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Published in final edited form as: J Phycol. 2020 Feb 10;56(3):649–661. doi: 10.1111/jpy.12971

Ecophysiological Changes and Spore Formation: Two Strategies in Response to Low-Temperature and High-Light Stress in Klebsormidium Cf. Flaccidum (Klebsormidiophyceae, Streptophyta) 1

Fátima Míguez 1,2, Andreas Holzinger 2, Beatriz Fernandez-Marin 3, José I García-Plazaola 4, Ulf Karsten 5, Lydia Gustavs 6
PMCID: PMC7612455  EMSID: EMS143663  PMID: 31957017

Abstract

Members of the cosmopolitan streptophycean genus Klebsormidium live in various habitats, including sand dunes and polar/alpine environments. To survive in these harsh conditions they must possess an array of adaptive physiological and structural mechanisms, for example, to deal with chilling and photochilling stresses. Since these mechanisms have not been studied in detail, the objectives of this study were (i) to determine the physiological and biochemical responses of Klebsormidium cf. flaccidum (K. cf. flaccidum) to chilling (low temperature [LT]) and photochilling (LT in combination with high light [HL]) stresses; and (ii) to understand the cross-link between biochemical parameters and cellular ultrastructural changes. The results indicated that 5°C is a temperature threshold (i.e., at 5°C) but not at higher temperatures, physiological changes were observed (Fv/Fm and ETR decreased and energy-partitioning distribution changed, with an increase in Y[NPQ] under LT and an increase in Y[NO] under HL-LT). Also, pigment contents changed significantly, with increased concentrations of photoprotective pigments such as antheraxanthin, zeaxanthin, and total carotenes. All of these responses occurred under LT and, to a greater extent, under LT-HL, indicating that the two stresses (temperature and light) are additive. The cold treatment applied here induced the formation of spores under both LL and HL. The degree of photoinhibition was higher in spores than in vegetative cells, indicating that spores are less susceptible to photodamage. This study demonstrated a broad acclimation potential in different developmental stages of K. cf. flaccidum, which helps to explain the ecological success of this genus.

Keywords: chilling, high light, Klebsormidium, low temperature, morphology, photochilling, pigment, spore, ultrastructure

Among abiotic environmental factors, temperature most strongly limits the establishment and growth of photosynthetic organisms on Earth. Sensitivity to low temperatures (LT) determines species distribution (Bannister and Neuner 2001, Körner 2012). The degree of sensitivity can vary among species, tissues (within a single individual), and even among seasons within a single tissue (Neuner 2014). It is widely recognized that acclimation processes are fundamental for the preservation of tissue integrity and plant survival under LT. Most studies of LT sensitivity have focused mainly on woody species although other photosynthetic organisms, such as terrestrial algae, must also face LT in their habitats. Under LT stress, vegetative cells of several microalgae transform to resting stages. Different terms have been applied to these stages; for example, “hypnoblasts” in snow algae (Remias et al. 2005), or “akinetes” in Zygnematophyceae (Pichrtová et al. 2014). The more general term “spores” is also used for these resting stages (Holzinger and Pichrtová 2016). These specialized cells have in common that they derive from vegetative stages that are morphologically and functionally converted or at least modified. In contrast, microalgae that maintain vegetative cells must counteract the high risk of photo-oxidation caused by the simultaneous light absorption by chlorophyll (Chl) and limited enzymatic activity. Photochilling specifically refers to the over-excitation of the photosynthetic apparatus under low but not freezing temperatures, in combination with high light (HL) intensities. To counteract the potentially deleterious effects of this energy imbalance, photosynthetic organisms have basically two (not mutually exclusive) alternatives. They can dissipate the excess absorbed energy, which is measurable as nonphotochemical quenching of Chl fluorescence (NPQ), or they can alleviate the excess absorbed energy after the water splitting (e.g., through cyclic electron transport and/or pathways for metabolic energy consumption; Öquist and Huner 2003). When these mechanisms are overloaded, light is sent to nonregulated processes that can induce photodamage in the photosynthetic apparatus (Lazár 2015).

The photochemistry of terrestrial algae under LT is insufficiently studied (Míguez et al. 2017a,b). Terrestrial green algae are a heterogeneous group and originate from several independent transitions from aquatic to terrestrial habitats. They are highly polyphyletic, originating from the classes Ulvophyceae, Chlorophyceae, and Trebouciophyecae (Chlorophyta) as well as Klebsormidiophyceae, Zygnemophyceae, and Chlorokybophyceae (Streptophyta). Although Míguez et al. (2017a,b) recently investigated photochilling in Chlorophyta, available information about early-branching Streptophyta is very sparse. Overall, down-regulation of photochemical processes is seen as a controlled mechanism that favors the dissipation of excess energy to protect the photosynthetic apparatus under LT conditions. This down-regulation, which can be assessed by monitoring the photochemical efficiency of photosystem II (Fv/Fm), is generally concomitant with a high de-epoxidation index in the xanthophyll cycle (V-cycle) pigments (Míguez et al. 2015). Míguez et al. (2017a, b) found that under LT stress, algae (as well as other photosynthetic groups) not only undergo changes in pigment/antioxidant content, but also accumulate soluble sugars.

Among streptophyte algae, the genus Klebsormidium (Klebsormidiophyceae, Streptophyta) has received much attention because of its worldwide distribution in terrestrial habitats such as soil or rock surfaces, as well as its abundance in biological soil crusts (Rindi 2011, Škaloud and Rindi 2013, Škaloud et al. 2014, Mikhailyuk et al. 2015, Ryšánek et al. 2015). Ecophysiological and biochemical acclimation to gradients of irradiation (PAR and UV), temperature, pH and water potential has been reported in Klebsormidium strains from all clades (Karsten et al. 2010, 2016, Holzinger et al. 2011, 2014, Kaplan et al. 2012, Kitzing et al. 2014, Škaloud et al. 2014). Nevertheless, investigations of cold tolerance in Klebsormidum are sparse (Nagao et al. 2008). In general, these studies revealed that the physiological response patterns to the different stresses can be quite different, depending on morphological and structural features such as strong or easily disintegrating filaments (Holzinger et al. 2011). Some of the underlying molecular mechanisms have been discussed in regard to the published genome of Klebsormidium nitens (erroneously determined as Klebsormidium flaccidum [K. flaccidum]) strain NIES-2285 (Hori et al. 2014), which was isolated from a freshwater habitat.

The strain Klebsormidium cf. flaccidum (WD-2-4; K. cf. flaccidum) investigated here is a member of the rare B-clade (Samolov et al. 2019), whose physiology and ecophysiological performance have been less studied than in other Klebsormidium species. This strain is distinct from the type species K. flaccidum, which belongs to the C clade sensu Rindi et al. (2011). Klebsormidium cf. flaccidum WD-2-4 is closely related to the strain K. flaccidum SAG 7.91, which has been subject to ecophysiological investigation by Karsten et al. (2016). For the type species of Klebsormidium, K. flaccidium, an adequate diagnosis is still lacking, affecting the circumscription of the genus Klebsormidium. In early phylogenies such as that of Rindi et al. (2011), strains morphologically assigned to K. flaccidum are polyphyletic and occur in three different clades (B, C, E). For a long period it was impossible to assign the type specimen to any of these clades. The original description by Kützing (1849) did not provide sufficient morphological information. Eventually, Mikhailyuk et al. (2015) proposed a strain of K. flaccidum isolated by Lokhorst (1996) and now preserved in the SAG algal collection (Sammlung für Algenkulturen, Göttingen, Germany; strain SAG 2307) as the epitype. This proposal was disputed by Škaloud et al. (2014), who argued that this strain was isolated far from the type locality, and in their study they were able to resample the original location, isolating several strains belonging to the C clade.

We hypothesized that down-regulation of photochemical activity (e.g., rise in dissipation of excess energy) combined with accumulation of sugars such as sucrose (which can act as both an osmolyte and as a carbon and energy reservoir) drive the acclimation of Klebsormidium under photochilling conditions. Specifically, we aimed to (i) elucidate which factor (HL or LT) plays a main role in the acclimation to photochilling, (ii) assess how photochemistry and a carbon reservoir (such as sucrose) interact in the acclimation process, and iii) understand the crosslink between photochemically measurable parameters and ultrastructural changes at the cellular level.

Materials and Methods

Strains and growth conditions

Klebsormidium cf. flaccidum (WD-2-4; for molecular phylogeny see Samolov et al. 2019) was collected in October 2012 in quartz sand from dunes on the Baltic Sea coast, Warnemünde, Germany (54°10′13.23″ N, 12°04′42.39″ E). Cultures of vegetative filaments of K. cf. flaccidum were grown in modified Bolds basal medium (3N-BBM+V; medium 26a; Schlösser 1997). Before the experiment, algal cultures were maintained for 5 weeks at room temperature (20°C), low light (LL; PFD 30 μmol photons · m−2 · s−1) and a photoperiod of 16:8 h (light:dark). Lamps used were Osram Daylight Lumilux Deluxe and radiation was measured with a Li-Cor LI-190-SB cosine-corrected sensor connected to a Li-Cor LI-1000 datalogger (Lambda Instruments, Lincoln, NE, USA; sensor error < 2%).

Stress induction: artificial winter season

Three days prior to the experiment, Klebsormidium cf. flaccidum replicates (three per treatment) in sterile cell culture flasks filled with 3-N BBM+V were transferred to custom-made incubators (Kunststoff-Technik Rostock, Rostock, Germany) for acclimation (Gustavs et al. 2009). The incubators were equipped with LED arrays (LED neutral white Ediline III 3.5 W COB Modul; Edison Opto Corp., Taipei, Taiwan) that allow precise light regulation.

During the experiment, (i) day length was progressively reduced from 16 to 8 h over a period of 2 d and (ii) temperature was reduced from 20°C (control) to 5°C (LT) over 4 days (see Fig. 1 for the description of the experimental design). Klebsormidium cf. flaccidum was subjected to LT (with a photoperiod of 8:16 h light:dark) for five additional days. During these cold days, samples were divided into two light treatments: LL (PFD 30 μmol photons · m−2 · s−1) and HL (PFD 300 μmol photons · m−2 · s−1). The PFD of the control was 30 μmol photons · m−2 · s−1. The samples for biochemical analyses were taken on (i) t0: day 1 of the experiment, before any cold acclimation (CA) process (control); (ii) t1: day 5 of the experiment (after 4 days under gradual cooling from 20 to 5°C, and 1 day at 5°C and LL or HL); and (iii) t2: the last day of the experiment (when the samples were held at 5°C during 5 consecutive days at LL or HL) (see Fig. 1 for sample collection times). This model of hardening was previously used by Míguez et al. (2017a, b).

Fig. 1.

Fig. 1

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Panel A: Cold-acclimation experimental design: Short and long dashed lines represent temperature and photoperiod reductions respectively. From day 0 to day 4, the temperature decreased from 20 to 5°C and was then maintained at 5°C until the end of the experiment. Panel B: Photon fluence rate (PPFD) during the experiment. On day 4 the samples were divided into two different treatments: low light, represented by a thin line (LL: 30 μmol photons · m−2 · s−1), and high light, represented by a thick line (HL: 300 μmol photons · m−2 · s−1). On the x-axis, arrows indicate the sampling times for pigment and sugar analyses. Circles indicate the days when fluorescence parameters were recorded.

Fluorescence analysis

Photosynthesis/irradiance curves (P/I curves) were determined as described by Míguez et al. (2017a,b). Briefly, samples filtered on filter discs were measured in a PAM-2500 fluorometer (Walz, Effeltrich, Germany) after 30 min of dark adaptation. Then, they were exposed to a continuously increasing light gradient consisting of 13 steps of 1 min each. PFDs increased from 12 to 489 μmol photons · m−2 · s−1. Fv/Fm was determined as (Fm − Fo)/Fm. Relative electron transport rate of PSII (ETR) was calculated according to Masojídek et al. (2001) at PFD 12 μmol photons · m−2 · s−1 (first light pulse of light curve). Both the ETR curves and Fv/Fm were measured during the first 5 d (from t0 to t4) to characterize the process of down-regulation of photosynthesis in response to the temperature decrease. Then, we again measured on days 7 and 9 in order to check if the curves recovered their initial shape. Because the down-regulation continued, we assumed that it was unnecessary to measure the fluorescence on each day (Fig. 1). The three components representing the light energy distribution, i.e., Y (II), effective photochemical quantum yield of PSII; Y(NPQ), non-photochemical quenching in PSII due to down-regulation of the light-harvesting function; and Y(NO), non-photo-chemical quenching in PSII other than that caused by down-regulation of the light-harvesting function, were also measured at 12 μmol photons · m−2 · s−1.

Fluorescence was also measured in each cell/spore individually after 5 days under LT (5°C) and under LL or LT and HL. Prior to fluorescence measurements, samples were dark-adapted for 30 min. Fluorescence was measured with a Microscopy Pulse Amplitude-Modulated (PAM) imaging fluorimeter (Walz, Effeltrich, Germany), coupled to an Axiostar Plus microscope (Carl Zeiss, Gottingen, Germany). A drop of Klebsormidium cf. flaccidum culture was placed on a slide and covered with a coverslip. The timing and settings control, as well as the fluorescence image processing were carried out with WinControl software.

Sample preparation for sugar and pigment determination

For biochemical analyses, algal cultures were centrifuged twice (5 min at 5°C, at 5,000 and 14,000g, respectively). The first centrifugation allowed us to reduce the culture total volume; after the second, the highly concentrated biomass was divided into several aliquots for the subsequent extractions. After centrifugation, the pellets were frozen in liquid nitrogen and stored at –80°C until extraction. Prior to sucrose and pigment determination, pellets were freeze-dried in a Savant SpeedVac (SPD 111V; Thermo Fisher Scientific, Waltham, MA, USA) connected to a Lyovac GT2 freeze-dryer (Steris, Cologne, Germany).

Sucrose determination

Dry algal samples (7–12 mg dry weight) were extracted with 70% ethanol-water (v/v) in capped centrifuge tubes at 70°C in a water bath for 4 h, according to Karsten et al. (1991). After centrifugation for 5 min at 5,000g, 700 μL of the supernatant was evaporated to dryness under vacuum (SpeedVac Concentrator SPD 100H). Dried extracts were dissolved again in 700 lL distilled water, vortexed for 30 s, and treated in an ultrasonic bath for 5 min (Bandelin Sonorex, Berlin, Germany). After centrifugation at 5,000 g for 5 min, the clear supernatant was pipetted into HPLC vials that were closed with membrane-equipped lids. Then, the sucrose was detected and quantified as in Karsten et al. (1991) and Míguez et al. (2017a,b).

Pigment analysis

Pigments were extracted from freeze-dried algal material as described by Míguez et al. (2017a,b) and were separated by HPLC with a reverse-phase C18 column (Waters Spherisorb ODS1, 4.6 x 250 mm; Milford, MA, USA). Pigments were detected and quantified with a photodiode array detector, following the method of García-Plazaola and Becerril (1999) modified after Garcia-Plazaola and Esteban (2012). The de-epoxidation rate of V-cycle pigments was estimated as (A+Z)/(V+A+Z), abbreviated as AZ/VAZ.

Light microscopy

For the morphological investigations, an Olympus BX-51 light microscope was used. The micrographs were taken with an Olympus UC 30 camera using the cellSens Entry imaging system (Olympus, Tokyo, Japan). A Zeiss Axiovert 200 M microscope was used for fluorescence photomicrographs, where a Zeiss filter set 01 (excitation band pass BP 365/12 nm, emission long pass LP 397 nm) was used. Images were captured with a Zeiss Mrc5 camera operated under Axiovision software (release 4.7).

Transmission electron microscopy

Klebsormidium cf. flaccidum cells were fixed for TEM with a standard chemical fixation protocol (2.5% glutaraldehyde, 1% OsO4) according to Holzinger et al. (2009). TEM ultrathin (60 nm) sections were prepared with a Leica Ultracut, counterstained with aqueous uranyl acetate and Reynold’s lead citrate for 10 min. Sections were examined with a Zeiss Libra 120 TEM at 80 kV. Images were captured digitally with a ProScan 2k SSCCD camera, controlled with OSIS iTEM software and further processed with Adobe Photoshop software.

Statistical analysis

Kolmogorov-Smirnov and Levene tests were used to test for the normality of data and homogeneity of variances, respectively. One-way ANOVA, with Duncan test as post hoc, was used to check for differences among treatments. All analyses were performed using the SPSS v24.0 statistical package (SPSS, Armonk, NY, USA).

Results

Effects of the interaction of light and low temperature on the physiology of Klebsormidium cf. flaccidum. The process of cold acclimation was monitored in Klebsormidium cf. flaccidum during 10 days of treatment (for details of the experimental design see Fig. 1). Despite the progressive temperature decrease, Fv/Fm remained unchanged between 20 and 7.5°C (Fig. 2A). Down-regulation of photosystem II activity occurred only at 5°C, suggesting that the transition from 7.5 to 5°C represents an important threshold to understand the physiological behavior of K. cf. flaccidum under LT stress. When LT was combined with HL, the Fv/Fm reduction was even higher, and, as occurred in the LT-LL treatment, the reduction occurred immediately after the shift in light and temperature conditions. Once the temperature reached 5°C, there was no further decrease in Fv/Fm (Fig. 2A). ETR followed basically the same pattern of changes (Fig. 2B), but with a more acute decrease under LT.

Fig. 2.

Fig. 2

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Photochemical efficiency of PSII (Fv/Fm; panel A) and electron transport rate (ETR; panel B) during the cold-acclimation interval (open bars) and under low-light (LL: grey bars) and high-light (HL: closed bars) treatments. Each bar represents the mean ± SE (n = 3). Different letters indicate significant differences between different treatments.

In parallel with Fv/Fm during the cold-acclimation process (between 20 and 7.5°C), light-energy partitioning remained constant (Fig. 3). Nevertheless, at 5°C, sudden changes were observed. In LT-LL, Y (NPQ) increased while Y(II) decreased. In contrast, in the LT-HL treatment, the decrease in Y(II) was higher while both Y(NPQ) and Y(NO) increased (Fig. 3).

Fig. 3.

Fig. 3

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Energy-partitioning distribution in Klebsormidium cf. flaccidum during winter simulation: Black sections of the columns represent quantum yield of PSII photochemistry (Y[II]); grey sections represent regulated thermal loss as heat (Y[NPQ]); and white sections represent non-regulated energy loss (Y[NO]). Each bar section (black, grey or white) corresponds to the percentage of each parameter with respect to the total light interception (n = 3 replicates).

The chilling treatment triggered a readjustment in the composition of the photosynthetic pigments in Klebsormidium cf. flaccidum (Fig. 4). Thus, while the total Chl content (Chl a+b) and Chl a/b ratio did not change significantly, a significant increase in the total carotenes (Tot Carots/Chl) in both treatments was observed, and was particularly noticeable in the LT-HL treatment (Fig. 4D). This change was explained mainly by a significant increase in the V-cycle pool (VAZ/Chl; Fig. 4E). In addition, the de-epoxidation cycle increased (Fig. 4, F and G). At a short timescale, the synthesis of zeaxanthin (Z) was triggered in parallel with the consumption of antheraxanthin (A; Fig. 4, F and G). At a longer timescale, the rise in A/VAZ was also observed, while the Z content remained unchanged (Fig. 4F). The sucrose content in K. cf. flaccidum temporarily rose during the cold acclimation but the sucrose was later consumed (Fig. 5). The photon fluence rate did not trigger any significant change in the sugar concentration.

Fig. 4.

Fig. 4

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Photosynthetic pigments and tocopherol contents in Klebsormidium cf. flaccidum in control (open bars), low-light (LL: grey bars) and high-light (HL: closed bars) treatments. Panel A: Chl a+b; Panel B: Chl a/b; Panel C: Tot Carots (total carotenoids); Panel D: Tot Carots/Chl; Panel E: Violaxanthin-cycle pigments (V (violaxanthin) + A (anteraxanthin) + Z (zeaxanthin)); Panel F: A/VAZ; Panel G: Z/VAZ; Panel H: Tot Toc/Chl (total tocopherol/chlorophyll). Bars are mean SE ± (n = 3). Different letters indicate significant differences shown by one-way ANOVA (P < 0.05). Missing letters indicate no significant differences.

Fig. 5.

Fig. 5

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Sucrose content in Klebsormidium cf. flaccidum in control (open bars), low-light (LL; grey bars) and high-light (HL; closed bars) treatments. Bars represent mean ± SE (n = 3), except for control (n = 1). One-way ANOVA indicated no significant differences (P < 0.05).

Spores and vegetative cells: two complementary structures with different physiological behaviors

Klebsormidium cf. flaccidium contained vegetative filaments and filaments enclosing round spores under LL (Fig. 6, A-C) and HL (Fig. 6, D-F) conditions when observed by LM. The round spores were retracted from the cell walls (Fig. 6B). In many cases they were separated from the filaments, forming individual propagules (Fig. 6E). When observed by fluorescence microscopy, living cells contained a bright-red chloroplast (Chl auto-fluorescence) and spores had a rounded appearance (Fig. 6, C and F, arrows), whereas damaged or dead cells showed turquoise autofluorescence (Fig. 6, C and >F).

Fig. 6.

Fig. 6

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Light micrographs of Klebsormidium cf. flaccidum cells under low-light (LL) (A–C) and high-light (HL; D–F) conditions. Panel A: vegetative filament and a filament containing spores (round); Panel B: filament with rounded spore; Panel C: Chl autofluorescence in rounded spores (arrow) next to dead or damaged cells (pale cells); Panel D: many vegetative filaments and filament with spores in the center; Panel E: individual spore (arrow); Panel F: fluorescence image with spores (arrows) and dead cells (pale cells). C, F, Zeiss Filter set 01. Scale bars: A, C, D, E 10 μm; B, E 5 μm. [Color figure can be viewed at wileyonlinelibrary.com]

In the TEM, vegetative cells with a normal appearance were found in both LL (Fig. 7, A-C) and HL (Fig. 7, D-F) treatment. Within each cell, a parietal chloroplast and a central nucleus were observed (Fig. 6, B and E). In the LL treatment, the pyrenoid was surrounded by plastoglobules, and only a few starch grains were observed (Fig. 7C). The pyrenoids in the HL-treated cells had a similar appearance, starch grains and a reduced number of plastoglobules were observed (Fig. 7, E and F). Numerous large mitochondria were observed in the HL-treated cells (Fig. 7E).

Fig. 7.

Fig. 7

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Transmission electron micrographs of Klebsormidium cf. flaccidum cells under low-light (LL; A–C) and high-light (HL; D–F) treatments. Panel A: overview of several filaments; Panel B: typical cell architecture with central nucleus, parietal chloroplast; Panel C: pyrenoid in chloroplast surrounded by plastoglobules; Panel D: filament with several newly divided cells, indicated by very thin cross-walls; Panel E: central area with nucleus, large mitochondria and peroxisome; Panel F: pyrenoid with tiny starch grains. Abbreviations: Chl chloroplast, P peroxisome, Py pyrenoid, M mitochondrion, N nucleus, S starch grain, V vacuole. Scale bars: 1 μm.

The morphology of spores formed under LL (Fig. 8, A and B) and HL (Fig. 8, C-E) conditions was compared by TEM. The spores contained an inner cell-wall layer (Fig. 8, A and B); the outer envelope of some was already ruptured and open (Fig. 8B, arrow). Several dead or aborted cells were observed in the filaments (white asterisks in Fig. 8, A and B). There was no structural difference between the spores, regardless of the LL or HL conditions to which they were exposed. Occasionally, under HL conditions, already-divided spores were found (Fig. 8E).

Fig. 8.

Fig. 8

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Transmission electron micrographs of spore formation in Klebsormidium cf. flaccidum cells under low-light (LL; Panels A–B) and high-light (HL; Panels C–E) treatments. Panel A: rounded spore, note the inner cell-wall layer; Panel B: mature spore, with the outer cell-wall layer already fractured; Panel C: overview with several dead cells and spores (arrows); Panel D: individual spore with cell-wall layer; Panel E: spore that has already divided again, indicated by a thin cross-wall (arrow). Abbreviations: Chl chloroplast, Py pyrenoid, N nucleus, V vacuole. Scale bars: 1 μm.

Chlorophyll fluorescence at single cell level

The photochemical responses of vegetative cells and spores were studied separately, using a PAM-fluorometer coupled to an epifluorescence microscope (Fig. 9). As observed in cultures, Fv/Fm decreased after cold acclimation; the extent of this decrease was dependent on the light treatment, being greater under HL than under LL (Fig. 2). In the LL treatment, this decrease was higher in spores than in vegetative cells, while no difference was observed in the HL treatment (Fig. 9).

Fig. 9.

Fig. 9

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Photochemical efficiency of PSII (Fv/Fm) in individual vegetative cells and spores under low temperature (LT). Low-light (LL) and high-light (HL) treatments are represented by open and closed bars respectively. Each bar represents the mean SE ± (n = 3). Different letters indicate significant differences between treatments.

Discussion

Klebsormidium cf. flaccidum is a widespread terrestrial alga with strains that are able to colonize highly contrasting habitats, from sand dunes to alpine environments (Karsten et al. 2013). In this study, we analyzed the effects of chilling and photochilling on K. cf. flaccidum (WD-2-4) obtained from a sand dune on the southern Baltic Sea coast.

Chilling and photochilling: mechanisms of Klebsormidium cf. flaccidum to deal with these stresses. One of the key targets of chilling and photochilling stresses is photosynthesis. Previous studies reported that members of Klebsormidiophyceae require LL, with PFDs of only 30 μmol photons · m−2 · s−1 for optimum growth (Karsten and Rindi 2010). Not only growth, but also the photosynthetic rate measured under experimental conditions were higher at lower irradiances (Holzinger and Pichrtová 2016). This LL preference was usually coupled with low photoinhibition under higher photon-fluence rates, and was interpreted as high photophysiological plasticity (Karsten et al. 2013, Pierangelini et al. 2017). Nevertheless, there are some exceptions within Klebsormidiophyceae; e.g., in Klebsormidium crenulatum the ETR started to decrease slightly at PFDs higher than 500 μmol photons · m−2 · s−1 (Karsten and Holzinger 2012). Previous studies have demonstrated the high photophysiological plasticity of members of Klebsormidiophyceae over a wide range of PPDs (Darienko et al. 2010). The present study extended these investigations, showing that under LT, 300 μmol photons · m−2 · s−1 was sufficient to provoke stress (Fig. 2). This indicated that both HL and LT stresses have a synergistic effect on the physiological response of K. cf. flaccidum.

Artificial algal cultures constitute an unrealistic environment in terms of nutrient availability and resource competition. Furthermore, the experimental set-ups tend to control abiotic factors under certain artificial conditions. Under natural conditions, the most typical situation involves the simultaneous interaction of multiple stressors affecting photosynthetic organisms. Coping with multiple stressors requires different adaptive mechanisms. The typical three-dimensional structure of Klebsormidium flaccidum biofilms (Karsten and Rindi 2010) helps to limit the light intensity, and since the requirements of Klebsormidium for optimal growth include LL, this multilayered mat-like filament structure probably contributes a high degree of self-shading of individual filaments in the population (Karsten et al. 2013). This might also explain the damaging effects of HL on the physiological performance of members of Klebsormidiaceae.

Apart from the ability to form specific growth structures, photosynthetic organisms must be able to down-regulate the photosynthetic ETR in order to face (photo)chilling stress. In the case of Klebsormidium cf. flaccidum, these adaptations are a prerequisite for life in inhospitable environments such as high mountains or coastal dunes with multiple stresses (LT, HL, dehydration, etc.) acting simultaneously (Holzinger et al. 2011). Consequently, protection of the photosynthetic apparatus through increasing thermal dissipation is crucial for their survival (Demmig-Adams and Adams 2014). In the strain of K. cf. flaccidum employed in the present study, the physiological readjustments to face chilling stress were activated between 7.5 and 5°C. The short-term effect of temperatures above 5°C was negligible at the photochemical level (Fig. 2). In essence, under LT the thermal dissipative processes, known as nonphotochemical quenching, gained in importance, as shown by the increase in Y(NPQ). In contrast, in the case of photochilling, a photophysical decay was observed, with an increase in Y(NO; Fig. 3). This indicates that although K. cf. flaccidium is able to regulate NPQ under chilling stress, photochilling involves not only an increase in thermal dissipation in comparison to control conditions, but also an uncontrolled increase in energy fluxes.

In algae, the activation of dissipative pathways under LT has often been described as a response to cold acclimation. This phenomenon involves an adjustment of the pigment composition (Fig. 4), in particular increases in the pools of V-cycle pigments and other carotenoids (Eggert et al. 2003). In fact, V-cycle is one of the main components of NPQ mechanisms (Jahns and Holzwarth 2012); hence, the size of the V-cycle pool may contribute substantially to a well-developed photoprotection process. In this way, the accumulation of these components permits greater dissipation of excessive irradiation under stress conditions (Mock and Hoch 2005, Stamenković et al. 2014). A previous study in which four terrestrial unicellular chlorophytes were exposed to the same (photo)chilling treatments observed that in all of them, the de-epoxidation state of the V-cycle increased with respect to the levels obtained under control conditions (Míguez et al. 2017a,b). As expected, after 24 h of both chilling and photochilling stress, Z was accumulated, indicating that in the short-term, the de-epoxidation process was completed. Nevertheless, after 6 days under stress, the xanthophyll accumulated was A, with consequent Z depletion. This again demonstrated the high capability of Klebsormidium cf. flaccidum of adapting to prolonged stresses.

In parallel with photochemical adjustments, the initial phase of LT acclimation was also characterized by a transient increase in sucrose content (the only soluble carbohydrate identified in Klebsormidium cf. flaccidium; Fig. 5). Control values (t0) obtained in this study were similar to those observed in previous studies of Klebsormidium sp. (Karsten and Rindi 2010, Kaplan et al. 2012). Not only chilling stress but also salinity induced an increase in sucrose content (Karsten and Rindi 2010), suggesting that this is a general stress response in Klebsormidium. This conclusion is supported by the ~2–3-fold up-regulation of transcripts for sucrose-phosphate synthase and sucrose synthase upon severe desiccation stress (Holzinger et al. 2014). A similar, but even more dramatic enhancement of sucrose was described by Nagao et al. (2008) for the same species, but in a slightly colder environment (2°C) than in the present study (5°C). These authors interpreted sucrose accumulation as a mechanism to increase the osmotic strength and to develop freezing tolerance. Alternatively, the accumulation of nonstructural carbohydrates in angiosperms has been interpreted as the cause of the LT-induced inhibition of photo-synthesis (Adams et al. 2013). However, in the present study, the initial sucrose accumulation after 1 day under LT stress was followed by depletion to prestress levels. The decrease in sucrose suggests an incomplete acclimation, or a metabolic shift toward other, undetected low-molecular-weight cryoprotectants such as the putative glycosides proposed by Nagao et al. (2008). The interaction of LT with HL did not involve any further enhancement of sucrose accumulation.

Spores and vegetative cells: two supplementary ways for living under chilling and photochilling stresses

Many species of algae from extreme habitats do not form a specialized cell stage to withstand stresses, and must cope with harsh environmental conditions in their vegetative state by using only ecophysiological adjustments within the limits of their physiological plasticity (Agrawal 2009). In general, vegetative cells under natural or experimentally induced stress conditions differ from nonstressed cells in many morphological traits at the cellular level. Usually, they are characterized by thicker cell walls, larger cytoplasmatic volumes, a smaller vacuole size, and a higher accumulation of products (e.g., starch), among others (Fujikawa et al. 1999). Previous studies observed increased starch accumulation in vegetative cells of Klebsormidium flaccidum under cold-stress conditions and assumed that this compound facilitates freezing tolerance in this species, protecting cellular components, especially the plasma membrane (Nagao et al. 2008). During cold acclimation in the presence of light, the starch content increased and was considered to be a precursor for synthesizing compounds involved in freezing tolerance (Nagao et al. 2008). The results of the present study confirmed these earlier findings, as the number of starch grains increased under HL-LT stress in comparison to the LL-LT treatment (Fig. 7). The occurrence of large mitochondria under HL-LT could be related to an effective respiration, which would be especially needed under this combined stress scenario. However, in Klebsormidium, mitochondria remain unchanged under desiccation stress, as documented by the DIOC6 staining protocol (Holzinger et al. 2011).

In Klebsormidium dissectum, akinetes can be formed as a consequence of prolonged desiccation stress (Morison and Sheath 1985). The term “akinete” (“hypnoblast”) was used because of the inclusion of the original cell wall of the former vegetative cell in the wall of the resting stage (Uzunov et al. 2012, Stoyneva-Gärtner et al. 2019). We decided to use the more neutral term “spore” for this resting stage, as we could not observe any inclusion of the “old” in the “new” spore wall. The rounded spores observed in the present study were initially located in the cell centre. In TEM, ruptured outer cell walls were occasionally visible and the newly formed spore was surrounded by a new cell wall (e.g., Fig. 8B). Asexual processes in Klebsormidium may also lead to the formation of flagellated “zoospores”, which are subsequently released (e.g., Škaloud 2006); but zoospore formation was not observed in the present study.

Our data indicate that spores exhibit higher photoinhibition under chilling stress compared to vegetative cells, probably because their main function is related to survival and dispersal, rather than to other physiological roles. Vegetative cells transform directly into spores, which have a rounded appearance, mostly because the neighboring cells are dead. At the TEM level, it became obvious that an inner cell-wall layer is formed and the outer layer is ruptured. This phenomenon is distinct from the formation of zoospores in Klebsormidium, where flagellated swarmers are formed inside a vegetative cell (Lokhorst 1996). The formation of spores with an extra cell-wall layer was also described by Mikhailyuk et al. (2014) for Klebsormdium and Interfilum. In the present study we observed that the outer cell-wall layer ruptured, which is a prerequisite for dispersal of spores.

Conclusions

In conclusion, the filamentous Klebsormidium cf. flaccidum WD-2-4 investigated in the present study exhibited high photosynthetic performance from 7.5 to 20°C. Chilling stress started at 5°C and was more pronounced when combined with HL conditions. The cold treatment induced the formation of spores, which showed a significantly reduced Fv/Fm compared to vegetative filaments under LL conditions. In contrast, under HL, vegetative cells and spores exhibited similarly reduced levels of Fv/Fm, indicating that the combination of HL and LT can be regarded as particularly stressful for K. cf. flaccidum. Future studies should be directed toward understanding the mechanisms involved in freezing stress, as there are indications in the literature that hardened Klebsormidium cells can even tolerate freezing temperatures (e.g., Elster et al. 2008). Such investigations require, besides the ecophysiological, biochemical, and ultrastructural aspects, a deeper evaluation of the underlying molecular processes.

Abbreviations

A

anteraxanthin

BBM

Bolds basal medium

ETR

electron transport rate

Fv/Fm

photochemical efficiency of PSII

HL

high light

LT

low temperature

M

mitochondrion

N

nucleus

NPQ

non-photochemical quenching

PAM

pulse amplitude modulated

PDA

photodiode array

P

peroxisome

Py

pyrenoid

SAG

Sammlung für Algenkulturen (SAG Algal Culture Collection, Göttingen)

SPD

Savant™SpeedVac™

S

starch grain

Tot Carots

total carotenoids

VAZ

violaxanthin cycle pigment pool (violaxanthin+anteraxanthin+zeaxanthin)

V-cycle

violaxanthin cycle

V

vacuole

Z

zeaxanthin

Acknowledgments

This study was supported by the Deutsche Forschungsgemeinschaft, grant number DFG GU 1278/1-1 (L.G.). Part of the present study was undertaken during the doctoral visit of F.M. at the University of Rostock (Germany), thanks to a PhD fellowship from the Basque Government as well as a travel grant that allowed a collaborative project between the University of the Basque Country (UPV/EHU) and the University of Rostock. F.M. is grateful for a post-doctoral fellowship from the University of the Basque Country (UPV/EHU). The study was also supported by FWF Project I 1951-B16 to A.H., Basque Government (research project UPV/EHU IT-1018-16) and the Spanish Ministry of Science, Innovation and Universities (MCIU/FEDER, UE) (PGC2018-093824-B-C44). We thank Klaus Herburger, PhD (present address: University of Copenhagen, Department of Plant and Environmental Science) and Sabrina Obwegeser, University of Innsbruck, for their help in TEM sample preparation and image generation.

Footnotes

The authors have no conflict of interest to declare.

Author Contributions

F.M., A.H., and L.G. contributed significantly to the development of the experiment and to writing and revising the manuscript. F.M., A.H., L.G., and B.F.M. were actively involved in the sample analysis and interpretation of the results. U.K., J.I.G.P., and L.G. contributed greatly to the conception of the study, revision and supervision of the manuscript. All authors meet all criteria for inclusion in the authorship.

Contributor Information

Fátima Míguez, Department of Plant Biology and Ecology, Faculty of Science and Technology, Leioa, Spain.

Andreas Holzinger, Department of Botany, University of Innsbruck, Sternwartestrasse 15, 6020 Innsbruck, Austria.

Beatriz Fernandez-Marin, Department of Plant Biology and Ecology, Faculty of Science and Technology, Leioa, Spain; Department of Botany, Ecology and Plant Physiology, University of La Laguna (ULL), 38200 La Laguna, Canarias, Spain.

José I. García-Plazaola, Department of Plant Biology and Ecology, Faculty of Science and Technology, Leioa, Spain

Ulf Karsten, Applied Ecology and Phycology, Institute of Biological Sciences, University of Rostock, Albert-Einstein-Str. 3, D-18051 Rostock, Germany.

Lydia Gustavs, Applied Ecology and Phycology, Institute of Biological Sciences, University of Rostock, Albert-Einstein-Str. 3, D-18051 Rostock, Germany; Project Management Julich, Schweriner Str. 44, D-18069 Rostock, Germany.

References

  1. Adams WW, Muller O, Cohu CM, Demmig-Adams B. May photoinhibition be a consequence, rather than a cause, of limited plant productivity? Photosynth Res. 2013;117:31–44. doi: 10.1007/s11120-013-9849-7. [DOI] [PubMed] [Google Scholar]
  2. Agrawal SC. Factors affecting spore germination in algae - review. Folia Biol. 2009;54:273–302. doi: 10.1007/s12223-009-0047-0. [DOI] [PubMed] [Google Scholar]
  3. Bannister P, Neuner G. Frost Resistance and the Distribution of Conifers. Springer; Dordrecht, Netherlands: 2001. pp. 3–21. [Google Scholar]
  4. Darienko T, Gustavs L, Mudimu O, Menendez CR, Schumann R, Karsten U, Friedl T, Pröschold T. Chloroidium, a common terrestrial coccoid green alga previously assigned to Chlorella (Trebouxiophyceae, Chlorophyta) Eur J Phycol. 2010;45:79–95. [Google Scholar]
  5. Demmig-Adams B, Adams WWIII. Non-Photochemical Quenching and Energy Dissipation in Plants, Algae and Cyanobacteria. Springer; Netherlands, Dordrecht: 2014. p. 649. [Google Scholar]
  6. Eggert A, van Hasselt PR, Breeman AM. Differences in thermal acclimation of chloroplast functioning in two ecotypes of Valonia utricularis (Chlorophyta) Eur J Phycol. 2003;38:123–31. [Google Scholar]
  7. Elster J, Degma P, Kováčik L, Valentová L, Šramková K, Batista Pereira A. Freezing and desiccation injury resistance in the filamentous green alga Klebsormidium from the Antarctic, Arctic and Slovakia. Biologia (Bratisl) 2008;63:843–51. [Google Scholar]
  8. Fujikawa S, Jitsuyama Y, Kuroda K. Determination of the role of cold acclimation-induced diverse changes in plant cells from the viewpoint of avoidance of freezing injury. J Plant Res. 1999;112:237–44. [Google Scholar]
  9. García-Plazaola JI, Becerril JM. A rapid high-performance liquid chromatography method to measure lipophilic antioxidants in stressed plants: Simultaneous determination of carotenoids and tocopherols. Phytochem Anal. 1999;10:307–13. [Google Scholar]
  10. Garcia-Plazaola JI, Esteban R. Determination of chloro-phylls and carotenoids by HPLC. PrometheusWiki; 2012. [accessed 23/02/2012]. Available at: http://prometheuswiki.publish.csiro.au/tikiindex.php?page=Determination+of+chlorophylls+and+carotenoids+by+HPLC . [Google Scholar]
  11. Gustavs L, Schumann R, Eggert A, Karsten U. In vivo growth fluorometry: accuracy and limits of microalgal growth rate measurements in ecophysiological investigations. Aquat Microb Ecol. 2009;55:95–104. [Google Scholar]
  12. Holzinger A, Kaplan F, Blaas K, Zechmann B, Komsic-Buchmann K, Becker B. Transcriptomics of desiccation tolerance in the streptophyte green alga Klebsormidium reveal a land plant-like defense reaction. PLoS ONE. 2014;9:e110630. doi: 10.1371/journal.pone.0110630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Holzinger A, Lütz C, Karsten U. Desiccation stress causes structural and ultrastructural alterations in the aeroterrestrial green alga Klebsormidium crenulatum (Klebsormidiophyceae, Streptophyta) isolated from an alpine soil crust. J Phycol. 2011;47:591–602. doi: 10.1111/j.1529-8817.2011.00980.x. [DOI] [PubMed] [Google Scholar]
  14. Holzinger A, Pichrtová M. Abiotic stress tolerance of Charophyte green algae: new challenges for omics techniques. Front Plant Sci. 2016;7:1–17. doi: 10.3389/fpls.2016.00678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Holzinger A, Tschaikner A, Remias D. Cytoarchitecture of the desiccation-tolerant green alga Zygogonium ericetorum . Protoplasma. 2009;243:15–24. doi: 10.1007/s00709-009-0048-5. [DOI] [PubMed] [Google Scholar]
  16. Hori K, Maruyama F, Fujisawa T, Togashi T, Yamamoto N, Seo M, Sato S, et al. Klebsormidium flaccidum genome reveals primary factors for plant terrestrial adaptation. Nat Commun. 2014;5:3978. doi: 10.1038/ncomms4978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jahns P, Holzwarth AR. The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II. Biochim Biophys Acta. 2012;1817:182–93. doi: 10.1016/j.bbabio.2011.04.012. [DOI] [PubMed] [Google Scholar]
  18. Kaplan F, Lewis LA, Wastian J, Holzinger A. Plasmolysis effects and osmotic potential of two phylogenetically distinct alpine strains of Klebsormidium (Streptophyta) Protoplasma. 2012;249:789–804. doi: 10.1007/s00709-011-0324-z. [DOI] [PubMed] [Google Scholar]
  19. Karsten U, Herburger K, Holzinger A. Living in biological soil crust communities of African deserts—Physiological traits of green algal Klebsormidium species (Streptophyta) to cope with desiccation, light and temperature gradients. J Plant Physiol. 2016;194:2–12. doi: 10.1016/j.jplph.2015.09.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Karsten U, Holzinger A. Light, temperature, and desiccation effects on photosynthetic activity, and drought-induced ultrastructural changes in the green alga Klebsormidium dissectum (Streptophyta) from a high alpine soil crust. Microb Ecol. 2012;63:51–63. doi: 10.1007/s00248-011-9924-6. [DOI] [PubMed] [Google Scholar]
  21. Karsten U, Luütz C, Holzinger A. Ecophysiological performance of the aeroterrestrial green alga Klebsormidium crenulatum (Charophyceae, Streptophyta) isolated from an alpine soil crust with an emphasis on desiccation stress. J Phycol. 2010;46:1187–97. doi: 10.1111/j.1529-8817.2011.00980.x. [DOI] [PubMed] [Google Scholar]
  22. Karsten U, Pröschold T, Mikhailyuk T, Holzinger A. Photosynthetic performance of different genotypes of the green alga Klebsormidium sp. (Streptophyta) isolated from biological soil crusts of the Alps. Arch Hydrobiol Suppl Algol Stud. 2013;142:45–62. [Google Scholar]
  23. Karsten U, Rindi F. Ecophysiological performance of an urban strain of the aeroterrestrial green alga Klebsormidium sp. (Klebsormidiales, Klebsormidiophyceae) Eur J Phycol. 2010;45:426–35. [Google Scholar]
  24. Karsten U, Thomas DN, Weykam G, Daniel C, Kirst GO. A simple and rapid method for extraction and separation of low molecular weight carbohydrates from macroalgae using high performance liquid chromatography. Plant Physiol Biochem. 1991;29:373–8. [Google Scholar]
  25. Kitzing C, Pröschold T, Karsten U. UV-induced effects on growth, photosynthetic performance and sunscreen contents in different populations of the green algae Klebsormidium fluitans (Streptophyta) from alpine soil crusts. Microb Ecol. 2014;67:327–40. doi: 10.1007/s00248-013-0317-x. [DOI] [PubMed] [Google Scholar]
  26. Körner C. In: Alpine Treelines. Körner C, editor. Springer Basel; Basel: 2012. Freezing and other forms of stress; pp. 131–49. [Google Scholar]
  27. Kützing FT. Species algarum. Lipsiae. 1849:1–922. [Google Scholar]
  28. Lazár D. Parameters of photosynthetic energy partitioning. J Plant Physiol. 2015;175:131–47. doi: 10.1016/j.jplph.2014.10.021. [DOI] [PubMed] [Google Scholar]
  29. Lokhorst GJC. Reasoning about actions and obligations in first-order logic. Stud Log. 1996;57:221–37. [Google Scholar]
  30. Masojídek J, Grobbelaar J, Pechar L, Koblizek M. Photosystem II electron transport rates and oxygen production in natural waterblooms of freshwater cyanobacteria during a diel cycle. J Plankton Res. 2001;23:57–66. [Google Scholar]
  31. Míguez F, Fernández-Marín B, Becerril JM, Garcia-Plazaola JI. Activation of photoprotective winter photoinhibition in plants from different environments: a literature compilation and meta-analysis. Physiol Plant. 2015;155:414–23. doi: 10.1111/ppl.12329. [DOI] [PubMed] [Google Scholar]
  32. Míguez F, Fernández-Marín B, Becerril JM, García-Plazaola JI. Diversity of winter photoinhibitory responses: a case study in co-occurring lichens, mosses, herbs and woody plants from subalpine environments. Physiol Plant. 2017a;160:282–96. doi: 10.1111/ppl.12551. [DOI] [PubMed] [Google Scholar]
  33. Míguez F, Schiefelbein U, Karsten U, García-Plazaola JI, Gustavs L. Unraveling the photoprotective response of lichenized and free-living green algae (Trebouxiophyceae, Chlorophyta) to photochilling stress. Front Plant Sci. 2017b;8:1–14. doi: 10.3389/fpls.2017.01144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mikhailyuk T, Glaser K, Holzinger A, Karsten U. Biodiversity of Klebsormidium (Streptophyta) from alpine biological soil crusts (Alps, Tyrol, Austria, and Italy) J Phycol. 2015;51:750–67. doi: 10.1111/jpy.12316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mikhailyuk T, Holzinger A, Massalski A, Karsten U. Morphology and ultrastructure of Interfilum and Klebsormidium (Klebsormidiales, Streptophyta) with special reference to cell division and thallus formation. Eur J Phycol. 2014;49:395–412. doi: 10.1080/09670262.2014.949308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mock T, Hoch N. Long-term temperature acclimation of photosynthesis in steady-state cultures of the polar diatom Fragilariopsis cylindrus. Photosynth Res. 2005;85:307–17. doi: 10.1007/s11120-005-5668-9. [DOI] [PubMed] [Google Scholar]
  37. Morison MO, Sheath RG. Responses to desiccation stress by Klebsormidium rivulare (Ulotrichales, Chlorophyta) from a Rhode Island stream. Phycologia. 1985;24:125–45. [Google Scholar]
  38. Nagao M, Matsui K, Uemura M. Klebsormidium flaccidum, a charophycean green alga, exhibits cold acclimation that is closely associated with compatible solute accumulation and ultrastructural changes. Plant, Cell Environ. 2008;31:872–85. doi: 10.1111/j.1365-3040.2008.01804.x. [DOI] [PubMed] [Google Scholar]
  39. Neuner G. Frost resistance in alpine woody plants. Front Plant Sci. 2014;5:654. doi: 10.3389/fpls.2014.00654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Öquist G, Huner NPA. Photosynthesis of overwintering evergreen plants. Annu Rev Plant Biol. 2003;54:329–55. doi: 10.1146/annurev.arplant.54.072402.115741. [DOI] [PubMed] [Google Scholar]
  41. Pichrtová M, Híjek T, Elster J. Osmotic stress and recovery in field populations of Zygnema sp. (Zygnemato-phyceae, Streptophyta) on Svalbard (High Arctic) subjected to natural desiccation. FEMS Microbiol Ecol. 2014;89:270–80. doi: 10.1111/1574-6941.12288. [DOI] [PubMed] [Google Scholar]
  42. Pierangelini M, Raven JA, Giordano M. The relative availability of inorganic carbon and inorganic nitrogen influences the response of the dinoflagellate Protoceratium reticu-latum to elevated CO2 . J Phycol. 2017;53:298–307. doi: 10.1111/jpy.12463. [DOI] [PubMed] [Google Scholar]
  43. Remias D, Lütz-Meindl U, Lütz C. Photosynthesis, pigments and ultrastructure of the alpine snow alga Chlamy-domonas nivalis . Eur J Phycol. 2005;40:259–68. [Google Scholar]
  44. Rindi F. In: Climate Change. Hodkinson T, Jones M, Waldren S, Parnell J, editors. Cambridge University Press, Ecology and Systematics; 2011. Terrestrial green algae: systematics, biogeography and expected responses to climate change; pp. 201–8. [Google Scholar]
  45. Rindi F, Mikhailyuk TI, Sluiman HJ, Friedl T, López-Bautista JM. Phylogenetic relationships in Interfilum and Klebsormidium (Klebsormidiophyceae, Streptophyta) Mol Phylogenet Evol. 2011;58:218–31. doi: 10.1016/j.ympev.2010.11.030. [DOI] [PubMed] [Google Scholar]
  46. Ryšánek D, Hrčková K, Škaloud P. Global ubiquity and local endemism of free-living terrestrial protists: Phylogeographic assessment of the streptophyte alga Klebsormidium . Environ Microbiol. 2015;17:689–98. doi: 10.1111/1462-2920.12501. [DOI] [PubMed] [Google Scholar]
  47. Samolov E, Mikhailyuk T, Lukešová A, Glaser K, Büdel B, Karsten U. Usual alga from unusual habitats: Biodiversity of Klebsormidium (Klebsormidiophyceae, Streptophyta) from the phylogenetic superclade G isolated from biological soil crusts. Mol Phylogenet Evol. 2019;133:236–55. doi: 10.1016/j.ympev.2018.12.018. [DOI] [PubMed] [Google Scholar]
  48. Schlösser UC. Additions to the culture collection of algae since 1994. Bot Acta. 1997;110:424–9. [Google Scholar]
  49. Škaloud P. Variation and taxonomic significance of some morphological features in European strains of Klebsormidium (Klebsormidiophyceae, Streptophyta) Nov Hedwigia. 2006;83:533–50. [Google Scholar]
  50. Škaloud P, Lukešová A, Malavasi V, Ryšánek D, Hrčková K, Rindi F. Molecular evidence for the polyphyletic origin of low pH adaptation in the genus Klebsormidium (Klebsormidiophyceae, Streptophyta) Plant Ecol Evol. 2014;147:333–45. [Google Scholar]
  51. Škaloud P, Rindi F. Ecological differentiation of cryptic species within an asexual protist morphospecies: A case study of filamentous green alga Klebsormidium (Streptophyta) J Eukaryot Microbiol. 2013;60:350–62. doi: 10.1111/jeu.12040. [DOI] [PubMed] [Google Scholar]
  52. Stamenković M, Bischof K, Hanelt D. Xanthophyll cycle pool size and composition in several Cosmarium strains (Zygnematophyceae, Streptophyta) are related to their geographic distribution patterns. Protist. 2014;165:14–30. doi: 10.1016/j.protis.2013.10.002. [DOI] [PubMed] [Google Scholar]
  53. Stoyneva-Gärtner M, Uzunov B, Gärtner G, Radkova M, Ata-nassov I, Atanasova R, Borisova C, Draganova P, Stoy-kova P. Review on the biotechnological and nanotechnological potential of the streptophyte genus Klebsormidium with pilot data on its phycoprospecting and polyphasic identification in Bulgaria. Biotechnol Biotechnol Equip. 2019;33:559–78. [Google Scholar]
  54. Uzunov BA, Gärtner G, Stoynkva MP. Notes on the akinete-forming strain of the green alga Klebsormidium dissectum (Streptophyta) from Pirin Mts, Bulgaria. Phyton - Ann Rei Bot. 2012;52:139–44. [Google Scholar]

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