Differential synthesis of sucrose and trehalose in
Euglena gracilis
cells during growth and salt stress
Andrea C. Porchia, Diego F. Fiol, Graciela L. Salerno *
Centro de In6estigaciones Biolo´gicas,Fundacio´n para In6estigaciones Biolo´gicas Aplicadas(F.I.B.A.),INBIOP(CONICET),C.C.1348,
7600Mar del Plata,Argentina
Received 10 March 1999; received in revised form 3 June 1999; accepted 19 July 1999
Abstract
The existence of sucrose and trehalose synthesizing activities as well as a differential accumulation of both disaccharides were demonstrated in Euglena gracilis Klebs Z strain during standard growth conditions and in the presence of NaCl. Toluene permeabilized cells were used to measure enzyme activities during the growth curve, by incubating with UDP-[14C]glc and fru-6P,
fru or glc-6P. While sucrose synthesis activity reached its maximum at a late log phase, that of trehalose was at its highest at the exponential phase. According to the results, sucrose and trehalose biosynthesis inE.gracilismay occur by the concomitant action of sucrose-phosphate synthase/sucrose-phosphate phosphatase and trehalose-phosphate synthase/trehalose-phosphate phos-phatase, respectively. The accumulation of the disaccharides was also studied along the growth curve and in the presence of NaCl. The level of trehalose was ten times higher at the late stationary than at the latency phase, while sucrose content reached its maximum at the late exponential phase. The addition of NaCl (50 – 150 mM) did not change sucrose content while trehalose level had a four- to tenfold increase. Taken together, these results suggest that trehalose and sucrose play different roles inE.gracilis
cell life: trehalose may be associated with nutrition and salt stress responses, while sucrose may be related to the normal cellular metabolism, with no apparent function in stress protection. © 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Euglena; Salt stress; Sucrose synthesis; Trehalose synthesis
www.elsevier.com/locate/plantsci
1. Introduction
Sucrose and trehalose are non-reducing disac-charides widespread in nature [1,2]. Sucrose plays a central role in plant life as a major product of photosynthesis and because of its function in translocation and storage [3]. It is also involved in plant responses to environmental stress [4] and as a regulator of cellular metabolism [5]. Sucrose synthesis in plants [3,6] and unicellular eukaryotic organisms [7] is generally considered to be cata-lyzed by sucrose-phosphate synthase (SPS,
UDP-glucose: D
-fructose-6-phosphate-2-glucosyltrans-ferase, EC 2.4.1.14) in conjunction with sucrose-phosphate phosphatase (SPP, EC 3.1.3.24), while sucrose synthase (SS, UDP-glucose: D
-fructose-2-glucosyltransferase, EC 2.4.2.13) [8] is largely in-volved in sucrose cleavage [3]. Recently, these enzymes were shown to be present in cyanobacte-ria [9 – 11]. Trehalose is a major storage carbohy-drate commonly found in fungi, invertebrates, and some bacteria that appears to act as a stress protectant [12,13]. It is also found in photosyn-thetic organisms such as some Rodophyceae, ferns, mosses, lichens and angiosperms [3,14]. It is remarkable that most vascular plants seem to lack trehalose, even when undergoing severe environ-mental conditions [14]. While recently, evidence was provided for the occurrence of genes encoding trehalose-6P synthase and a specific trehalose-6P Part of this research was conducted by A.C. Porchia in fulfilment
of the requirements for her Licentiate degree and by D.F. Fiol in partial fulfilment of the requirements for Ph.D. degree of Universidad Nacional de Mar del Plata, Mar del Plata, Argentina.
* Corresponding author. Tel.: +54-223-474 8257; fax: + 54-223-475-7120.
E-mail address:[email protected] (G.L. Salerno)
phosphatae in Arabidopsis, it remans to be eluci-dated where and when the enzymes, as well as their putative products are actually present in plant tissues [15,16]. The fact that trehalose is found in a few plants and in very low concentra-tions suggests that it can not have an important function related to stress protection [17]. Trehalose biosynthesis, extensively studied in yeast has a two-step pathway, involving the action of tre-halose-6-phosphate synthase (TPS, UDP-glucose:
D-glucose-6-phosphate-1-glucosyltransferase, EC
2.4.1.15) and a specific phosphatase (TPP, tre-halose-6-phosphate phosphohydrolase, EC 3.1.3.12) [18]. Trehalose hydrolysis may be pro-duced by different enzymes: in Euglena gracilis and Pichia fermentans it is achieved by trehalose phosphorylase (TP, a-a-trehalose phosphorylase, E.C. 2.4.1.64) [19,20], in Bacillus popilliae and E. coli by trehalose-6-P hydrolase (TPH, a-a -phos-photrehalase, E.C. 3.2.1.93) [21,22], and in other bacteria, plants, fungi, and animals by trehalase (TH, a-a-trehalase E.C. 3.2.1.28) [14,23]. It was suggested that the presence of trehalase activity in plant tissues prevents the accumulation of tre-halose. However, the function of trehalase in plants remains unclear [17].
The occurrence of sucrose as a photosynthesis product [24] and of trehalose during the utilization of [14C]-acetate in non-photosynthetic conditions [25] and under salt or osmotic stresses [26], was reported in E. gracilis, a protozoan living in fresh water. Thus, this organism is potentially appropri-ate to study the transition from organisms that respond to environmental stresses accumulating trehalose to higher plants which have lost this ability [27]. It has also been suggested that sucrose might have replaced trehalose as a stress response molecule in plants [17].
The present work shows a difference in sucrose and trehalose accumulation as well as in their biosynthetic activities in E. gracilis cells at differ-ent growth states or submitted to a salt stress. The results indicate distinct roles for each disaccharide.
2. Materials and methods
2.1. Biological material
Axenic cultures of E. gracilis Klebs strain Z
were grown at 2891°C in Erlenmeyer flasks con-taining 2/10 of their volume of Hutner medium [28], and under orbital shaking. Continuous illu-mination was provided by banks of fluorescent lamps. Cells were harvested at intervals by cen-trifugation at 5000×g for 5 min and were washed three times with 25 mM Hepes – NaOH (pH 7.0), containing 1 mM EDTA and 5 mM 2-mercap-toethanol. Salt treatment was performed by adding different NaCl amounts to cell cultures at the late exponential phase. Cells were collected 24 h after the treatment.
2.2. Sucrose and trehalose determination
Packed and washed cells (approximately 1 g) were lyophilized and extracted with 80% (v/v) ethanol at 80°C three times [29]. Sugars were separated by paper chromatography and eluted as indicated [7]. Sucrose was estimated by measuring fructose and glucose after hydrolysis with inver-tase (SIGMA, St. Louis), and trehalose, by quan-tifying glucose after hydrolysis with trehalase (SIGMA, St. Louis) as previously described [19], and by HPLC (BIO-RAD Aminex HPX-87C column (7.8×300 mm) equilibrated with water, flow rate 0.5 ml min−1).
2.3. Cell permeabilization
Cell suspensions (1 ml, 80 mg fresh weight (FW)) in 10 mM Hepes – NaOH (pH 7.5) contain-ing 5 mM 2-mercaptoethanol were permeabilized by shaking for 2 min in 2% toluene [29].
2.4. Disaccharide synthesis determination in permeabilized cells
chro-matography. Standard sugars were chro-matographed in parallel strips [7].
2.5. Enzyme extracts
All operations were carried out at 2 – 4°C unless otherwise stated. Different methods of cell disrup-tion were tested (sonicadisrup-tion, passage through a French press at different conditions, and mortar disruption in the presence of glass powder). Maxi-mal enzyme activities were obtained when packed cells were resuspended in eight volumes of 100 mM Hepes – NaOH (pH 7.5) containing 20 mM 2-mercaptoethanol, 2 mM EDTA, 2% ethylengly-col and 0.5 mM phenylmetylsulfonylfluoride, and were broken by sonication at 40 W applied for four times during 15 s. Cell debris were removed by two successive centrifugations at 40 000×g for 30 min. The supernatant was absorbed onto a
DEAE-Sephacel column (1×15 cm) that had been preequilibrated with 20 mM Hepes – NaOH buffer (pH 8.0), containing 1 mM EDTA, 10 mM MgCl2, 5 mM 2-mercaptoethanol and 20% glycerol. After washing, the enzyme was eluted with a linear NaCl gradient (0 – 0.5 M, total volume 100 ml), in the same equilibrium buffer. Fractions were analyzed for SS, SPS and TPS activities. Proteins were estimated according to Bradford [30].
2.6. Enzyme assays
SPS and SS activities were assayed by incuba-tion in a total volume of 0.05 ml, 10mM UDP-glc, 10 mM MgCl2, fru-6P or fru (respectively), 100 mM Hepes – NaOH buffer (pH 7.0), and variable amounts of enzyme preparations. Activities were measured by quantifying sucrose-6P or sucrose by the thiobarbituric acid method [31]. TPS was as-sayed in a similar reaction mixture but using UDP-glc and glc-6P as substrates. The product was measured by the anthrone method [32]. TP was measured in the direction or trehalose phos-phorolysis according to Mare´chal and Belocopi-tow [19].
3. Results
3.1. Sucrose and trehalose synthesis in E. gracilis permeabilized cells
Following our studies on sucrose enzymes in unicellular organisms, we investigated sucrose biosynthesis in flagelated eukaryotes. Toluene per-meabilized E. gracilis cells were incubated in the presence of SPS or SS substrates using UDP-[14C]glc as described [29]. Three radioactive peaks were detected when the cells were incubated with fru-6P (Fig. 1A). The areas corresponding to each peak were eluted for further identification. Each eluate was rechromatographed and analyzed as indicated below. Peak 1 sugar was characterized as trehalose, peak 2 as sucrose, and peak 3 as glu-cose. When the incubation was performed with fru, only radioactive glc was obtained (Fig. 1B). No phosphorylated radioactive sugar was ob-tained when the mixtures of the three incubations described in Fig. 1 were analyzed in a parallel chromatography (data not shown).
Fig. 1. Chromatographic analysis of the products of incubat-ing permeabilizedE.graciliscells with sucrose enzymes sub-strates. Cells were incubated for 30 min with: (A) UDP-[14C]glc+fru-6P; (B) UDP-[14C]glc+fru; and (C)
UDP-[14C]glc. Standard sugars were localized in parallel
Table 1
Sucrose and trehalose content inE.gracilisa
Phase mmol g−1FW Exponential 0.2590.06
0.7690.05 0.4390.09
Late exponential
Stationary 0.2890.05 1.1190.12 2.4990.18 n.d.b
Late stationary
aCells were harvested at different growth phases. bn.d., not detected.
all the phases tested. It was always higher than [14C]sucrose formation, reaching its maximum at the late log phase. [14C]Sucrose was preferentially synthesized in the presence of fru-6P or glc-6P, reaching its highest level at the end of the culture growth. The addition of fru did not show any change in sucrose or trehalose synthesis as com-pared against the control incubation mixture (no sugar addition).
3.3. Enzyme acti6ities in E. gracilis cell free extracts
The presence of SPS, SS and TPS was investi-gated in E. gracilis cell free extracts. It was not possible to measure sucrose enzyme activities even when different protein extracting methods were assayed (data not shown). Conversely, trehalose production (but not trehalose-P) was obtained when crude extracts or DEAE-Sephacel fractions (Fig. 2) were incubated with TPS substrates. The absence of phosphorylated disaccharide was confi-rmed by paper chromatography and incubation with alkaline phosphatase.
Enzyme activities involved in trehalose metabolism (TPS and TP) were measured in crude extracts of cells harvested at different growth phases (Fig. 3). TPS activity markedly increased along the growth curve (maximum at the late stationary phase). While TP was at its highest during the late exponential phase, it decreased at 3.2. Sucrose and trehalose synthesis in E. gracilis
cells at different growth stages
Sucrose and trehalose contents were determined during the growth of E. gracilis (Table 1). The sucrose level reached its maximum at the late exponential phase (ca. 0.43 mmol g−1 FW). Con-versely, the accumulation of trehalose increased along the growth curve, being ten times higher (ca. 2.5 mmol g−1 FW) at the late stationary than at the latency phase. Synthetic enzyme activities were also estimated in permeabilized cells harvested at different growth phases. Cells were incubated with TPS, SPS and SS substrates. Radioactive products were quantified after separation by paper chro-matography (Table 2). [14C]Trehalose synthesis was similar in the presence of glc-6P or fru-6P in
Table 2
Sucrose and trehalose synthesizing activites during growth of E.gracilisa
cpm
Exponential 2900 190 450
240
Late exponential 4200 660 830
200 300
Sucrose 520 500
Trehalose 4600
Early stationary 3600 790 710
Sucrose 510 480 460 490
3500 3500
Trehalose
Stationary 320 430
Sucrose 610 670 220 200
Trehalose 4300
Late stationary 3700 300 360
Sucrose 1820 2000 900 1050
aToluene permeabilized cells were incubated in the presence of UDP-[14C]glc+glc-6P, UDP-[14C]glc+fru-6P or UDP-[14C]glc+
fru.
Fig. 2. Analysis of sucrose and trehalose synthetic activities in the fractions of a DEAE-Sephacel chromatography of E.
gracilisextracts. Enzyme activities: TPS ( — ); SPS ( — ); protein ( ). The broken line represents the
NaCl gradient.
mM NaCl. Consequently, the effect of NaCl on the biosynthesis of trehalose and sucrose was in-vestigated by incubating permeabilized E. gracilis cells in the presence of SPS or TPS substrates (Fig. 5). Both synthetic activities increased as a response to enhanced salt concentrations. The production of [14C]-sucrose was about threefold higher at 150 mM NaCl, while [14C]-trehalose only showed a mild increase. When TPS and TP activities were
Fig. 4. Effect of NaCl on disaccharide contents in E.gracilis
cells. Trehalose (open bars) and sucrose (dotted bars) were quantified 24 h after salt addition. Values represent the aver-age of three independent experiments; S.D. did not exceed 8%.
Fig. 3. Trehalose synthesis and degradation inE.graciliscells at different growth phases. TPS/TPP (open bars) and TP (dotted bars) activities measured in crude extracts. Values are the means of four replicates; S.D. did not exceed 6%.
Fig. 5. Effect of NaCl on sucrose and trehalose synthetic activities in E. gracilis permeabilized cells. Different NaCl amounts were added to the culture medium of cells at the late exponential phase. Cells were toluene permeabilized 24 h after the addition and incubated in the presence of UDP-[14C]glc+
glc-6P (A), or UDP-[14C]glc+fru-6P (B), or UDP-[14C]glc
(C). Sucrose synthesis (open bars) and trehalose synthesis (dotted bars). Values are the means of three replicates; S.D. did not exceed 10%.
the stationary phase. The trehalose synthesis/
degradation ratios (TPS/TP activities) calculated at the late exponential and late stationary phases were 0.6 and 2.3, respectively.
3.4. Effect of salt stress on sucrose and trehalose metabolism
assayed in crude extracts from control and salt-treated cells, the TPS/TP ratios were about 4 and 2, respectively.
4. Discussion
Although trehalose and sucrose seem to have similarities regarding their biosynthetic pathways, stress-induced accumulation and cellular protective effects, it is important to investigate the pattern of distribution of both disaccharides in living organ-isms [1 – 6,14]. Sucrose is synthesized only in photo-synthetic eukaryotes and cyanobacteria while trehalose is present throughout the animal, fungal, protista and plant kingdoms. Consequently, it was suggested that sucrose evolutionary origin proba-bly occurred much later than that of trehalose [17]. Euglenoids, which appear to have diverged early in evolution, might probably use ancestral metabolic mechanisms [33].E.gracilis, a photosynthetic flage-lated eukaryote, became a crucial organism for analyzing the presence and biosynthesis of both disaccharides.
A different accumulation pattern of sucrose and trehalose was shown during E. gracilis growth. Maximal sucrose level, reached at the late exponen-tial phase, was fivefold lower than that of trehalose. On the other hand, trehalose accumulation in-creased along the growth curve reaching its maxi-mum at the stationary phase (Table 1). This accumulation pattern was not paralleled by both disaccharide synthetic activities shown in permeabi-lizedE.graciliscells from UDP-[14C]glc and fru-6P or glc-6P (Table 2). The use of permeabilized cells was an effective tool to show sucrose synthetic activity, which was not measurable in crude ex-tracts. However, product formation was obviously influenced by other intracellular enzyme activities. For example, trehalose was also formed in the presence of fru-6P, which could likely be due to the action of an intracellular phosphoglucoisomerase activity (Fig. 1B). That neither sucrose-6P nor trehalose-6P could be detected, suggests the pres-ence of endogenous phosphatase activities, either TPP and/or unspecific phosphatases. Thus, sucrose and trehalose biosynthesis in E. gracilis might be ascribed to the concomitant action of SPS/SPP, and TPS/TPP as described earlier (i.e. in plants and yeasts) [1,2]. While trehalose synthetic activity could be measured in homogenates and partially
purified fractions (Fig. 2), sucrose enzymes (SPS/
SPP) were only shown in permeabilized cells (Table 2). It was already demonstrated in Chlorella 6 ul-gariscells that sucrose enzyme activities were four-fold higher in permeabilized cells than in homogenates. In particular, SPS was shown to be very sensitive to cell disruption and susceptible to inactivation [29,34]. On the other hand, the detec-tion of SS activity in E. gracilispermeabilized cells may have been impaired by the possible presence of a relatively large pool of intracellular sugars (the amount of radioactivity incorporated to sucrose in the presence of fru was similar to that obtained with UDP-[14C]glc alone) (Table 2). The fact that there is no trehalase activity in crude extracts [19] and TP is believed to act in6i6oin the disaccharide break-down direction, it is possible to think that trehalose degradation inE.gracilisis due to the action of TP activity (Fig. 3).
The difference in sucrose and trehalose accumu-lation during the growth curve (Table 1) suggests that both of them play a distinct role in the cell growth cycle. While sucrose is related to the stage of maximal cellular division, a significant trehalose accumulation due to a decrease in its degradation (Table 2, Fig. 3) is important when cells are sub-jected to limited nutritional conditions. Addition-ally, variations in both disaccharide levels as a response to salinity proved to be dramatically different. While trehalose content strongly in-creased in the presence of salt, sucrose level did not change (Figs. 4 and 5), suggesting that trehalose may be acting as a compatible solute equilibrating the inner osmolarity and protecting structures inE. graciliscells. In view of the low amount of sucrose present in E. gracilis in comparison with that of trehalose it seems unlikely that sucrose may have an important function in stress protection. The role of sucrose in E. gracilis, an organism with photosyn-thetic and carbon fixation mechanisms similar to those in higher plants is still unknown. The finding that sucrose biosynthesis inE.gracilisis functional and not stress-related will bring about a lot of scientific interest. Thus, E. gracilisis an important model for understanding the molecular and bio-chemical mechanisms involved in photosynthesis and sugar metabolism.
Acknowledgements
de Investigaciones Cientı´ficas y Tecnolo´gicas (CONICET) and FIBA. GS is a Career Investiga-tor of CONICET. We are grateful to Professor Horacio G. Pontis and Dr Darı´o Bernacchi for critical reading of the manuscript.
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![Fig. 1. Chromatographic analysis of the products of incubat-ing permeabilizedUDP-[ E. gracilis cells with sucrose enzymes sub-strates.Cellswereincubatedfor30minwith:(A)UDP-[14C]glc+fru-6P;(B)UDP-[14C]glc+fru;and(C)14C]glc](https://thumb-ap.123doks.com/thumbv2/123dok/1036007.929185/3.612.38.255.322.626/chromatographic-analysis-products-incubat-permeabilizedudp-gracilis-sucrose-cellswereincubatedfor.webp)
