Unique Dynamics of Paramylon Storage in the Marine Euglenozoan

Protist, Vol. 171, 125717, xx 2020

http://www.elsevier.de/protis

Published online date 7 February 2020

ORIGINAL PAPER

Unique Dynamics of Paramylon Storage

in the Marine Euglenozoan Diplonema papillatum

a,b a a,c

Ingrid Skodová-Sverákováˇ , Galina Prokopchuk , Priscila Pena-Diaz˜ ,

a,c d b d a,e,1

Kristína Záhonová , Martin Moos , Anton Horváth , Petr Simekˇ , and Julius Lukesˇ

a ˇ

Institute of Parasitology, Biology Centre, Czech Academy of Sciences, Ceské Budejoviceˇ

(Budweis), Czech Republic

Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia

Faculty of Science, Charles University, BIOCEV, Vestec, Czech Republic

d ˇ

Institute of Entomology, Biology Centre, Czech Academy of Sciences, Ceské Budejoviceˇ

(Budweis), Czech Republic

e ˇ

Faculty of Sciences, University of South Bohemia, Ceské Budejoviceˇ (Budweis), Czech

Republic

Submitted September 10, 2019; Accepted February 2, 2020

Monitoring Editor: Michael Ginger

Diplonemids belong to the most diverse and abundant marine protists, which places them among

the key players of the oceanic ecosystem. Under in vitro conditions, their best-known representa-

tive Diplonema papillatum accumulates in its cytoplasm a crystalline polymer. When grown under the

nutrient-poor conditions, but not nutrient-rich conditions, D. papillatum synthesizes a ␤-1,3-glucan

polymer, also known as paramylon. This phenomenon is unexpected, as it is in striking contrast to the

accumulation of paramylon in euglenids, since these related ﬂagellates synthesize this polymer solely

under nutrient-rich conditions. The capacity of D. papillatum to store an energy source in the form of

polysaccharides when the environment is poor in nutrients is unexpected and may contribute to the

wide distribution of these protists in the ocean.

Key words: ␤-1,3-glucan; paramylon; trehalose; diplonemids; paramylon synthase.

Introduction 2015). The capacity of protists to inhabit virtually

every niche in the world ocean is reﬂected in their

metabolism, which is often highly complex and ver-

Protists represent the bulk of extant eukaryotic

satile due to their gene-rich genomes (Keeling et al.

diversity, yet their abundance and ecological sig-

2014; Keeling and del Campo 2017).

niﬁcance in the oceans is being appreciated only

One of the prominent groups are diplonemids, a

recently (de Vargas et al. 2015; Worden et al.

sister clade of the mostly parasitic kinetoplastids

and the free-living euglenids, all belonging to the

1 phylum Euglenozoa (Adl et al. 2018). Diplonemids

Corresponding author; th

are the most diverse and the 6 most species-rich

e-mail [email protected] (J. Lukes).ˇ

https://doi.org/10.1016/j.protis.2020.125717

2 I. Skodová-Sveráková, G. Prokopchuk, P. Pena-Diaz˜ et al.

group of marine protists (de Vargas et al. 2015; survival in the mammalian host (Ralton et al. 2003;

Flegontova et al. 2016). The available sequence Sernee et al. 2006). Thus, the polymer synthesis

data shows that they are non-photosynthetic, which and remodeling mostly depend on the parasite life

is consistent with their preference for mesopelagic stage, rather than on carbon availability.

and bathypelagic habitats (Flegontova et al. 2016; Out of the diverse euglenozoans with lifestyles

Lara et al. 2009). So far, the only diplonemid exper- ranging from parasitism to photosynthesis (Adl et al.

imentally studied to some extent is Diplonema 2018), only the euglenophytes carrying the sec-

papillatum, best-known for the unprecedented ondary plastid are known to deposit glucose via

␤

complexity of its mitochondrial genome and tran- -1,3-glycosidic linkage, forming a storage polysac-

scriptome (Valach et al. 2017). Moreover, studies charide known as paramylon (Kiss and Triemer

implementing genetic modiﬁcations in D. papillatum 1988). However, outside of the euglenozoans, lin-

have begun to turn this ﬂagellate into a genetically ear ␤-1,3-glucans are widely distributed in bacteria,

tractable organism (Kaur et al. 2018). haptophytes, fungi, lichens and some plants, where

D. papillatum has also been the subject of the the polymer carries different names, such as curd-

only metabolic study performed on diplonemids so lan, pachyman, laricinan or callose (Archibald et al.

far, which revealed that they compartmentalize ﬁve 1963; Aspinall and Kessler 1957; Clarke and Stone

of the ﬁrst seven enzymes of the gluconeogenic 1960; Harada et al. 1968; Warsi and Whelan 1957).

pathway into peroxisomes, which are thus reminis- Paramylon is an insoluble, linear ␤-1,3-glucan of

cent of glycosomes (Morales et al. 2016). While high molecular mass, which occurs naturally in the

hexokinase and phosphofructokinase do not seem crystalline form (Booy et al. 1981). Euglenids accu-

to be expressed, D. papillatum was shown to per- mulate paramylon in granules, which may be widely

form gluconeogenesis. Consequently, it has been distributed in the cytoplasm, may form caps over

hypothesized that this protist is incapable of per- the pyrenoids or be packed together (Monﬁls et al.

forming glycolysis, with the gluconeogenic ﬂux 2011). The various types of paramylon were sepa-

originating from the metabolism of amino acids rated into six morphological categories, all present

being routed through the pentose phosphate path- in the diverse euglenids (Monﬁls et al. 2011).

way (Morales et al. 2016). In trypanosomes and The formation of this polysaccharide follows

related ﬂagellates, the ﬁrst seven of 10 glycolytic a general pathway where a uridine diphosphate

enzymes are also compartmentalized in glyco- glucose (UDP-Glc) donates the glucose moiety

somes, a hallmark of these protists (Bringaud et al. that is subsequently linked to form the grow-

2006; Opperdoes and Borst 1977). They metab- ing polysaccharide. Glucose for the UDP-Glc

olize glucose and amino acids, with proline and precursor originates from various sources and

threonine being essential for energy production the formation of the sugar nucleotide depends

(Coustou et al. 2003; Millerioux et al. 2013). Gluco- on UTP:␣-D-glucose uridylyltransferase or UDP-

neogenesis has been reported from trypanosomes Glc pyrophosphorylase (Muchut et al. 2018). In

as a ﬂux that may be directed through the pen- euglenids, paramylon formation requires the activ-

tose phosphate pathway for the regeneration of ity of paramylon synthase (Bäumer et al. 2011;

cytosolic NADPH (Allmann et al. 2013). This unique Marechal and Goldemberg 1964; Tanaka et al.

compartmentalization of glycolytic enzymes has 2017), which is a membrane-bound enzyme com-

been proposed as an adaptation to their special- plex of approximately 670 kDa belonging to the

ized lifestyles (Haanstra et al. 2016; Szöor et al. eukaryotic family of glycosyltransferases 48 (GT48)

2014). (Bäumer et al. 2011). So far, two glucan synthase-

Although trypanosomes lack the capability of like genes, called EgGSL1 and EgGSL2, were

storing carbohydrates (Bakker et al. 2000), they found in Euglena gracilis. They code for proteins

may store carbon sources in the form of lipid of 304 and 258 kDa, with 15 and 19 transmem-

droplets (Allmann et al. 2014; Smith et al. 2017). brane domains, respectively (Tanaka et al. 2017).

Moreover, members of the genus Leishmania dif- The synthesis of paramylon, however, relies on the

fer from trypanosomes in that they synthesize a activity of EgGSL2, which forms a complex with

carbohydrate polymer that resembles a mannose 37 and 54 kDa UDP-binding proteins (Tanaka et al.

polymer or mannan (Blum 1993). Leishmania mex- 2017). The activity of paramylon synthase is associ-

icana was subsequently shown to synthesize and ated with the membrane fraction that surrounds the

␤

store -1,2-mannan, a polymer of mannose. How- paramylon granules (Bäumer et al. 2011). Degra-

ever, glycopolymers in Leishmania species are dation of ␤-1,3-glucan involves the action of endo-

linked to the assembly of the glycocalyx coat and and exo-␤-1,3-glucanases found in various glyco-

have an essential function in their infectivity and

Paramylon in Diplonema 3

side hydrolase families (Henrissat 1991; Henrissat medium. Since tryptone is the main carbon source

and Bairoch 1996; Henrissat and Davies 1997). in this medium, and to a lower extent, of nitro-

Paramylon has been extensively studied in gen, hereafter we refer to them as carbon-rich and

E. gracilis, where its synthesis is usually trig- carbon-poor media. Both media had 3.6% salinity

gered by growth under the nutrient-rich conditions and contained 1% (v/v) horse serum. Inoculation

(Calvayrac et al. 1981), represented by either pho- of the cells into the carbon-poor medium resulted

tosynthetic carbon assimilation or heterotrophic in a signiﬁcant growth retardation and a switch

growth on a suitable carbon source, such as sug- of the cells into what may be considered a per-

ars or organic acids (Briand and Calvayrac 1980). manent stationary phase (Fig. 1A), as compared

The dynamics of paramylon synthesis in E. gracilis to the carbon-rich culture condition, under which

is inﬂuenced by light, as the metabolite accumu- the cells grew exponentially for 11 days (264 hrs)

lated during the dark phase may be consumed (Fig. 1A). A signiﬁcant accumulation of cytosolic

for chlorophyll synthesis in the presence of light vesicles occurred in cells grown in the carbon-poor

(Calvayrac et al. 1981; Dwyer and Smillie 1970; medium (Fig. 1B). When viewed with a light micro-

Schwartzbach et al. 1975). When E. gracilis is cul- scope, diplonema cells appeared to have higher

tured under carbon starvation, the breakdown of motility in the carbon-poor medium as compared

paramylon is triggered by light, while this process to the carbon-rich one, with their morphology unal-

is slower in cells grown in the dark (Dwyer and tered (Fig. 1B).

Smillie 1970). Paramylon hydrolysis may also be

triggered by several factors other than metabolic Characterization of Paramylon

requirements, such as osmotic stress or environ-

The carbohydrate polymer isolated from approx-

mental adaptations (Takenaka et al. 1997).

6 7

imately 8 × 10 and 4 × 10 E. gracilis and D.

Recent studies demonstrated the usefulness of

papillatum cells, respectively, were subjected to

dietary paramylon for therapeutic purposes, namely

acid hydrolysis in order to obtain monomers. Fol-

its activity against fatty liver disease and its antitu-

lowing this treatment, the samples were submitted

mor activity in mice, where it signiﬁcantly reduced

to thin layer chromatography (TLC) to assess

colon cancer (Nakashima et al. 2019; Watanabe

their composition (Fig. 2A). The monosaccha-

et al. 2013). It has also been tested as a dietary

ride proﬁles of D. papillatum cultivated in the

supplement in mouse models, as it reduces devel-

carbon-poor medium and E. gracilis cultivated

opment of adipocyte differentiation in human cell

in the complete medium, which was used as

tissues and enhances the immune system (Kondo

control, comprise of a single band co-migrating

et al. 1992; Nakashima et al. 2019; Okouchi et al.

with the glucose standard (Fig. 2A). The total

2019; Sugimoto et al. 2018).

monosaccharide proﬁles of D. papillatum grown

In this report, we isolated and analyzed a car-

either in the carbon-rich or carbon-poor media

bohydrate polymer bearing ␤-1,3-glucan linkages

(Fig. 2B) display more complexity and consisted

from D. papillatum grown under carbon-poor condi-

of galactose, glucose, ribose, rhamnose and one

tions. The isolated polymer has the same properties

monosaccharide that did not match any of the

as paramylon from E. gracilis. We show that

used standards (ribose, mannose, glucose, rham-

accumulation of this polymer depends on speciﬁc

nose, galactose, arabinose). The ratio between the

physiological conditions encountered by diplone-

determined monosaccharides strongly depended

mids during their life cycle and possibly contributes

on the cultivation medium, as was shown by the

to their success in the least habitable zones of the

TLC and gas chromatography–mass spectrometry

world oceans.

(GC–MC) analyses of saccharides. Indeed, both

methods revealed an increase in glucose and other

Results sugars levels, except fructose and myo-inositol

(Fig. 2B; Table 1). Accumulation of sugars was

Cell Growth in Carbon-depleted noticeably lower in cells grown in the carbon-rich

Conditions medium (Fig. 2B).

We assessed the viability of D. papillatum in two dif- Identiﬁcation of ˇ-1,3-glucan Linkage

ferent media: (1) a carbon-rich medium, or standard Within the Cells

growth medium, and (2) a carbon-poor medium,

in which the concentration of tryptone is 100× To determine the presence of a polymer harbouring

lower than that present in the standard growth a ␤-1,3-glucan linkage within the cells, D. papil-

4 I. Skodová-Sveráková, G. Prokopchuk, P. Pena-Diaz˜ et al.

Figure 1. Growth (A) and morphology (B) of D. papillatum under different cultivation conditions. 5 × 10 cells

were inoculated into the carbon-rich (full line) and carbon-poor media (dashed line). The dynamics of growth

was monitored every 24 hours over 18 days, when the number of cells dropped to initial concentration. (B)

Morphology of cells was examined by light microscopy at 24, 72 and 240 hours (see black arrows in A). Scale

bar – 10 ␮m.

Figure 2. TLC analysis of monosaccharides. (A) Hydrolyzed ␤-1,3-polymers from E. gracilis and D. papillatum.

(B) Total monosaccharide proﬁle of D. papillatum grown in the carbon-rich (rich) and carbon-poor (poor) media.

Individual monosaccharides were identiﬁed using standards resolved on TLC along with the samples. *, does

not match to any of the available standards.

Paramylon in Diplonema 5

Figure 3. Detection of ␤-1,3-glucan by immunoﬂuorescence, in D. papillatum grown in the carbon-rich (A) and

carbon-poor conditions (B), Euglena gracilis (C) and Trypanosoma brucei (D). E. gracilis and T. brucei served

as positive and negative controls, respectively. Cells were treated at 24 (R24, P24, E24 and T24), 72 (R72, P72,

E72 and T72) and 240 hours (R240, P240 and E240) with anti-␤-1,3-glucan monoclonal antibody and visualized

using red-ﬂuorescent Alexa Fluor 555 goat anti-mouse antibody. DAPI was used to stain the nucleus. Ph, phase

contrast. Scale bar – 5 ␮m.

Table 1. GC-MS analysis of polyols and saccharides medium within the ﬁrst 24 hours of growth, with the

in D. papillatum. intensity of staining decreasing with the cultivation

time. Trypanosoma brucei, which lacks the capac-

Medium Rich Poor

ity to synthesize paramylon and hence does not

Concentration [nmol/6 × 10 cells]

show any signal with the above antibody, served

as a negative control (Fig. 3).

Ribose 0.022 ± 0.002 0.032 ± 0.007

Arabinitol 0.031 ± 0.004 0.100 ± 0.027

± ±

Ribitol 0.016 0.001 0.021 0.009 Dynamics of Paramylon Accumulation

Fructose 0.050 ± 0.010 0.036 ± 0.008

Cells were also subjected to the analysis of paramy-

Glucose 0.058 ± 0.018 0.067 ± 0.020

lon accumulation at three time points, namely at 24,

Myo-inositol 0.366 ± 0.052 0.359 ± 0.053

72, and 240 hours after inoculation of the culture

Trehalose 0.110 ± 0.056 0.223 ± 0.093

(Fig. 1A). For each time point, 3 × 10 cells were

The concentration [nmol] of polyols and saccharides

collected from the carbon-rich and carbon-poor

detected in 6 10 cells. Cells were harvested after media and subsequently lyophilized. The polysac-

72 hours of cultivation in the carbon-rich (rich) and

charide was then isolated and hydrolyzed to its

carbon-poor (poor) media, washed in 3.6% sea salt

monomers. We detected paramylon-derived glu-

solution and analyzed by GC-MS.

cose by spectrophotometric measurement only at

the 72 hours time point. Glucose concentration was

more than four times lower in cells grown in the

latum was grown in carbon-rich and carbon-poor

carbon-rich medium as compared to cells derived

media and harvested at different time points (24, 72,

from the carbon-poor medium (Fig. 4A).

and 240 hours). The cells were subsequently ﬁxed

and labeled with a monoclonal antibody speciﬁc Trehalose

for ␤-1,3-glucan. Cells cultured in the carbon-poor

medium stained strongly, particularly after 24 and Under certain cultivation conditions, paramylon

72 hours of growth (Fig. 3), whereas the immunoﬂu- degradation in E. gracilis is linked with trehalose

orescence signal became much weaker in a 10 synthesis (Takenaka et al. 1997). Hence, we

days-old culture. In cells grown in the carbon- focused on this disaccharide, known to be a stress

rich medium, the anti-␤-1,3-glucan antibody usually factor. Trehalose was extracted from 3 × 10 cells

recognized just a single cytosolic vesicle of a kept for 24, 72, and 240 hours in both the carbon-

variable size (Fig. 3). The E. gracilis cells, used rich and carbon-poor media. Sugars obtained from

as a positive control for ␤-1,3-glucan staining, the acetone cell fraction were resolved by TLC,

showed intense ﬂuorescence signal in the complete where amongst two visualized signals, one co-

6 I. Skodová-Sveráková, G. Prokopchuk, P. Pena-Diaz˜ et al.

Figure 4. Glucose concentration. Paramylon (A) and trehalose (B) were isolated and hydrolyzed to their

monomers. Glucose concentration was then speciﬁed by spectrophotometric measurement. Values of one

of three independent experiments are shown. x axis – time points of cultivation prior to the sugar extraction; y

axis – ␮g of glucose per 3 × 10 cells. Rich – carbon-rich medium; poor – carbon-poor medium.

Figure 5. Accumulation of trehalose. After 24, 72 and 240 hours of cultivation in the carbon-rich (rich) or carbon-

poor (poor) media, 3 × 10 cells were subjected to the TLC analysis of trehalose.

migrated with the trehalose standard (Fig. 5). 72 hours in carbon-poor medium, when compared

Trehalose seemed to be the only variable signal on to those in carbon-rich conditions (Table 1).

the TLC proﬁle, as in cells grown in carbon-poor To determine the origin of glucose forming

medium, the amount of trehalose increased with paramylon and trehalose, cells were fed with

14 14

time. In contrast, a stably low level of this metabo- radiolabeled C-glucose and C-proline. When

lite was observed in cells grown in the carbon-rich C-proline was used, incorporation of the labeled

medium (Fig. 5). The same trend was observed at carbon into paramylon and trehalose increased

the concentration of glucose obtained by hydrolysis when cells were cultivated in the carbon-poor

of trehalose isolated from the same cells (Fig. 4B). medium (Tables 2 and 3). When C-glucose was

Through GC–MS analysis, we observed two times used, only a small trace of radiolabeled trehalose

higher concentration of trehalose in cells kept for was observed in the carbon-poor medium (Table 3).

Paramylon in Diplonema 7

Table 2. Paramylon labeling with C[U] substrates.

C[U]- Sample Radioactivity Total radioactivity

taken up

dpm %

rich 60 0

Proline

poor 24900 0,3

rich 0 0

Glucose

poor 0 0

5 × 10 cells were cultivated for 24 hours in the

carbon-rich (rich) and carbon-poor (poor) media sup-

14 14

plemented with C-proline or C-glucose. Radioac-

tivity in the isolated paramylon was quantiﬁed by

Liquid Scintilation Counter; dpm – disintegration per minute.

Table 3. Trehalose labeling with C[U] substrates.

C[U]- Sample Radioactivity Total radioactivity

taken up

dpm %

rich 564800 11,3

Proline

poor 1596000 44,9

rich 0 0

Glucose

poor 25600 0,4

5 × 10 cells were cultivated for 24 hours in the

carbon-rich (rich) and carbon-poor (poor) media sup-

plemented with radioactively labeled C-proline or

C-glucose. Radioactivity in the trehalose containing

acetone fraction was quantiﬁed by Liquid Scintilation

Counter; dpm – disintegration per minute.

To clarify whether proline is the actual precursor

used for glucose synthesis, labeled monosaccha-

rides obtained from 5 10 D. papillatum cells Figure 6. Detection of labeled monosaccharides.

grown in both types of media were puriﬁed, sep- Autoradiogram of labeled monosaccharides obtained

arated on TLC and exposed overnight on an X-ray from 5 × 10 cells grown in the carbon-rich (rich) and

ﬁlm. The autoradiogram showed high intensity carbon-poor media (poor), both supplemented with

of glucose labeling in the carbon-poor medium C[U]-proline. Labeled monosaccharides were iden-

(Fig. 6). Moreover, other signals that did not tiﬁed using cold standards. *,** may represent either

match the used standards (Fig. 6) were still some- phosphorylated monosaccharides or amino acids.

what higher than those in the carbon-rich medium ***,**** identity of these monosaccharides could not

(Fig. 6). Unidentiﬁed signals may represent either be established using the available standards.

monosaccharides or amino acids. Mobility of the

labeled proline was evaluated using a set of

standards. Despite the fact that galactose and Enzymes of Paramylon Metabolism in

ribose were among the prominent sugar monomers Euglenozoans

together with glucose (Fig. 2C), no labeling of

these sugars was intercepted on autoradiogram

We have analyzed the sequences of glucan syn-

after 24 hours of labeling (Fig. 6).

thase from the genome of D. papillatum and

8 I. Skodová-Sveráková, G. Prokopchuk, P. Pena-Diaz˜ et al.

Figure 7. Phylogenetic analysis of glucan synthase proteins. Diplonemid sequences represent two paralogs

inside the highly supported euglenozoan clade. For simplicity, broader clades were collapsed and are repre-

sented by triangles. Protein domains identiﬁed by InterProScan are mapped onto the tree and explained in the

graphical legend below the tree. Bootstrap support values are given when ≥75. A full version of the tree is

provided as Supplementary Material Figure S1.

other diplonemids available in culture (Prokopchuk homologous to the glucanases, which may serve in

et al. 2019; Tashyreva et al. 2018a, b). In these the hydrolysis of paramylon.

genomes, three paralogues of glucan synthase A search for glucanases in diplonemids revealed

were found, except for D. papillatum, which pos- only glucanase domains fused to glucan syn-

sesses only two of them. Diplonemid sequences thases. No other glucanase sequences were

form a highly supported monophyletic clade along- retrieved from the diplonemid dataset, except for

side other euglenozoan sequences (Bodo saltans Diplonema japonicum, which possesses a glu-

representing kinetoplastids, and Euglena longa and canase without any additional domain, and is

E. gracilis representing euglenids) (Fig. 7; Sup- unrelated to those synthases with fused architec-

plementary Material Fig. S1). Interestingly, the ture yet shows similarity to a sequence retrieved

sequences of glucan synthases exhibited an extra from B. saltans (Supplementary Material Fig. S2).

domain at their N-termini (Fig. 7). One diplonemid Although the posterior mean site frequency method

paralog contains an extra glucanosyltransferase was employed to build the phylogenetic tree (Wang

domain (GH72, IPR004886; lacking in D. papil- et al. 2018), which should prevent the long-branch

latum), while the other two paralogs each bear attraction phenomenon, we cannot rule out that the

a glycosyl hydrolase domain (GH81, IPR040720) clustering of these two sequences may represent

Paramylon in Diplonema 9

an artifact. Surprisingly, we observed an expansion the ﬁrst 24 hours of growth in a carbon-rich medium

of the glucanase protein family in euglenids, as they (Briand and Calvayrac 1980). On the contrary, in D.

encode 12 paralogs of this enzyme. Two paralogs papillatum the polysaccharide biogenesis is stimu-

form a monophyletic group with diplonemids, while lated by the depletion of carbon and nitrogen in the

the other paralogs are related to stramenopiles medium. The dynamics of polymer accumulation is

(Supplementary Material Fig. S2). also distinct, with the culmination at 72 hours, fol-

␤

E. gracilis encodes also -1,3-glucan phospho- lowed by a decline. Such dynamics were conﬁrmed

rylase, another enzyme for paramylon degradation by two independent methods, namely by measur-

(Kuhaudomlarp et al. 2018). We failed to iden- ing glucose concentration in the isolated polymer,

tify this enzyme in B. saltans genome and all of and by immunodetection of the ␤-1,3-linkage within

the diplonemid datasets even when HMMER, a the cells. To the best of our knowledge, the synthe-

more sensitive method than BLAST, was employed. sis of storage macromolecules was documented

␤

Thus, -1,3-glucan phosphorylase was apparently solely under the nutrient-rich conditions (Barsanti

acquired in the Euglenid lineage, and the retrieved et al. 2001; Pagni et al. 1992; van Loosdrecht et al.

glucanases are the only enzymes involved in 1997), making D. papillatum unique in this respect.

paramylon degradation in diplonemids. Hence, we can only speculate what advantage D.

papillatum gains from the accumulation of paramy-

lon under carbon-poor conditions. The possibility of

a stress response to a shift from glucose-rich con-

Discussion

ditions to the poor ones can be excluded since the

cells used in this study were priorly adapted to long-

Although the synthesis of storage polysaccha- term cultivation in carbon-poor medium. Moreover,

ride(s) is a highly beneﬁcial metabolic adaptation, cultivation in different media was accompanied by

it is not as frequent as one would intuitively pronounced changes in cell motility. The lack of

expect. Indeed, within the widespread and eco- nutrients induced the formation of an extremely

logically highly relevant euglenozoan protists, only motile form apparently adapted to active swimming,

euglenids are known to be capable of the anabolic most probably in order to move into more favor-

pathway of paramylon synthesis. Paramylon is the able conditions. Another notable feature restricted

simplest ␤-1,3-glucan, insoluble in water, with a to the starving cells was the formation of vacuo-

linear unbranched structure (Gottlieb 1850). The lar structures in their cytoplasm. Diatoms, another

side-branching changes the physical properties prominent group of marine protists, use extensive

of the polymer from water-insoluble to water- formation of vacuoles as a tool for maintaining nutri-

soluble (Westerlund et al. 1993) and hence, not tion and energetic balance under limited resources

all crystalline glucose polymers may be labeled as (Raven 1997). However, in D. papillatum the accu-

paramylon-like structures (Kiss and Triemer 1988). mulation of paramylon is not in direct correlation

Here we show that a protocol for the isolation of with the cytoplasmic vacuolization in the carbon-

paramylon from E. gracilis applied to D. papilla- poor media, since vacuoles remained abundant

tum yielded a polymer with identical properties. throughout the 10 days-long period of observation,

It is insoluble in water and responsive to acid whereas the amount of paramylon dropped.

hydrolysis, which produces glucose monomers. It has been previously shown that the uptake of

Importantly, D. papillatum grown in media with dif- glucose by D. papillatum is negligible in carbon-

ferent tryptone content modiﬁed the intensity of rich medium (Morales et al. 2016). Similar results

staining with the antibody against the ␤-1,3-linkage. were also obtained in our experiments, where radi-

This bond connects the glucose monomers, sug- olabeled glucose failed to be incorporated into

gesting that the amount of the polymer is subject the isolated paramylon. Feeding D. papillatum with

to signiﬁcant ﬂuctuations triggered by different con- C-glucose resulted in trace labeling of trehalose

centrations of tryptone, which in itself is a mixture under carbon-poor condition, while no labeling was

of (non)essential amino acids and longer peptides. detected in the isolated polymer. C-proline was

This observation links the dynamics of storage to abundantly incorporated into trehalose and to a

carbon and nitrogen availability. much smaller extent into paramylon. Therefore,

Paramylon was detected predominantly in cells it seems unlikely that D. papillatum synthesizes

grown in the carbon-poor medium. Interestingly, paramylon directly from glucose. Our results indi-

this observation is in striking contrast with what cate that trehalose is synthesized independently

has been described for the well-studied E. gracilis, of paramylon degradation. This situation is differ-

where the paramylon synthesis is most active in ent from what is known for E. gracilis, where the

10 I. Skodová-Sveráková, G. Prokopchuk, P. Pena-Diaz˜ et al.

radioactivity of C-paramylon was stoichiometri- While the carbohydrate synthesis is usually a

cally incorporated into trehalose (Takenaka et al. consequence of an abundant food supply, D. papil-

1997). This observation is in line with previous ﬁnd- latum seems to initiate this pathway under low

ings, according to which the gluconeogenic ﬂux nutrition conditions. Here we propose that paramy-

is achieved via the metabolism of amino acids lon, in which glucose monomers are linked via the

␤

associated with re-routing of carbons through the -1,3-glycosidic bond, provides a hitherto unknown

glucan synthase activity, eventually leading to the function, which may represent one of the key adap-

formation of a carbohydrate polymer (Morales et al. tations that are behind the enormous success of

2016). the enigmatic diplonemid ﬂagellates in the world’s

Expectedly, in phylogenetic analysis glucan oceans.

synthases of diplonemids, kinetoplastids and

euglenids form a monophyletic clade rather distinct

from other eukaryotes. However, a striking differ- Concluding Remarks

ence between the glucan synthases of the marine

D. papillatum and freshwater E. gracilis exists,

Diplonemids successfully inhabit the various

as the former exhibit an extra N-terminal domain

depths of the world oceans. Their life strategy is

homologous to glucanases. A similar branching

still unknown although several scenarios have been

order of diplonemid sequences in both glucan syn-

proposed, each of which may be valid under cer-

thase and glucanase phylogenies suggests that a

tain conditions. The ability of D. papillatum, a model

fusion of these two domains in the common ances-

species of the group, to synthesize storage polysac-

tor of diplonemids was followed by a duplication

charides under nutrient-low conditions may point

event. Hence, these two paralogs bear both syn-

at the phenomenon of a unique metabolic adap-

thetic and degrading activities.

tation that allows diplonemids to occupy virtually

So far, the carbohydrate metabolism in D. papil-

every niche in the world’s oceans including the least

latum studied under the carbon-rich conditions

habitable zones.

seemed to lack hexokinase and phosphofructok-

inase, key enzymes in the phosphorylation of Methods

glucose (Morales et al. 2016). This situation was

interpreted as leading to a gluconeogenic ﬂux

Cell cultivation: D. papillatum cells were inocu-

that is further channeled into the pentose phos-

lated into carbon-rich and carbon-poor media, at a

phate pathway, making use of the synthesized 5

concentration of 5 × 10 cells/ml. The carbon-rich

glucose. Our experiments support this view. The

medium contained 36 g/l sea salts, 1 g/l tryptone

most plausible scenario postulates an alternative

and 1% (v/v) horse serum, while the carbon-

route using amino acids as gluconeogenic sub-

poor medium consisted of 36 g/l sea salts, 0.01 g/l

strate, the product of which is stored in the form

tryptone and 1% (v/v) horse serum. Cells were sub-

of a polysaccharide that maintains viability when

jected to long-term cultivation in these media types

◦

the cells are depleted of a constant carbon sup-

at 15 C stationary, with subculturing every seven

ply. Such interpretation of the available data implies

days. Both cultures were kept in this manner for

the existence of several intriguing features: (1) a

at least 18 days prior to the experiments, and the

quorum-sensing system that allows maximum cell

growth was monitored on a daily basis. For polymer

growth using gluconeogenesis under the carbon-

and sugars isolation, cells were harvested on the

rich conditions; (2) a secondary system for the

third day. For the study of the dynamics of polymer

re-routing of the gluconeogenic ﬂux towards the

accumulation, cells were collected at three selected

activity of glucan synthase, which leads to the sta-

time points (24, 72 and 240 hours).

tionary growth phase and paramylon synthesis,

E. gracilis cells were cultivated axenically in Hut-

◦

and, ﬁnally (3) glycolysis activated only under cer-

ner liquid medium (Hutner et al. 1966) at 27 C

tain conditions, particularly when amino acids are 2 1

under permanent light conditions (10 ␮m/m s )

not available and the cells contain stored carbon.

and constant shaking. The procyclic stage of T. bru-

◦

An alternative for the third requirement is the usage

cei (strain 29-13) was cultured at 27 C in SDM79

of glucose for the synthesis of trehalose, a non-

medium containing 10% (v/v) heat-inactivated fetal

reducing disaccharide found in most eukaryotes

bovine serum and 2.5 mg/ml hemin (Changmai

except mammals (Avonce et al. 2006). We suggest

et al. 2013).

that in the carbon-poor medium, trehalose functions

Light microscopy: For light microscopy imag-

as a protectant against protein degradation.

ing, a drop with live cells at three time points

Paramylon in Diplonema 11

(24, 72 and 240 hours of cultivation) was trapped in the mixture of ethyl acetate:pyridine:acetic

between a glass slide and a coverslip and exam- acid:water (6:3:1:1). Saccharides were visualized

ined at 100 under the Olympus BX53 microscope as described above, while trehalose was extracted

equipped with differential interference contrast from the acetone fraction obtained after acetone

(DIC). Recordings were taken with a DP72 dig- sonication (see Polysaccharide isolation). Ace-

ital camera at 1600 1200-pixel resolution using tone was dried under N2 and trehalose was

CellSens software v. 1.11 (Olympus). The images re-extracted from the pellet by a mixture of chloro-

were processed using Image J v. 1.51 software. form:methanol:water (4:2:1). The water phase was

Polysaccharide isolation: For polysaccharide dried using a vacuum concentrator and the samples

extractions, 4 10 cells were harvested from were resuspended in distilled water and developed

72 hours-old cultures grown in carbon-rich and together with the trehalose standard by TLC in

carbon-poor media. Cells were collected by cen- butanol:methanol:water (5:3:2). Visualization was

trifugation at 1000 g for 10 min, washed in 3.6% sea performed as described above. Three independent

salts and freeze-dried (Alpha 1-2 LDplus Freeze experiments were performed.

Dryer). Polymer was extracted as described else- Determination of glucose concentration:

where (Tanaka et al. 2017). Brieﬂy, the dried cells Concentration of glucose in 3 × 10 cells at three

were defatted by dual sonication in acetone and time points (24, 72 and 240 hours of cultivation) was

samples were spun at 5000 g for 5 min. Ace- identiﬁed for paramylon and trehalose hydrolyzed

tone was retained for further analysis. To remove in 2 M triﬂuoroacetic acid by spectrophotometric

proteins from the defatted cells, these were resus- method for hexoses at 490 nm in sulfuric acid and

pended in 1% (w/v) SDS and boiled for 30 min in a phenol (Dubois et al. 1956).

water-bath. The polymer was collected by centrifu- Immunoﬂuorescence analysis: Cells were

gation at 5000 g for 5 min and rinsed once with 0.1% ﬁxed for 30 min with 4% paraformaldehyde in either

(w/v) SDS and twice with distilled water, undergoing artiﬁcial sea water for D. papillatum or PHEM buffer

centrifugation under the same conditions between (Schliwa and van Blerkom 1981) for E. gracilis and

these steps. phosphate buffer (PBS) for T. brucei, and allowed

Determination of polysaccharide composi- to adhere onto poly-L-lysine coated glass slides.

tion: Isolated polysaccharide was hydrolyzed with The bound cells were permeabilized with 100%

500 ␮l of 2 M triﬂuoroacetic acid for 2 hours at ice-cold methanol or 0.1% Igepal in case of E.

◦ ◦

120 C to release monomers. After cooling down gracilis for 20 min at 4 C, washed and incubated

to room temperature, samples were dried under in the blocking solution (5% nonfat milk, 0.05%

nitrogen gas (N2). Residual triﬂuoroacetic acid Tween-20 in PBS or PHEM containing 3% BSA) for

was removed from pellets by two subsequent 45 min. The slides were then incubated overnight

washes with methanol and distilled water and dried with monoclonal mouse anti-␤-1,3-glucan antibod-

under N2 between washes. Saccharide monomers ies (Biosupplies) at a dilution of 1:500, washed

were extracted with 1 ml of chloroform:water (1:1). with PBS and incubated for 1 hour at room tem-

The water phase was evaporated using a vac- perature with goat anti-mouse secondary antibody

uum concentrator (CentriVap) and the obtained conjugated to Alexa Fluor 555, diluted 1:1000. The

monosaccharides were dissolved in water and ana- samples were subsequently mounted in ProLong

lyzed by thin layer chromatography (TLC) (Kieselgel Gold antifade reagent (Life Technology) containing

60, Merck). Samples were developed twice in ethyl 4 ,6-diamidino-2-phenylindole (DAPI) and exam-

acetate:pyridine:acetic acid:water (6:3:1:1). Stan- ined by Olympus BX53 ﬂuorescence microscope.

dards of 200 nmol, each of rhamnose, ribose, Gas chromatography–mass spectrometry

arabinose, mannose, glucose, and galactose were (GC–MS): For GC-MS analysis, 6 × 10 cells were

run in the same fashion and visualized by ␣- collected from carbon-rich and carbon-poor media

◦

naphthol with subsequent heating at 120 C for on the third day after inoculation. Pellets were

10 min. This process was repeated three times on washed with a salts solution mimicking seawater

independent samples. (450 mM NaCl; 10 mM KCl; 9 mM CaCl2; 30 mM

Total sugars and trehalose extraction: Total MgC12 × 6H2O; 16 mM MgSO4 × 7H2O), collected

monosaccharide proﬁles were obtained from in an 1.5 ml tube and immediately extracted with

× 7 ␮

4 10 cells cultivated for 72 hours in both carbon- 100 l cold methanol:acetonitrile:water (2:2:1)

␣

rich and carbon-poor media, and samples were containing methyl- -D-glucopyranoside (0.5 ng/␮l)

processed as described above. The hydrolysates as an internal standard. The content was homoge-

were subjected to TLC analysis on silica gel nized in an ultrasonic bath at 50 Hz for 5 min and

◦

plates (Kieselgel 60, Merck) developed twice kept at −18 C. The mixture was centrifuged at

12 I. Skodová-Sveráková, G. Prokopchuk, P. Pena-Diaz˜ et al.

◦

4490 g at 4 C for 5 min and the supernatant was described above. In order to identify radiolabeled

transferred into another vial. The extraction step sugars, the samples were hydrolyzed in 2 M triﬂu-

◦

was repeated with 20 ␮l of the same extraction oroacetic acid at 120 C for 2 hours, dried under

medium and the supernatants were combined and N2, washed twice with methanol, and partitioned

evaporated by a vacuum concentrator (Jouan RC between hexane and distilled water. Subsequently,

10.10 and RCT 60). hexane was removed and discarded, and the aque-

The sample preparation and GC–MS analysis ous phase was dried in a vacuum evaporator

was conducted as described elsewhere (Kostálˇ (CentriVap). Repeated washes of the hydrolyzed

et al. 2007). Brieﬂy, keto and enol functional groups samples with deionized water removed the residual

␮

were oximated with 25 l O-methylhydroxylamine triﬂuoroacetic acid. The hydrolysates were sub-

(20 mg/ml pyridine) in 30 ␮l dimethylformamide at jected to the TLC analysis on silica gel plates

◦

80 C for 30 min, and hydroxyls were silylated developed twice in ethyl acetate:pyridine:acetic

␮ ␮

with 30 l trimethylsilylimidazol in 70 l dimethylfor- acid:water (6:3:1:1). Radioactivity was visualized

◦

mamide at 80 C for 30 min. After re-extraction into by the exposure of the TLC plates to Kodak X-

◦

␮ ␮

100 l isooctane, 1 l aliquot was injected into a Omat AR ﬁlm at −70 C. Unlabeled standards were

DSQ GC–MS single quadrupole mass spectrome- visualized by ␣-naphthol as described above.

× ×

ter (Thermo Fisher). A 30 m 0.25 mm 0.25 mm Sequence searches and phylogenetic analy-

DB – 1MS capillary column Agilent was used ses: Glucan synthase sequences were retrieved

for separation of the metabolites. The instrument from NCBI and CryptoDB based on a previous

settings were: helium ﬂow-rate 1.1 ml/min; inlet study (Huang et al. 2018). Sequences from E. gra-

◦

temperature, 250 C; injection mode, splitless; split cilis were reported previously (Tanaka et al. 2017).

ﬂow, 20 ml/min; splitless time, 1.3 min; temper- Sequences from E. longa and D. papillatum were

◦ ◦

ature program, 80 C hold 1 min, 20 C/min to found by tBLASTn searches in the recently pub-

◦ ◦ ◦ ◦ ◦

180 C, 5 C/min to 200 C, 25 C/min to 300 C lished transcriptomic data (Záhonová et al. 2018)

◦

hold for 3 min; transfer-line temperature 280 C and and in an unpublished genome assembly, respec-

◦

electron-ion source temperature, 220 C, ionization tively, using E. gracilis sequences as queries.

energy 70 eV. D. papillatum sequences served as queries for

14 14

Labeling with C[U]-proline or C[U]- searches in the transcriptomic data of other

glucose: Five × 10 cells were inoculated into diplonemid species, namely Diplonema japonicum,

1 ml of carbon-rich and carbon-poor media on a Rhynchopus humris, Lacrimia laniﬁca, Sulcionema

24-well plate. Each medium was supplemented specki, Artemidia motanka, and Namystynia kary-

14 14

with C[U]-proline or C[U]-glucose at a ﬁnal oxenos. Sequences from all organisms were

␮

concentration 1 Ci/ml and the cells were culti- submitted to InterProScan and identiﬁed protein

vated for 24 hours at a stationary phase. Cells domains were mapped onto the phylogenetic tree.

were collected at 1000 g for 10 min, followed Glucanase sequences were retrieved by BLAST

with two washes in 36 g/l sea salt solution. The searches at NCBI. Identiﬁed glucanase domains in

pellets underwent acetone sonication. Next, the diplonemids were used as queries for searches in

pellets and acetone supernatants were processed their transcriptomes. Glucanases from E. gracilis

following the polysaccharide and trehalose extrac- reported previously (Novák Vanclová et al. 2020)

tions described above. Both polysaccharide and were used as queries for searches in the E. gracilis

trehalose were resuspended in 100 ␮l of distilled and E. longa transcriptomes.

␤

water. Radioactive material was subjected to liquid -1,3-Glucan phosphorylase was searched in all

scintillation spectrometry (Perkin Elmer), quantiﬁed diplonemid data using E. gracilis sequence identi-

as disintegrations per min (dpm) and evaluated ﬁed previously (Kuhaudomlarp et al. 2018). Since

in percentage where the amount of dpm in cell no homolog was retrieved in any other euglenozoan

culture was taken as 100%. Each experiment was dataset, a more sensitive method based on pro-

performed in triplicate. ﬁle hidden Markov models, HMMER (Eddy 2009),

D. papillatum cultures at a concentration of was employed. To build an HMM proﬁle, all ␤-1,3-

5 × 10 cells/ml were inoculated into 3 ml of carbon- glucan phosphorylase sequences available in the

rich and carbon-poor media supplemented with CAZy database (Lombard et al. 2014) were aligned

C[U]-proline at a ﬁnal concentration of 1 ␮Ci/ml. by MAFFT tool (Katoh and Standley 2013).

Both cultures were kept at stationary phase for Since glucan synthase sequences also bear

24 hours and the labeled cells were then collected additional domains, these were manually trimmed

by centrifugation at 1000 g for 10 min, followed by and only glucan synthase domains were aligned.

two sea salts washes and acetone sonication as In case of diplonemid glucanases, glucan syn-

Paramylon in Diplonema 13

thase domains were trimmed and only glucanase Zlatogursky V, Zhang Q (2018) Revisions to the classiﬁcation,

nomenclature, and diversity of eukaryotes. J Eukaryot Microbiol

domains were aligned with glucanase proteins

66:4–119

from other organisms. Both alignments were pro-

duced by the MAFFT tool and are available upon Allmann S, Mazet M, Ziebart N, Bouyssou G, Fouillen L,

Dupuy JW, Bonneu M, Moreau P, Bringaud F, Boshart M

request from the corresponding author. To remove

(2014) Triacylglycerol storage in lipid droplets in procyclic Tr y-

poorly aligned positions, the trimAL tool was used

panosoma brucei. PLoS ONE 9:e114628

(Capella-Gutiérrez et al. 2009). Maximum likelihood

Allmann S, Morand P, Ebikeme C, Gales L, Biran M, Hubert

trees were inferred from the alignments using the

J, Brennand A, Mazet M, Franconi JM, Michels PA, Portais

LG + C20+F + G model and the posterior mean site

JC, Boshart M, Bringaud (2013) Cytosolic NADPH homeosta-

frequency method (Wang et al. 2018) in the IQ-

sis in glucose-starved procyclic Trypanosoma brucei relies on

TREE software (Nguyen et al. 2015) and employing malic enzyme and the pentose phosphate pathway fed by glu-

the strategy of rapid bootstrapping followed by a coneogenic ﬂux. J Biol Chem 288:18494–18505

“thorough” maximum likelihood search with 1000

Archibald AR, Cunningham WL, Manners DJ, Stark JR,

bootstrap replicates. Ryley JF (1963) Metabolism of the protozoa, X. The

molecular structure of the reserve polysaccharides from

Ochromonas malhamensis and Peranema trichophorum.

Biochem J 88:444–451

Conﬂict of Interest

Aspinall GO, Kessler G (1957) The structure of callose from

The authors declare no conﬂict of interest. the grape vine. Chem Ind (London) 1296

Avonce N, Mendoza-Vargas A, Morett E, Iturriaga G (2006)

Insights on the evolution of trehalose biosynthesis. BMC Evol

Acknowledgements Biol 19:109

Bakker BM, Mensonides FI, Teusink B, van Hoek P, Michels

We highly appreciate access to the genome

PA, Westerhoff HV (2000) Compartmentation protects try-

assembly of D. papillatum kindly provided by panosomes from the dangerous design of glycolysis. Proc Natl

Gertraud Burger (University of Montreal). We Acad Sci USA 97:2087–2092

acknowledge computation resources provided by

Barsanti L, Vismara R, Passarelli V, Gualtieri P (2001)

CERIT-SC and MetaCentrum Brno. This work Paramylon (␤-1,3-glucan) content in wild type and WZSL

was supported by the Czech Grant Agency grant mutant of Euglena gracilis. Effects of growth conditions. J Appl

Phycol 13:59–65

18-15962S, ERC CZ LL1601, and the ERD

Funds projectOPVVV0000759 (to JL), the Grant Bäumer D, Preisfeld A, Ruppel HG (2011) Isolation and

Agency of the Slovak Ministry of Education and characterization of paramylon synthase from Euglena gracilis

(Euglenophyceae). J Phycol 37:38–46

the Academy of Sciences 1/0387/17, the Slovak

Research and Development Agency grant APVV- Blum JJ (1993) Intermediary metabolism of Leishmania. Par-

0286-12 (to AH) and the Grant Agency of the Slovak asitol Today 9:118–122

Ministry of Education and the Academy of Sciences

Booy FP, Chanzy H, Boudet A (1981) An electron diffraction

1/0781/19. study of paramylon storage granules from Euglena gracilis. J

Microsc 12:133–140

Briand J, Calvayrac R (1980) Paramylon synthesis in het-

Appendix A. Supplementary Data erotrophic and photoheterotrophic Euglena (Euglenophyceae).

J Phycol 16:234–239

Supplementary material related to this arti-

Bringaud F, Rivière L, Coustou V (2006) Energy metabolism

cle can be found, in the online version, at

of trypanosomatids: adaptation to available carbon sources.

doi:https://doi.org/10.1016/j.protis.2020.125717. Mol Biochem Parasitol 149:1–9

Calvayrac R, Laval-Martin D, Briand J, Farineau J (1981)

Paramylon synthesis by Euglena gracilis photoheterotrophically

References grown under low O2 pressure: Description of a mitochloroplast

complex. Planta 153:6–13

Adl SM, Bass D, Lane Ch E, Lukesˇ J, Schoch CL, Smirnov

Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T (2009)

A, Agatha S, Berney C, Brown MW, Burki F, Cárdenas

trimAl: a tool for automated alignment trimming in large-scale

P, Cepiˇ ckaˇ I, Chistyakova L, del Campo J, Dunthorn M,

phylogenetic analyses. Bioinformatics 25:1972–1973

Edvardsen B, Eglit Y, Guillou L, Hampl V, Heiss AA, Hoppen-

rath M, James TY, Karnkowska A, Karpov S, Kim E, Kolisko Changmai P, Horáková E, Long S, Cernotíková-Stˇ ríbrnáˇ E,

M, Kudryavtsev A, Lahr DJG, Lara E, Le Gall L, Lynn DH, McDonald LM, Bontempi EJ, Lukesˇ J (2013) Both human

Mann DG, Massana R, Mitchell EAD, Morrow C, Park JS, ferredoxins equally efﬁciently rescue ferredoxin deﬁciency in

Pawlowski JW, Powell MJ, Richter DJ, Rueckert S, Shad- Trypanosoma brucei. Mol Microbiol 89:135–151

wick L, Shimano S, Spiegel FW, Torruella G, Youssef N,

14 I. Skodová-Sveráková, G. Prokopchuk, P. Pena-Diaz˜ et al.

Clarke AE, Stone BA (1960) Structure of the paramylon from Katoh K, Standley DM (2013) MAFFT multiple sequence align-

Euglena gracilis. Biochim Biophys Acta 44:161–163 ment software version 7: improvements in performance and

usability. Mol Biol Evol 30:772–780

Coustou V, Besteiro Biran M, Diolez P, Bouchaud V, Voisin

P, Michels PA, Canioni P, Baltz T, Bringaud F (2003) ATP Kaur B, Valach M, Pena-Diaz˜ P, Moreira S, Keeling PJ,

generation in the Trypanosoma brucei procyclic form: cytosolic Burger G, Lukesˇ J, Faktorová D (2018) Transformation of

substrate level is essential, but not oxidative phosphorylation. J Diplonema papillatum, the type species of the highly diverse

Biol Chem 278:49625–49635 and abundant marine microeukaryotes Diplonemida (Eugleno-

zoa). Environ Microbiol 20:1030–1040

de Vargas C, Audic S, Henry N, Decelle J, Mahé F, Logares

R, Lara E, Berney C, Le Bescot N, Probert I, Carmichael M, Keeling PJ, del Campo J (2017) Marine protists are not just

Poulain J, Romac S, Colin S, Aury J-M, Bittner L, Chaffron big bacteria. Curr Biol 27:541–549

S, Dunthorn M, Engelen S, Flegontova O, Guidi L, Horák

Keeling PJ, Burki F, Wilcox HM, Allam B, Allen EE, Amaral-

A, Jaillon O, Lima-Mendez G, Lukesˇ J, Malviya S, Morard R,

Zettler LA, Armbrust EV, Archibald JM, Bharti AK, Bell

Mulot M, Scalco E, Siano R, Vincent F, Zingone A, Dimier C,

CJ, Beszteri B, Bidle KD, Cameron CT, Campbell L, Caron

Picheral M, Searson S, Kandels-Lewis S, Tara Oceans Coor-

DA, Ann Cattolico RA, Collier JL, Coyne K, Davy SK,

dinators, Acinas SG, Bork P, Bowler C, Gorsky G, Grimsley

Deschamps P, Dyhrman ST, Edvardsen B, Gates RD, Gob-

N, Hingamp P, Iudicon D, Not F, Ogata H, Pesant S, Raes

ler CJ, Greenwood SJ, Guida SM, Jacobi JL, Jakobsen

J, Sieracki ME, Speich D, Stemmann L, Sunagawa S, Weis-

KS, James ER, Jenkins B, John U, Johnson MD, Juhl

senbach J, Wincker P, Karsenti E (2015) Eukaryotic plankton

AR, Kamp A, Katz LA, Kiene R, Kudryavtsev A, Leander

diversity in the sunlit ocean. Science 348:1261605

BS, Lin S, Lovejoy C, Lynn D, Marchetti A, McManus G,

Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F Nedelcu AM, Menden-Deuer S, Miceli C, Mock T, Montre-

(1956) Colorimetric method for determination of sugars and sor M, Moran MA, Murray S, Nadathur Nagai S, Ngam PB,

related substances. Anal Chem 28:350–356 Palenik B, Pawlowski J, Petroni G, Piganeau G, Posewitz

MC, Rengefors K, Romano G, Rumpho ME, Rynearson T,

Dwyer MR, Smillie RM (1970) A light-induced beta-1,3-glucan

Schilling KB, Schroeder DC, Simpson AGB, Slamovits CH,

breakdown associated with the differentiation of chloroplasts in

Smith DR, Smith GJ, Smith SR, Sosik HM, Stief P, The-

Euglena gracilis. Biochim Biophys Acta 216:392–401

riot E, Twary SN, Umale PE, Vaulot D, Wawrik B, Wheeler

GL, Wilson WH, Xu Y, Zingone A, Worden AZ (2014) The

Eddy SR (2009) A new generation of homology search tools

marine microbial eukaryote transcriptome sequencing project

based on probabilistic inference. Genome Inform 23:205–211

(MMETSP): Illuminating the functional diversity of eukaryotic life

Flegontova O, Flegontov P, Malviya S, Audic S, Wincker P, in the oceans through transcriptome sequencing. PLoS Biol 12:

de Vargas C, Bowler C, Lukesˇ J, Horák A (2016) Extreme e1001889

diversity of diplonemid eukaryotes in the ocean. Curr Biol

26:3060–3065 Kiss JZ, Triemer RE (1988) A comparative study of the storage

carbohydrate granules from Euglena (Euglenida) and Pavlova

Gottlieb J (1850) Ueber eine neue, mit Starkmehl isomere (Prymnesiida). J Eukaryot Microbiol 35:237–241

Substanz. Ann Chem Pharm 75:51–61

Kondo Y, Kato A, Hojo H, Nozoe S, Takeuchi M, Ochi

Haanstra JR, González-Marcano EB, Gualdrón-López M, K (1992) Cytokine-related immunopotentiating activities of

Michels PA (2016) Biogenesis, maintenance and dynamics of paramylon, a beta-(1→3)-D-glucan from Euglena gracilis. J

glycosomes in trypanosomatid parasites. Biochim Biophys Acta Pharmacobiodyn 15:617–621

1863:1038–1048

Kostálˇ V, Zahradnickováˇ H, Simekˇ P, Zeleny´ J (2007) Multiple

Harada T, Misaki A, Saito H (1968) Curdlan: a bacterial gel-

component system of sugars and polyols in the overwinter-

forming ␤-D-1,3-glucan. Arch Biochem Biophys 124:292–298

ing spruce bark beetle, Ips typographus. J Insect Physiol

53:580–586

Henrissat B (1991) A classiﬁcation of glycosyl hydrolases

based on amino acid sequence similarities. Biochem J

Kuhaudomlarp S, Patron NJ, Henrissat B, Rejzek M,

280:309–316

Saalbach G, Field RA (2018) Identiﬁcation of Euglena gra-

cilis ␤-1,3-glucan phosphorylase and establishment of a

Henrissat B, Bairoch A (1996) Updating the sequence-based

new glycoside hydrolase (GH) family GH149. J Biol Chem

classiﬁcation of glycosyl hydrolases. Biochem J 316:695–696

293:2865–2876

Henrissat B, Davies G (1997) Structural and sequence-based

Lara E, Moreira D, Vereshchaka A, Lopez-Garcia P (2009)

classiﬁcation of glycoside hydrolases. Curr Opin Struct Biol

7:637–644 Panoceanic distribution of new highly diverse clades of deep-

sea diplonemids. Environ Microbiol 11:47–55

Huang W, Haferkamp I, Lepetit B, Molchanova M, Hou S,

Lombard V, Golaconda Ramulu H, Drula E, Coutinho

Jeblick W, Río Bártulos C, Kroth PG (2018) Reduced vac-

PM, Henrissat B (2014) The carbohydrate-active

uolar ␤-1,3-glucan synthesis affects carbohydrate metabolism

enzymes database (CAZy) in 2013. Nucleic Acids Res

as well as plastid homeostasis and structure in Phaeodactylum

42(D1):D490–D495

tricornutum. Proc Natl Acad Sci USA 115:4791–4796

Marechal LR, Goldemberg SH (1964) Uridine diphosphate

Hutner SH, Zahalsky AC, Aaronson S, Baker H, Frank

glucose-beta-1,3-glucan beta-3-glucosyltransferase from

HO (1966) Culture media for Euglena gracilis. Meth Cell Biol

2:217–228 Euglena gracilis. J Biol Chem 239:3163–3167

Paramylon in Diplonema 15

Millerioux Y, Ebikeme C, Biran M, Morand P, Bouyssou G, Schliwa M, van Blerkom J (1981) Structural interaction of

Vincent IM, Mazet M, Riviere L, Franconi JM, Burchmore cytoskeletal components. J Cell Biol 90:222–235

RJ, Moreau P, Barrett MP, Bringaud F (2013) The threonine

Schwartzbach SD, Schiff JA, Goldstein NH (1975) Events

degradation pathway of the Trypanosoma brucei procyclic form:

surrounding the early development of Euglena chloroplasts.

the main carbon source for lipid biosynthesis is under metabolic

Plant Physiol 56:313–317

control. Mol Microbiol 90:114–129

Sernee MF, Ralton JE, Dinev Z, Khairallah GN, O’Hair RA,

Monﬁls AK, Triemer RE, Bellairs EF (2011) Character-

Williams SJ, McConville MJ (2006) Leishmania beta-1,2-

ization of paramylon morphological diversity in photosyn-

mannan is assembled on a mannose-cyclic phosphate primer.

thetic euglenoids (Euglenales, Euglenophyta). Phycologia

50:156–169 Proc Natl Acad Sci USA 103:9458–9463

Smith TK, Bringaud F, Nolan DP, Figueiredo LM (2017)

Morales J, Hashimoto M, Williams TA, Hirawake-Mogi H,

Metabolic reprogramming during the Trypanosoma brucei life

Makiuchi T, Tsubouchi A, Kaga N, Taka H, Fujimura T, Koike

cycle. F1000Res 6:683

M, Mita T, Bringaud F, Concepción JL, Hashimoto T, Embley

TM, Nara T (2016) Differential remodeling of peroxisome func-

Sugimoto R, Ishibashi-Ohgo N, Atsuji K, Miwa Y, Iwata O,

tion underpins the environmental and metabolic adaptability of

Nakashima A, Suzuki K (2018) Euglena extract suppresses

diplonemids and kinetoplastids. Proc Biol Sci 283:20160520

adipocyte-differentiation in human adipose-derived stem cells.

PLoS ONE 13:e0192404

Muchut RJ, Calloni R, Herrera FE, Garay SA, Arias DG, Igle-

sias AA, Guerrero SA (2018) Elucidating paramylon and other

Szöor B, Haanstra JR, Gualdrón-López M, Michels PA

carbohydrate metabolism in Euglena gracilis: Kinetic charac-

(2014) Evolution, dynamics and specialized functions of gly-

terization, structure and cellular localization of UDP-glucose

cosomes in metabolism and development of trypanosomatids.

pyrophosphorylase. Biochimie 154:176–186

Curr Opin Microbiol 22:79–87

Nakashima A, Sugimoto R, Suzuki K, Shirakata Y,

Takenaka S, Kondo T, Nazeri S, Tamura Y, Tokunaga M,

Hashiguchi T, Ch Yoshida, Nakano Y (2019) Anti-ﬁbrotic

Tsuyama S, Miyatake K, Nakano Y (1997) Accumulation

activity of Euglena gracilis and paramylon in a mouse model

of trehalose as a compatible solute under osmotic stress in

of non-alcoholic steatohepatitis. Food Sci Nutr 7:139–147

Euglena gracilis Z. J Eukaryot Microbiol 44:609–613

Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ (2015) IQ-

Tanaka Y, Ogawa T, Maruta T, Yoshida Y, Arakawa K,

TREE: a fast and effective stochastic algorithm for estimating

Ishikawa T (2017) Glucan synthase-like 2 is indispens-

maximum-likelihood phylogenies. Mol Biol Evol 32:268–274

able for paramylon synthesis in Euglena gracilis. FEBS Lett

591:1360–1370

Novák Vanclová AMG, Zoltner M, Kelly S, Soukal P,

Záhonová K, Füssy Z, Ebenezer E, Lacová Dobáková E,

Tashyreva D, Prokopchuk G, Votypka´ J, Yabuki A, Horák

Eliásˇ M, Lukesˇ J, Field MC, Hampl V (2020) Metabolic quirks

A, Lukesˇ J (2018a) Life cycle, ultrastructure, and phylogeny

and the colourful history of the Euglena gracilis secondary plas-

of new diplonemids and their endosymbiotic bacteria. mBio

tid. New Phytol 225:1578–1592 9:e02447–17

Okouchi R, E S, Yamamoto K, Ota T, Seki K, Imai M, Ota

Tashyreva D, Prokopchuk G, Yabuki A, Kaur B, Faktorová

R, Asayama Y, Nakashima A, Suzuki K, Tsuduki T (2019)

D, Votypka´ J, Kusaka C, Fujikura K, Shiratori T, Ishida KI,

Simultaneous intake of Euglena gracilis and vegetables exerts

Horák A, Lukesˇ J (2018b) Phylogeny and morphology of new

synergistic anti-obesity and anti-inﬂammatory effects by modu-

diplonemids from Japan. Protist 169:158–179

lating the gut microbiota in diet-induced obese mice. Nutrients

11:E204 Valach M, Moreira S, Hoffmann S, Stadler PF, Burger G

(2017) Keeping it complicated: Mitochondrial genome plasticity

Opperdoes FR, Borst P (1977) Localization of nine glycolytic

across diplonemids. Sci Rep 7:14166

enzymes in a microbody-like organelle in Trypanosoma brucei:

the glycosome. FEBS Lett 80:360–364 van Loosdrecht MCM, Pot MA, Heijnen JJ (1997) Importance

of bacterial storage polymers in bioprocceses. Water Sci Tech

Pagni M, Beffa T, Isch C, Aragno M (1992) Linear growth and 35:41–47

poly(beta-hydroxybutyrate) synthesis in response to pulse-wise

addition of the growth-limiting substrate to steady-state het- Wang HC, Minh BQ, Susko E, Roger AJ (2018) Modeling

erotrophic continuous cultures of Aquaspirillum autotrophicum. site heterogeneity with posterior mean site frequency pro-

J Gen Microbiol 138:429–436 ﬁles accelerates accurate phylogenomic estimation. Syst Biol

67:216–235

Prokopchuk G, Tashyreva D, Yabuki A, Horák A, Masarováˇ

P, Lukesˇ J (2019) Morphological, ultrastructural, motility Warsi SA, Whelan WJ (1957) Structure of pachyman, the

and evolutionary characterization of two new Hemistasiidae polysaccharide component of Poria cocos. Chem Ind (London)

species. Protist 170:259–282 1573

Ralton JE, Naderer T, Piraino HL, Bashtannyk TA, Watanabe T, Shimada R, Matsuyama A, Yuasa M, Sawamura

␤

Callaghan JM, McConville MJ (2003) Evidence that intra- H, Yoshida E, Suzuki K (2013) Antitumor activity of the -

cellular beta1-2 mannan is a virulence factor in Leishmania glucan paramylon from Euglena against preneoplastic colonic

parasites. J Biol Chem 278:40757–40763 aberrant crypt foci in mice. Food Funct 4:1685–1690

Raven JA (1997) The vacuole: a cost beneﬁt analysis. Adv Bot Westerlund E, Andersson R, Åman P (1993) Isolation and

Res 35:59–86 chemical characterization of water soluble mixed-linked beta-

16 I. Skodová-Sveráková, G. Prokopchuk, P. Pena-Diaz˜ et al.

glucans and arabinoxylans in oat milling fractions. Carbohydr Záhonová K, Füssy Z, Bircákˇ E, Novák Vanclová AMG,

Polym 20:115–123 Klimesˇ V, Vesteg M, Krajcoviˇ cˇ J, Oborník M, Eliásˇ M (2018)

Peculiar features of the plastids of the colourless alga Euglena

Worden AZ, Follows MJ, Giovannoni SJ, Wilken S, Zimmer-

longa and photosynthetic euglenophytes unveiled by transcrip-

man AE, Keeling PJ (2015) Environmental science. Rethinking

tome analyses. Sci Rep 8:17012

the marine carbon cycle: factoring in the multifarious lifestyles

of microbes. Science 347:1257594

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