, E.T. and C.B.; formal analysis, R.I. and
E.T.; funding acquisition, E.T.; investigation, R.I., C.B., S.B., N.Y., E.Z. (Elizaveta Zamyatkina), E.G.,
E.Z. (Ekaterina Zuy) and E.T.; methodology, E.T. and C.B.; project administration, E.T.; resources, E.T.
and C.B.; supervision, E.T.; validation, E.T. and C.B.; visualization, R.I. and E.T.; writing—original
draft, R.I. and E.T.; writing—review and editing, C.B. and E.T. All authors have read and agreed to
the published version of the manuscript.
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Funding: This research was funded by the Russian Science Foundation, grant № 22-24-20039, https:
//rscf.ru/en/project/22-24-20039/ (accessed on 23 May 2023) and St. Petersburg Science Foundation,
grant № 35/2022 from 14 April 2022.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets generated and/or analyzed in this study are available
from the corresponding author upon reasonable request.
Acknowledgments: We thank Marine Biological Station “UNB Belomorskaya” and Research Park
of St. Petersburg State University for providing facilities, and the mass spectrometry core facility at
the Faculty of Chemistry and Mineralogy at Leipzig University, MS-UL for carrying out the GC-MS
analyses. We also thank Elena Sharova for providing one of key reagents and Tatiana Bilova and
Elena Stepchenkova for fruitful discussions and technical support.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Davison, M.W.; Pearson, G.A. Stress tolerance in intertidal seaweeds. J. Phycol. 1996, 32, 197–211. [CrossRef]
Wahl, M.; Jormalainen, V.; Eriksson, B.K.; Coyer, J.A.; Molis, M.; Schubert, H.; Dethier, M.; Karez, R.; Kruse, I.; Lenz, M.; et al.
Stress ecology in Fucus: Abiotic, biotic and genetic interactions. Adv. Mar. Biol. 2011, 59, 37–105. [CrossRef] [PubMed]
Lalegerie, F.; Stengel, D.B. Concise review of the macroalgal species Pelvetia canaliculata (Linnaeus) Decaisne & Thuret. J. Appl.
Phycol. 2022, 34, 2807–2825. [CrossRef]
Kingham, D.L.; Evans, L.V. The Pelvetia-Mycosphaerella Interrelationship. In The Biology of Marine Fungi; Moss, S.T., Ed.; Cambridge
University Press: Cambridge, UK, 1986; pp. 177–187.
Konovalova, O.P.; Bubnova, E.N.; Sidorova, I.I. Biology of Stigmidium ascophylli—Fungal symbiont of fucoids in Kandalaksha bay,
White Sea. Mikol. Fitopatol. 2012, 46, 353–360. (In Russian)
Pfetzing, J.; Stengel, D.; Cuffe, M.; Savage, A.; Guiry, M. Effects of temperature and prolonged emersion on photosynthesis,
carbohydrate content and growth of the brown intertidal alga Pelvetia canaliculata. Bot. Mar. 2000, 43, 399–407. [CrossRef]
Martins, M.; Soares, C.; Figueiredo, I.; Sousa, B.; Torres, A.C.; Sousa-Pinto, I.; Veiga, P.; Rubal, M.; Fidalgo, F. Fucoid macroalgae
have distinct physiological mechanisms to face emersion and submersion periods in their southern limit of distribution. Plants
2021, 10, 1892. [CrossRef] [PubMed]
Sampath-Wiley, P.; Neefus, C.D.; Jahnke, L.S. Seasonal effects of sun exposure and emersion on intertidal seaweed physiology:
Fluctuations in antioxidant contents, photosynthetic pigments and photosynthetic efficiency in the red alga Porphyra umbilicalis. J.
Exp. Mar. Biol. Ecol. 2008, 361, 83–91. [CrossRef]
Flores-Molina, M.R.; Thomas, D.; Lovazzano, C.; Núñez, A.; Zapata, J.; Kumar, M.; Correa, J.A.; Contreras-Porcia, L. Desiccation
stress in intertidal seaweeds: Effects on morphology, antioxidant responses and photosynthetic performance. Aquat. Bot. 2014,
113, 90–99. [CrossRef]
Benes, K.M.; Bracken, M.E.S. Nitrate uptake varies with tide height and nutrient availability in the intertidal seaweed Fucus
vesiculosus. J. Phycol. 2016, 52, 863–876. [CrossRef]
Bischof, K.; Rautenberger, R. Seaweed responses to environmental stress: Reactive oxygen and antioxidative strategies. In Seaweed
Biology; Wiencke, C., Bischof, C., Eds.; Springer: Berlin/Heidelberg, Germany, 2012; pp. 109–132.
Connan, S.; Deslandes, E.; Gall, E.A. Influence of day–night and tidal cycles on phenol content and antioxidant capacity in three
temperate intertidal brown seaweeds. J. Exp. Mar. Biol. Ecol. 2007, 349, 359–369. [CrossRef]
Bidwell, R.G.S.; McLachlan, J. Carbon nutrition of seaweeds: Photosynthesis, photorespiration and respiration. J. Exp. Mar. Biol.
Ecol. 1985, 86, 15–46. [CrossRef]
Kawamitsu, Y.; Boyer, J.S. Photosynthesis and carbon storage between tides in a brown alga, Fucus vesiculosus. Mar. Biol. 1999,
133, 361–369. [CrossRef]
Connan, S.; Stengel, D.B. Impacts of ambient salinity and copper on brown algae: 2. Interactive effects on phenolic pool and
assessment of metal binding capacity of phlorotannin. Aquat. Toxicol. 2011, 104, 1–13. [CrossRef]
Lemesheva, V.; Tarakhovskaya, E. Physiological functions of phlorotannins. Biol. Commun. 2018, 63, 70–76. [CrossRef]
Michel, G.; Tonon, T.; Scornet, D.; Cock, J.M.; Kloareg, B. Central and storage carbon metabolism of the brown alga Ectocarpus
siliculosus: Insights into the origin and evolution of storage carbohydrates in Eukaryotes. New Phytol. 2010, 188, 67–81. [CrossRef]
Dittami, S.M.; Gravot, A.; Renault, D.; Goulitquer, S.; Eggert, A.; Bouchereau, A.; Boyen, C.; Tonon, T. Integrative analysis of
metabolite and transcript abundance during the short-term response to saline and oxidative stress in the brown alga Ectocarpus
siliculosus. Plant Cell Environ. 2011, 34, 629–642. [CrossRef]
Tarakhovskaya, E.; Lemesheva, V.; Bilova, T.; Birkemeyer, C. Early embryogenesis of brown alga Fucus vesiculosus L. is characterized by significant changes in carbon and energy metabolism. Molecules 2017, 22, 1509. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 10626
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
19 of 21
Birkemeyer, C.; Osmolovskaya, N.; Kuchaeva, L.; Tarakhovskaya, E. Distribution of natural ingredients suggests a complex
network of metabolic transport between source and sink tissues in the brown alga Fucus vesiculosus. Planta 2019, 249, 377–391.
[CrossRef]
Jones, A.L.; Harwood, J.L. Lipid metabolism in the brown marine algae Fucus vesiculosus and Ascophyllum nodosum. J. Exp. Bot.
1993, 44, 1203–1210. [CrossRef]
Andrade, P.B.; Barbosa, M.; Matos, R.P.; Lopes, G.; Vinholes, J.; Mouga, T.; Valentão, P. Valuable compounds in macroalgae
extracts. Food Chem. 2013, 138, 1819–1828. [CrossRef] [PubMed]
Schmid, M.; Stengel, D.B. Intra-thallus differentiation of fatty acid and pigment profiles in some temperate Fucales and Laminariales. J. Phycol. 2015, 51, 25–36. [CrossRef] [PubMed]
Dring, M.J.; Brown, F.A. Photosynthesis of intertidal brown algae during and after periods of emersion: A renewed search for
physiological causes of zonation. Mar. Ecol. Prog. Ser. 1982, 8, 301–308. [CrossRef]
Barr, H.D.; Weatherley, P.E. A re-examination of the relative turgidity technique for estimating water deficit in leaves. Aust. J. Biol.
Sci. 1962, 15, 413–428. [CrossRef]
Kalariya, K.A.; Singh, A.L.; Chakroborty, K.; Patel, C.B.; Zala, P.V. Relative water content as an index of permanent wilting in
groundnut under progressive water deficit stress. J. Environ. Sci. 2015, 8, 17–22.
Lange, O.L. Moisture content and CO2 exchange of lichens: I. Influence of temperature on moisture-dependent net photosynthesis
and dark respiration in Ramalina maciformis. Oecologia 1980, 45, 82–87. [CrossRef] [PubMed]
Farrant, J.M. A comparison of mechanisms of desiccation tolerance among three angiosperm resurrection plant species. Plant
Ecol. 2000, 151, 29–39. [CrossRef]
Wilce, R.T.; Webber, E.E.; Sears, J.R. Petroderma and Porterinema in the New World. Mar. Biol. 1970, 5, 119–135. [CrossRef]
Sanders, W.B.; Moe, R.L.; Ascaso, C. Ultrastructural study of the brown alga Petroderma maculiforme (Phaeophyceae) in the
free-living state and in lichen symbiosis with the intertidal marine fungus Verrucaria tavaresiae (Ascomycotina). Eur. J. Phycol.
2005, 40, 353–361. [CrossRef]
Gueidan, C.; Thüs, H.; Pérez-Ortega, S. Phylogenetic position of the brown algae-associated lichenized fungus Verrucaria tavaresiae
(Verrucariaceae). Bryologist 2011, 114, 563–569. [CrossRef]
Sanders, W.B.; Moe, R.L.; Ascaso, C. The intertidal marine lichen formed by the pyrenomycete fungus Verrucaria tavaresiae
(Ascomycotina) and the brown alga Petroderma maculiforme (Phaeophyceae): Thallus organization and symbiont interaction. Am.
J. Bot. 2004, 91, 511–522. [CrossRef]
Valenzuela, A. The biological significance of malondialdehyde determination in the assessment of tissue oxidative stress. Life Sci.
1991, 48, 301–309. [CrossRef]
Cheeseman, J.M. Hydrogen peroxide and plant stress: A challenging relationship. Plant Stress 2007, 1, 4–15.
Tarakhovskaya, E.R.; Bilova, T.E.; Maslov, Y.I. Hydrogen peroxide content and vanadium-dependent haloperoxidase activity in
thalli of six species of Fucales (Phaeophyceae). Phycologia 2015, 54, 417–424. [CrossRef]
Costa, M.M.; Barrote, I.; Silva, J.; Olivé, I.; Alexandre, A.; Albano, S.; Santos, R.O.P. Epiphytes modulate Posidonia oceanica
photosynthetic production, energetic balance, antioxidant mechanisms and oxidative damage. Front. Mar. Sci. 2015, 2, 111.
[CrossRef]
Kumar, A.; AbdElgawad, H.; Castellano, I.; Lorenti, M.; Delledonne, M.; Beemster, G.T.S.; Asard, H.; Buia, M.C.; Palumbo, A.
Physiological and biochemical analyses shed light on the response of Sargassum vulgare to ocean acidification at different time.
Front. Plant. Sci. 2017, 8, 570. [CrossRef]
Lemesheva, V.; Birkemeyer, C.; Garbary, D.; Tarakhovskaya, E. Vanadium-dependent haloperoxidase activity and phlorotannin
incorporation into the cell wall during early embryogenesis of Fucus vesiculosus (Phaeophyceae). Eur. J. Phycol. 2020, 55, 275–284.
[CrossRef]
Graiff, A.; Karsten, U. Antioxidative properties of Baltic Sea keystone macroalgae (Fucus vesiculosus, Phaeophyceae) under ocean
warming and acidification in a seasonally varying environment. Biology 2021, 10, 1330. [CrossRef]
Collén, J.; Davison, I.R. Reactive oxygen production and damage in intertidal Fucus spp. (Phaeophyceae). J. Phycol. 1999, 35, 54–61.
[CrossRef]
Sáez, C.A.; Roncarati, F.; Moenne, A.; Moody, A.J.; Brown, M.T. Copper-induced intra-specific oxidative damage and antioxidant
responses in strains of the brown alga Ectocarpus siliculosus with different pollution histories. Aquat. Toxicol. 2015, 159, 81–89.
[CrossRef]
Cruces, E.; Huovinen, P.; Gómez, I. Phlorotannin and antioxidant responses upon short-term exposure to UV radiation and
elevated temperature in three south Pacific kelps. Photochem. Photobiol. 2012, 88, 58–66. [CrossRef]
Schelbert, S.; Aubry, S.; Burla, B.; Agne, B.; Kessler, F.; Krupinska, K.; Hörtensteiner, S. Pheophytin pheophorbide hydrolase
(pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis. Plant Cell 2009, 21, 767–785.
[CrossRef] [PubMed]
Smith, B.M.; Morrissey, P.J.; Guenther, J.E.; Nemson, J.A.; Harrison, M.A.; Allen, J.F.; Melis, A. Response of the photosynthetic
apparatus in Dunaliella salina (green algae) to irradiance stress. Plant Physiol. 1990, 93, 1433–1440. [CrossRef] [PubMed]
Pratap, V.; Sharma, Y.K. Impact of osmotic stress on seed germination and seedling growth in black gram (Phaseolus mungo).
J. Environ. Biol. 2010, 31, 721–726. [PubMed]
Int. J. Mol. Sci. 2023, 24, 10626
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
20 of 21
Bezzubova, E.M.; Drits, A.V.; Mosharov, S.A. Effect of mercury chloride on the chlorophyl a and pheophytin content in marine
microalgae: Measuring the flow of autotrophic phytoplankton using sediment traps data. Oceanology 2018, 58, 479–486. [CrossRef]
Salgado, L.T.; Cinelli, L.P.; Viana, N.B.; de Carvalho, R.T.; De Souza Mourão, P.A.; Teixeira, V.L.; Farina, M.; Filho, G.M.A.
A vanadium bromoperoxidase catalyzes the formation of high-molecular-weight complexes between brown algal phenolic
substances and alginates. J. Phycol. 2009, 45, 193–202. [CrossRef]
Shah, J. The salicylic acid loop in plant defense. Curr. Opin. Plant Biol. 2003, 6, 365–371. [CrossRef]
Song, Y.; Cui, X.S.; Chen, J.J.; Yang, R.; Yan, X. The profiling of eleven phytohormones in Pyropia haitanensis under different
high-temperature environments. J. Fish. China 2017, 41, 1578–1587. [CrossRef]
Zhang, T.; Shi, M.; Yan, H.; Li, C. Effects of salicylic acid on heavy metal resistance in eukaryotic algae and its mechanisms. Int. J.
Environ. Res. Public Health 2022, 19, 13415. [CrossRef]
Wang, Q.; Li, X.; Tang, L.; Fei, Y.; Pan, Y.; Sun, L. Paper-based electroanalytical devices for in situ determination of free 3indoleacetic acid and salicylic acid in living Pyropia haitanensis thallus under various environmental stresses. J. Appl. Phycol. 2020,
32, 485–497. [CrossRef]
Zhou, B.; Tang, X.; Wang, Y. Salicylic acid and heat acclimation pretreatment protects Laminaria japonica sporophyte (Phaeophyceae)
from heat stress. Chin. J. Oceanol. Limnol. 2010, 28, 924–932. [CrossRef]
Zhu, Z.B.; Sun, X.; Xu, N.J.; Luo, Q.J. Effects of salicylic acid on the resistance of Gracilaria/Gracilariopsis lemaneiformis to high
temperature. J. Fish. China 2012, 36, 1304–1312. [CrossRef]
Onofrejová, L.; Vasícková, J.; Klejdus, B.; Stratil, P.; Misurcová, L.; Krácmar, S.; Kopecký, J.; Vacek, J. Bioactive phenols in algae:
The application of pressurized-liquid and solid-phase extraction techniques. J. Pharm. Biomed. Anal. 2010, 51, 464–470. [CrossRef]
Gupta, V.; Kumar, M.; Brahmbhatt, H.; Reddy, C.R.; Seth, A.; Jha, B. Simultaneous determination of different endogenetic plant
growth regulators in common green seaweeds using dispersive liquid-liquid microextraction method. Plant Physiol. Biochem.
2011, 49, 1259–1263. [CrossRef]
Peng, Y.; Yang, J.; Li, X.; Zhang, Y. Salicylic acid: Biosynthesis and signaling. Annu. Rev. Plant Biol. 2021, 72, 761–791. [CrossRef]
Del Mondo, A.; Sansone, C.; Brunet, C. Insights into the biosynthesis pathway of phenolic compounds in microalgae. Comput.
Struct. Biotechnol. J. 2022, 20, 1901–1913. [CrossRef]
Zhao, F.; Wang, P.; Lucardi, R.D.; Su, Z.; Li, S. Natural sources and bioactivities of 2,4-di-tert-butylphenol and its analogs. Toxins
2020, 12, 35. [CrossRef]
Collén, J.; Davison, I.R. Seasonality and thermal acclimation of reactive oxygen metabolism in Fucus vesiculosus (Phaeophyceae).
J. Phycol. 2001, 37, 474–481. [CrossRef]
Birkemeyer, C.; Lemesheva, V.; Billig, S.; Tarakhovskaya, E. Composition of intracellular and cell wall-bound phlorotannin
fractions in fucoid algae indicates specific functions of these metabolites dependent on the chemical structure. Metabolites 2020,
10, 369. [CrossRef]
Meshalkina, D.; Tsvetkova, E.; Orlova, A.; Islamova, R.; Grashina, M.; Gorbach, D.; Babakov, V.; Francioso, A.; Birkemeyer, C.;
Mosca, L.; et al. First insight into the neuroprotective and antibacterial effects of phlorotannins isolated from the cell walls of
brown algae Fucus vesiculosus and Pelvetia canaliculata. Antioxidants 2023, 12, 696. [CrossRef] [PubMed]
Steevensz, A.J.; Mackinnon, S.L.; Hankinson, R.; Craft, C.; Connan, S.; Stengel, D.B.; Melanson, J.E. Profiling phlorotannins in
brown macroalgae by liquid chromatography-high resolution mass spectrometry. Phytochem. Anal. 2012, 23, 547–553. [CrossRef]
[PubMed]
Sanchez, D.H.; Siahpoosh, M.R.; Roessner, U.; Udvardi, M.; Kopka, J. Plant metabolomics reveals conserved and divergent
metabolic responses to salinity. Physiol. Plant. 2008, 132, 209–219. [CrossRef] [PubMed]
Han, P.P.; Yuan, Y.J. Metabolic profiling as a tool for understanding defense response of Taxus cuspidata cells to shear stress.
Biotechnol. Prog. 2009, 25, 1244–1253. [CrossRef]
Wang, Y.; Xu, L.; Shen, H.; Wang, J.; Liu, W.; Zhu, X.; Wang, R.; Sun, X.; Liu, L. Metabolomic analysis with GC-MS to reveal
potential metabolites and biological pathways involved in Pb & Cd stress response of radish roots. Sci. Rep. 2015, 5, 18296.
[CrossRef]
Li, Z.; Yu, J.; Peng, Y.; Huang, B. Metabolic pathways regulated by abscisic acid, salicylic acid and γ-aminobutyric acid in
association with improved drought tolerance in creeping bentgrass (Agrostis stolonifera). Physiol. Plant. 2017, 159, 42–58.
[CrossRef]
Wamelink, M.M.C.; Kerick, M.; Kirpy, A.; Lehrach, H.; Jakobs, C.; Ralser, M. The pentose phosphate pathway is a metabolic redox
sensor and regulates transcription during the antioxidant response. Antioxid. Redox Signal. 2011, 15, 311–324. [CrossRef]
Sharkey, T.D. Pentose phosphate pathway reactions in photosynthesizing cells. Cells 2021, 10, 1547. [CrossRef]
Kornecki, J.F.; Carballares, D.; Tardioli, P.W.; Rodrigues, R.C.; Berenguer-Murcia, Á.; Alcántara, A.R.; Fernandez-Lafuente, R.
Enzyme production of D-gluconic acid and glucose oxidase: Successful tales of cascade reactions. Catal. Sci. Technol. 2020,
10, 5740–5771. [CrossRef]
Igamberdiev, A.U.; Eprintsev, A.T. Organic acids: The pools of fixed carbon involved in redox regulation and energy balance in
higher plants. Front. Plant Sci. 2016, 7, 1042. [CrossRef]
Madsen, T.V.; Maberly, S.C. A comparison of air and water as environments for photosynthesis by the intertidal alga Fucus spiralis
(Phaeophyta). J. Phycol. 1990, 26, 24–30. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 10626
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
21 of 21
Hamid, S.S.; Wakayama, M.; Soga, T.; Tomita, M. Drying and extraction effects on three edible brown seaweeds for metabolomics.
J. Appl. Phycol. 2018, 30, 3335–3350. [CrossRef]
Hossain, A.H.; Hendrikx, A.; Punt, P.J. Identification of novel citramalate biosynthesis pathways in Aspergillus niger. Fungal Biol.
Biotechnol. 2019, 6, 19. [CrossRef]
Umino, M.; Onozato, M.; Sakamoto, T.; Koishi, M.; Fukushima, T. Analyzing citramalic acid enantiomers in apples and
commercial fruit juice by liquid chromatography–tandem mass spectrometry with pre-column derivatization. Molecules 2023,
28, 1556. [CrossRef]
Sugimoto, N.; Engelgau, P.; Jones, A.D.; Song, J.; Beaudry, R. Citramalate synthase yields a biosynthetic pathway for isoleucine
and straight- and branched-chain ester formation in ripening apple fruit. Proc. Natl. Acad. Sci. USA 2021, 118, e2009988118.
[CrossRef]
Nitschke, U.; Connan, S.; Stengel, D.B. Chlorophyll a fluorescence responses of temperate Phaeophyceae under submersion and
emersion regimes: A comparison of rapid and steady-state light curves. Photosynth. Res. 2012, 114, 29–42. [CrossRef] [PubMed]
Winter, K.; Smith, J.A.C. CAM photosynthesis: The acid test. New Phytol. 2022, 233, 599–609. [CrossRef]
Lüttge, U. Day-night changes of citric-acid levels in crassulacean acid metabolism: Phenomenon and ecophysiological significance.
Plant Cell Environ. 1988, 11, 445–451. [CrossRef]
Gawronska, K.; Niewiadomska, E. Participation of citric acid and isocitric acid in the diurnal cycle of carboxylation and
decarboxylation in the common ice plant. Acta Physiol. Plant. 2015, 37, 61. [CrossRef]
González, L.; González-Vilar, M. Determination of relative water content. In Handbook of Plant Ecophysiology Techniques; Reigosa
Roger, M.J., Ed.; Springer: Dordrecht, The Netherlands, 2001; pp. 207–212. [CrossRef]
Wolff, S.P. Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Methods
Enzymol. 1994, 233, 182–189. [CrossRef]
Gay, C.; Gebicki, J.M. A critical evaluation of the effect of sorbitol on the ferric-xylenol orange hydroperoxide assay. Anal. Biochem.
2000, 284, 217–220. [CrossRef]
Velikova, V.; Yodanov, I.; Edreva, A. Oxidative stress and some antioxidant systems in acid rain-treated bean plants. Plant Sci.
2000, 151, 59–66. [CrossRef]
Lorenzen, K. Determination of chlorophyll and pheo-pigments: Spectrophotometric equations. Limnol. Oceanogr. 1967, 12, 343–346.
[CrossRef]
Lichtenthaler, H.K.; Buschmann, C. Chlorophylls and carotenoids: Measurement and characterization by UV-VIS spectroscopy.
Curr. Protoc. Food Anal. Chem. 2001, 1, 1–8. [CrossRef]
Ritchie, R.J. Consistent sets of spectrophotometric chlorophyll equations for acetone, methanol and ethanol solvents. Photosynth.
Res. 2006, 89, 27–41. [CrossRef]
Koivikko, R.; Loponen, J.; Honkanen, T.; Jormalainen, V. Contents of cytoplasmic, cell-wall-bound and exudes phlorotannins in
the brown alga Fucus vesiculosus, with implications on their ecological functions. J. Chem. Ecol. 2005, 31, 195–209. [CrossRef]
Koivikko, R.; Loponen, J.; Pihlaja, K.; Jormalainen, V. High-performance liquid chromatographic analysis of phlorotannins from
the brown alga Fucus vesiculosus. Phytochem. Anal. 2007, 18, 326–332. [CrossRef] [PubMed]
Cicco, N.; Lanorte, M.T.; Paraggio, M.; Viggiano, M.; Lattanzio, V. A reproducible, rapid and inexpensive Folin–Ciocalteu
micro-method in determining phenolics of plant methanol extracts. Microchem. J. 2009, 91, 107–110. [CrossRef]
Hutschenreuther, A.; Kiontke, A.; Birkenmeier, G.; Birkemeyer, C. Comparison of extraction conditions and normalization
approaches for cellular metabolomics of adherent growing cells with GCMS. Anal. Methods 2012, 4, 1959–1963. [CrossRef]
Kopka, J.; Schauer, N.; Krueger, S.; Birkemeyer, C.; Usadel, B.; Bergmüller, E.; Dörmann, P.; Weckwerth, W.; Gibon, Y.; Willmitzer,
M.S.L.; et al. GMD@CSB.DB: The Golm Metabolome Database. Bioinformatics 2005, 21, 1635–1638. [CrossRef]
Pang, Z.; Chong, J.; Zhou, G.; Morais, D.; Chang, L.; Barrette, M.; Gauthier, C.; Jacques, P.E.; Li, S.; Xia, J. MetaboAnalyst 5.0:
Narrowing the gap between raw spectra and functional insights. Nucl. Acids Res. 2021, 49, W388–W396. [CrossRef]
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