Approximating subcellular organisation of carbohydrate metabolism during cold acclimation in different natural accessions of Arabidopsis thaliana

Approximating subcellular organisation of carbohydratemetabolism during cold acclimation in different naturalaccessions of Arabidopsis thaliana

Thomas N€agele and Arnd G. Heyer

Institute of Biology, Department of Plant Biotechnology, University of Stuttgart, 70569, Stuttgart, Germany

Author for correspondence:Arnd G. HeyerTel: +49 711 685 65050

Email: [email protected]

Received: 27 November 2012Accepted: 27 January 2013

New Phytologist (2013) 198: 777–787doi: 10.1111/nph.12201

Key words: Arabidopsis thaliana,carbohydrate compartmentation, coldacclimation, mathematical modeling,steady-state analysis.

Summary

� Accessions of Arabidopsis thaliana originating from climatically different habitats show

different levels of cold acclimation when exposed to low temperatures. The central carbohy-

drate metabolism plays a crucial role during this acclimation. Subcellular distribution of carbo-

hydrates over the compartments cytosol, vacuole and plastids, and putative interactions of

the compartments, are analyzed in three differentially cold-tolerant accessions of Arabidopsis

thaliana, originating from the Iberian Peninsula (C24), Russia (Rschew) and Scandinavia

(Tenela), respectively.� Subcellular carbohydrate concentrations were determined by applying the nonaqueous

fractionation technique. Mathematical modeling and steady-state simulation was used to

analyse the metabolic homeostasis during cold exposure.� In all accessions, the initial response to cold exposure was a significant increase of plastidial

and cytosolic sucrose concentrations. Raffinose accumulated in all cellular compartments of

cold-tolerant accessions with a delay of 3 d, indicating that raffinose accumulation is a long-

term component of cold acclimation. Minimal rates of metabolite transport permitting steady-

state simulations of metabolite concentrations correlated with cold tolerance, indicating an

important role of subcellular re-distribution of metabolites during cold acclimation.� A highly regulated interplay of enzymatic reactions and intracellular transport processes

appears to be a prerequisite for maintaining carbohydrate homeostasis during cold exposure

and allowing cold acclimation in Arabidopsis thaliana.

Introduction

Many temperate herbaceous plants species, including the modelplant Arabidopsis thaliana, can grow at low temperature and evensurvive freezing (Hurry et al., 1995; Zhen & Ungerer, 2008).Exposure to low but nonfreezing temperatures induces a multifac-eted and complex process termed cold acclimation, by whichplants are able to increase their cold tolerance. During cold accli-mation, numerous physiological and biochemical changes occurenabling plants to tolerate temperatures several degrees lower thanbefore cold exposure (Xin & Browse, 2000; Hannah et al., 2006).Many studies have shown that cold acclimation capacity is amultigenic trait that is influenced by a variety of factors includingregulation of gene expression, enzyme activity and metabolite con-centrations (Stitt & Hurry, 2002; Cook et al., 2004; Davey et al.,2009). Reprogramming of the central carbohydrate metabolism,comprising the regulation of photosynthetic activity and concen-trations of soluble sugars, was shown to play a crucial role duringcold acclimation (Wanner & Junttila, 1999; Stitt & Hurry,2002). In particular, the concentrations of sucrose and raffinosewere shown to correlate with cold tolerance in Arabidopsis (Klotke

et al., 2004). In a cytosolic reaction, raffinose is synthesized fromsucrose and myo-inositol by raffinose synthase and may thenbe transported into chloroplasts to function as a protectant(Schneider & Keller, 2009). However, the accumulation ofsoluble sugars during cold exposure is insufficient to fully explainthe process of cold acclimation (Hincha et al., 1996).

In Arabidopsis thaliana grown at low temperature, a repro-gramming of carbon metabolism was shown to result in a shift inpartitioning of fixed carbon into sucrose rather than starch(Strand et al., 1997, 1999). Additionally, the overexpression ofsucrose phosphate synthase in Arabidopsis leads to an improvedphotosynthetic activity and an increased flux of fixed carbon intosucrose, associated with an increase in cold tolerance comparedto wild-type plants (Strand et al., 2003). Besides the finding thatsucrose may act as a cryoprotectant for membrane systems inplants (Hincha et al., 2003), it may also serve as a substrate forthe synthesis of other cryoprotective compounds, such as raffi-nose. Analyzing the cold acclimation potential of two geneticallydistinct Arabidopsis accessions, C24 and Columbia (Col-0), thebasic cold tolerance and the capacity to cold acclimate werefound to correlate with tissue raffinose concentrations (Klotke

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et al., 2004). Recently, it was demonstrated that raffinose specifi-cally acts to protect the photosystems located in the thylakoidmembranes of plastids from damage during freeze-thaw cycles(Knaupp et al., 2011).

The analysis of regulatory instances involved in reprogram-ming of the central carbohydrate metabolism is complex. First,this is due to the large number of cellular metabolite interactions,which frequently include nonlinear enzyme kinetics. In case ofallosteric effectors, metabolites are part of regulatory circuitsthemselves, for example, by inhibiting or activating enzymes. Sec-ondly, metabolism of photosynthetically active leaf cells is highlycompartmentalized with substantial redundancy of metabolicpathways among the compartments (depicted schematically inFig. 1). While CO2 is fixed in chloroplasts, sucrose is synthesizedin the cytosol and then either exported to sinks or transportedinto the vacuole or chloroplast. Alternatively, it is cleaved bycytosolic invertase or consumed during synthesis of raffinose.Hexoses, which are products of sucrose cleavage, can either betransported across the tonoplast and the chloroplast envelope orthey can be phosphorylated by hexokinase thereby supplying sub-strates for re-synthesis of sucrose. Considering metabolic cyclesand the numerous metabolite transport processes across intracel-lular membrane systems (an overview given in (Linka & Weber,2010)), it becomes obvious that principles of regulation allowingestablishment of metabolic homeostasis are difficult to unravel.One attractive method for studying complex networks is mathe-matical modeling and simulation, which was already applied suc-cessfully to various biochemical networks (Ross & Arkin, 2009).A frequently used approach of mathematical modeling is the rep-resentation of biological networks by ordinary differential equa-tions (ODEs) describing time-dependent changes in metaboliteconcentrations. The changes result from mass input and output,as well as enzyme-catalyzed metabolite interconversions. Consid-ering compartment-specific metabolite concentrations, changesmay also occur due to transport processes. ODE-based modeling

approaches have been successfully applied to simulate variouscomplex processes in plants, for example photosynthetic CO2

fixation (Pettersson & Ryde-Pettersson, 1988; Laisk et al., 2006),dynamics of the circadian clock in Arabidopsis (Zeilinger et al.,2006) and the diurnal regulation of central carbohydrateinterconversions (N€agele et al., 2010; Henkel et al., 2011).

Carbohydrate dynamics during cold acclimation of Arabidopsisthaliana are different for cold-sensitive vs -tolerant accessions andindicate a complex interplay of plastidial and cytosolic reactions(N€agele et al., 2011, 2012). Based on this finding, we performeda detailed analysis of the compartmental localization of carbohy-drates during cold acclimation. We used nonaqueous fraction-ation to resolve carbohydrate concentrations in chloroplasts,cytosol and vacuole of the three differentially cold-tolerant acces-sions C24, Rsch and Te, where C24 represents a cold-sensitiveaccession, followed by the cold-tolerant accession Rsch and themost tolerant accession Te. The different levels of cold toleranceof these accessions, originating from the Iberian Peninsula (C24),Russia (Rsch) and Scandinavia (Te), were determined in previousstudies (Hannah et al., 2006; Mishra et al., 2011), and the devel-opment of cold tolerance during a time course of 7 d of coldexposure was investigated in this study. Rates of net photosynthe-sis and activities of rate-limiting enzymes were incorporated intoa steady-state modeling approach. The simulation of metabolitefluxes revealed distinct regulatory strategies for sucrose–hexoseinteractions and their compartmentation, allowing discrimina-tion between cold-sensitive (C24) and –tolerant, as well asbetween tolerant (Rsch) and highly tolerant (Te) accessions.

Materials and Methods

Plant material

Arabidopsis thaliana (L.) Heynh (Brassicaceae), accessions C24,Rschew (Rsch) and Tenela (Te), were grown in GS90 soil

Chloroplast Cytosol Vacuole

Starch

CO2

Sucpl

Hexpl

Rafpl

Succyt

Hexcyt

Rafcyt

Sucvac

Hexvac

Rafvac

Sucexp

r6

r2

r3r4r5

r1

r7

t1f

t2f

t4f

t5f

t6ft3f

t7 Sinks

t1r

t2r

t3r

t5r

t6r

t4r

Fig. 1 Schematic representation ofcarbohydrate compartmentation in leaf cellsof Arabidopsis thaliana. Reaction rates rrepresent central steps of metaboliteinterconversion. Transport processes t acrossthe chloroplast envelope and the tonoplastare subdivided into forward (tf) and reverse(tr) reactions. Suc, Sucrose; Hex, Hexoses;Raf, Raffinose; pl, plastidial; cyt, cytosolic;vac, vacuolar; exp, exported.

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and vermiculite (1 : 1) with three plants per 10 cm pot in agrowth chamber at 8 h light (50 lmol m�2 s�1; 22°C) : 16 hdark (16°C) for 4 wk and then transferred to a glasshousewith a temperature of 22°C during the day (16 h) and 16°Cduring the night (8 h). In the glasshouse, natural light wassupplemented to an intensity of at least 80 lmol m�2 s�1.The relative humidity was 70%. Plants were watered dailyand fertilized with standard nitrogen-phosphorus-potassiumfertilizer immediately after transfer to long-day conditions anda second time 2 wk later. At the bolting stage, that is, c. 3 wkafter transfer to long-day conditions, a set of plants was har-vested and another set was shifted to a growth chamber witha 16 h : 8 h light : dark regime of 4/4°C and a light intensityof 50 lmol m�2 s�1. Each sample consisted of three plantrosettes taken from three different pots in a random designbefore and after 1, 3 and 7 d of exposure to 4°C. Sampleswere taken at the mid-point of the light period. Because theaerial parts of the plants were composed exclusively of rosetteleaves, metabolite and enzyme concentrations could be directlycompared to CO2 exchange data. Samples were immediatelyfrozen in liquid nitrogen and stored at �80°C.

Electrolyte leakage measurement

Cold tolerance was analyzed according to the electrolyte leakagemethod as described (N€agele et al., 2011). The cooling rate wasset to 4°C h�1 and samples were taken at 2°C intervals over atemperature range of 0°C to �18°C. Conductivity was measuredusing an inoLab740 conductivity meter (WTW GmbH,Weilheim, Germany) and the multilabPilot software. The 50%lethality temperature (LT50) values were calculated as the logEC50 value of sigmoidal dose response curves fitted to the mea-sured leakage values using Graphpad Prism 3 software(Graphpad Software Inc., La Jolla, CA, USA).

Gas exchange measurement

The exchange rates of CO2 were measured using an infrared gasanalysis system (Uras 3 G, Hartmann & Braun AG, Frankfurtam Main, Germany). A whole-rosette cuvette design was used asdescribed in (N€agele et al., 2010). Gas exchange was measured inthe glasshouse and the growth chamber shortly before plant har-vest. Means of raw data for gas exchange were converted to fluxrates per gram DW obtained at the end of the exposure by freezedrying and weighing complete rosettes.

Carbohydrate analysis

Freeze dried leaf samples were ground to a fine powder. Thehomogenate was extracted twice in 400 ll of 80% ethanol at80°C. Extracts were dried and dissolved in 500 ll of distilledwater. Contents of glucose, fructose, sucrose and raffinose wereanalysed by high-performance anion exchange chromatography(HPAEC) using a CarboPac PA-1 column on a Dionex (Sunny-vale, CA, USA) DX-500 gradient chromatography systemcoupled with pulsed amperometric detection by a gold electrode.

For starch extraction, pellets of the ethanol extraction were solu-bilized by heating them to 95°C in 0.5 N NaOH for 45 min.After acidification with 1 N CH3COOH the suspension wasdigested for 2 h with amyloglucosidase. The glucose content ofthe supernatant was then determined and used to assess the starchcontent of the sample.

Measurement of enzyme activities

Enzyme activities were determined in crude extracts of freezedried leaf samples. To assess activities of soluble acid invertaseand neutral invertase, c. 10 mg of dried leaf tissue werehomogenized in 50 mM HEPES-KOH (pH 7.4), 5 mMMgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 5 mMdithiothreitol (DTT), 0,1% Triton-X-100 and 10% glycerol.Suspensions were centrifuged at 2800 g for 25 min at 4°C andsupernatants were desalted using a G-25 Sephadex gel filtrationmedium. Soluble acid invertase was assayed in 20 mM Na-Ace-tate buffer (pH 4.7) using 100 mM sucrose as a substrate.Neutral invertase was assayed in 20 mM HEPES-KOH (pH7.5) using also 100 mM sucrose as substrate. The control ofeach assay was boiled immediately for 5 min. Reactions wereincubated for 30 min at 30 and 4°C, stopped by boiling for5 min, and the concentration of glucose was determinedphotometrically.

Activity of glucokinase and fructokinase was measured asdescribed in (Wiese et al., 1999) at ambient temperature (22°C)and 4°C. Synthesized glucose-6-phosphate was converted to6-phosphogluconolactone by glucose-6-phosphate-dehydroge-nase and could be measured photometrically as a change in con-centration of the reduced co-substrate NADPH. For isomerisationof fructose-6-phosphate, phosphogluco-isomerase was added.

Nonaqueous fractionation

Sub-cellular fractionation was based on the procedure describedby (Iftime et al., 2011). Approximately 100 mg of freeze driedleaf homogenate were suspended in 10 ml heptane–tetrachlor-ethylene (q = 1.34 g cm�3) and repeatedly sonified on ice for5 s with pauses of 15 s over a time course of 12 min (BransonSonifier 250, Branson, Hannover, Germany). The sonified sus-pension was passed through 30-lm pore nylon gauze (Eckert,Waldkirch, Germany) and centrifuged. The sediment was sus-pended in heptane–tetrachlorethylene (q = 1.34 g cm�3) andloaded on a linear gradient of heptane–tetrachlorethylene(q = 1.34 g cm�3) to tetrachlorethylene (q = 1.6 g cm�3). Afterultracentrifugation at 121 000 g for 3 h, the gradient was frac-tionated into nine 1-ml fractions that were divided into threesub-fractions of 0.3 ml. These were dried under vacuum. Oneof these fractions was used for marker enzyme determinationand another for sugar analysis using HPAEC. Alkalinepyrophosphatase was measured as a marker for the plastidialcompartment as described (Jelitto et al., 1992), UGPase wasmeasured as cytosolic marker as described by (Zrenner et al.,1993). Acid phosphatase was used as marker for the vacuolarcompartment (Boller & Kende, 1979).


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A detailed description of the calculation process of the sub-cellular metabolite distribution is provided in SupportingInformation Notes S1. The relative distribution of metabolitesacross the compartments as well as the total sugar concentrationsare provided in Tables S1 and S2.

Mathematical modeling and simulation

A mathematical model of the central carbohydrate metabolismwas developed comprising metabolite interconversions as well astransport processes, as indicated in Fig. 1. The model was basedon a system of ordinary differential equations which is describedin detail within the model structure given in Notes S2. Solublecarbohydrate concentrations of each accession after 0, 1, 3 and7 d at 4°C were assumed to be at steady state (d/dt = 0). At steadystate, the rate of raffinose synthesis (r5) became 0. This assump-tion was based on the finding that there was only minor enzy-matic capacity for raffinose turnover in Arabidopsis thaliana(N€agele et al., 2012). Starch was assumed to be synthesised with aconstant rate (r2) leading to the measured starch concentrationsafter 8 h in the light phase (14:00 h):

r2 ¼ c Starch;14:00=8 h ½mM C6h�1�

Rates of net photosynthesis (rNPS) were calculated as the aver-age rate of carbon uptake during the first half of the light phase(n = 8 h):

rNPS ¼

Rn

i¼1

xNPSi

n½mM C6 h

�1�;

(xNPSi, the integral of carbon uptake per hour).The flux rate of carbon into sucrose synthesis (r1) was calcu-

lated as the difference between the rate of net photosynthesis andstarch synthesis:

r1 ¼ rNPS � r2 ½mM C6h�1�

At steady state, r1 becomes equivalent to the rate of sucroseexport to sinks (t7).

The rate of cytosolic, plastidial and vacuolar sucrose cleavage(r3, r6, r7), catalysed by invertase, was modeled by a Michaelis–Menten enzyme kinetic including competitive productinhibition. Thus, rates of sucrose cleavage depended on substrateconcentration, maximum activity of invertase and the enzymespecific substrate affinity, expressed by KM. It also depended onthe concentration of the reaction product as well as the dissocia-tion constant Ki for inhibitor binding.

The rate of hexose phosphorylation (r4) was described by themass action rate law assuming the reaction rates to depend onsubstrate concentration and a rate constant.

Rates of metabolite transport were modeled by a mass actionrate law as described for hexose phosphorylation.

Specific metabolite concentrations in cellular compartmentswere calculated assuming the volume proportions of chloroplasts,cytosol and vacuole to be similar to those described by Winter

et al. (1994) for mesophyll cells in spinach leaves (Plastids: 10%;Cytosol: 5%; Vacuole: 80%). The remaining 5% of volume wereassumed to consist of other cellular compartments, for example,mitochondria and nucleus. Experimentally determined metabo-lite concentrations, rates of net photosynthesis and enzyme activi-ties were determined with respect to 1 g dry weight. The volumeof 1 g of freeze-dried plant material was determined to be c. 10 mlwhich allowed for the calculation of concentrations in mM.

The identification of kinetic parameter sets enabling a steady-state simulation of metabolite concentrations (d/dt = 0, t?∞)was performed using a particle swarm pattern search method forbound constrained global optimization (Vaz & Vicente, 2007).Experimentally determined maximum rates of invertase andhexokinase (means� SD) were set as upper and lower bounds forparameter identification.

The model was implemented in the numerical softwareMATLAB (R2009b) with the software packages Systems BiologyToolbox2 and the SBPD Extension Package (Schmidt &Jirstrand, 2006).

Results

Cold acclimation and low temperature-induced changes innet photosynthesis and enzyme acitvities of hexokinaseand invertase

The development of cold tolerance during 1 wk of cold exposurewas assayed using the well-established electrolyte leakage methodthat yields the 50% lethality temperature LT50 (Fig. 2). The LT50

of C24 leaves was significantly higher at all time points of coldacclimation when compared to Rsch and Te (P < 0.01). Basic tol-erance of C24 was �3.54� 0.1°C and increased to�5.3� 0.2°C during 7 d at 4°C. Basic tolerance in Rsch and Tewas �4.9� 0.1°C and �5.3� 0.2°C, respectively. Te displayedan almost linear increase of cold tolerance until day 3 at 4°C(�8.0� 0.15°C) and gained a further 1.2°C of tolerance, reach-ing �9.2� 0.12°C after 1 wk. In Rsch, the LT50 decreased to�6.3� 0.3°C within 3 d and ended up at �8.1� 0.2°C after7 d in the cold. During the first 24 h of cold acclimation induced

0 1 3 7−10

−9

−8

−7

−6

−5

−4

−3

−2

−1

0

Time of exposure at 4°C (d)

LT50

[°C

]

Fig. 2 Cold tolerance expressed as LT50 of Arabidopsis thaliana, C24 (blueline), Rsch (red line) and Te (green line) over time of exposure to 4°C.Filled circles represent means� SD of six replicates (n = 6).



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by the 4°C treatment, rates of net photosynthesis (rNPS) were sig-nificantly reduced in all accessions (P < 0.05; Fig. 3). In C24,rNPS further decreased between days 1 and 3 of cold exposure(P < 0.05) and was significantly lower than in Rsch (P < 0.05).After 7 d at 4°C, there was no significant difference of rNPS

between all accessions.In all accessions, the activities of hexokinase, as well as of solu-

ble neutral and acid invertase were significantly (P < 0.001)decreased at 4°C due to thermodynamic effects on the enzymeactivity (Fig. 4a–c). While hexokinase activities went up signifi-cantly between days 1 and 7 of cold exposure (P < 0.01; Fig. 4a),activities of both, neutral and acid invertase, remained low ordecreased even further in the cold (Fig. 4b,c). In nonacclimatedTe, maximum activities of acid invertase were significantly higheras compared to C24 and Rsch (P < 0.001; Fig. 4c).

Starch content and subcellular concentration changes forsoluble carbohydrates during cold exposure

The content of transitory leaf starch was not significantly alteredduring the first 24 h of cold exposure in all accessions andremained constant until day 7 in Rsch, while it decreased signifi-cantly (P < 0.05) in C24 and Te (Fig. 5). Light intensities at22°C (80 lmol m�2 s�1) and 4°C (50 lmol m�2 s�1, seeMaterials and Methods) were set to restrict starch synthesis to theso-called programmed or baseline mode (Sun et al., 1999) inorder to minimize differences in diurnal starch tunover at bothgrowth regimes, because such differences would have biasedmodel simulations for soluble sugars (N€agele et al., 2012).

Plastidial sucrose content significantly increased during thefirst 24 h of cold exposure in all accessions (P < 0.01; Fig 6a). InRsch, this increase was most pronounced, reaching a calculatedconcentration of 17.5� 1 mM. In the cold-tolerant accessionsTe and Rsch, plastidial sucrose content declined between days 3and 7, while in C24 it stayed constant. Cold-induced dynamicsof cytosolic sucrose concentrations were similar to those in plast-ids except for Te, where a peak value of 51.7� 2.8 mM wasreached after 3 d of cold exposure (Fig. 6b). Additionally, cyto-solic sucrose concentrations were significantly higher than in

plastids and vacuole both before and during cold exposure. Cold-induced dynamics of vacuolar sucrose concentration were mostpronounced in Te, again reaching the highest concentration after3 d at 4°C (Fig. 6c). Concentrations of free hexoses showed largefluctuations and increased in plastids of C24 and Rsch signifi-cantly during the first 24 h of cold exposure, while this increasewas observed after 3 d in Te (Fig. 6d). After 7 d, C24 had thehighest hexose concentrations in plastids (P < 0.01), while cyto-solic hexoses were highest in Rsch (Fig. 6e). The dynamics ofcytosolic hexose concentrations revealed a significant increase at

µmol

C6

h–1

gDW

–1

Fig. 3 Rates of net photosynthesis rNPS during cold exposure to 4°C inArabidopsis thaliana, C24 (blue bars), Rsch (red bars) and Te (green bars).Bars represent means of measurements� SD (n = 3).

(a)

(b)

(c)

µmol

Hex

oses

h–1

gD

W–1

µmol

Suc

rose

h–1

gD

W–1

µmol

Suc

rose

h–1

gD

W–1

Fig. 4 Maximum rates of cytosolic hexose phosphorylation (a), sucrosecleavage catalyzed by neutral invertase (b), and sucrose cleavagecatalyzed by acid invertase (c) during cold exposure in Arabidopsis

thaliana, C24 (blue bars), Rsch (red bars) and Te (green bars).Measurements of nonacclimated plants (non-acc) were performed atambient temperature, those of cold-acclimated plants (1 d acc, 3 d acc, 7 dacc) were performed at 4°C. Bars, measurements� SD (n = 5).





day 1 in C24, while Rsch and Te showed peak values at day 3.Vacuolar hexose concentration peaked at day 1 in Rsch, at day 3in C24 and continually increased until day 7 in Te. However, theconcentrations were similar in Rsch and Te, but far lower in C24(Fig. 6f).

Raffinose concentration in plastids rose significantly duringthe entire cold treatment in the tolerant accessions Rsch and Te(P < 0.001) but increased only slightly in C24 (Fig. 6g). Whileaccumulation in Rsch was strongest between days 1 and 3, it wasalmost linear in Te. Concentrations of plastidial raffinose after7 d at 4°C were 0.3� 0.07 mM in C24, 2.1� 0.2 mM in Rschand 2.6� 0.45 mM in Te. During 7 d of cold exposure, cytosolicconcentrations of raffinose rose significantly in all accessions(P < 0.01; Fig. 6h). However, concentrations were significantlyhigher in Rsch and Te than in C24 from the beginning of thecold exposure (P < 0.05). Vacuolar content of raffinose was alsosignificantly elevated during cold exposure in all accessions, butaccumulation was stronger in cold-tolerant accessions than inC24 (Fig. 6i).

Simulated rates of intracellular membrane transport andmetabolite interconversion

Based on the experimentally determined metabolite concentra-tions and enzyme activities, a steady-state simulation of metabo-lite fluxes in C24, Rsch and Te was performed for nonacclimatedplants as well as for plants exposed to 4°C for 1, 3 and 7 d.Although numerous intracellular metabolite transporters inplants have been identified, only a few have been biochemicallywell characterized (Linka & Weber, 2010), and thus it wasimpossible to comprehensively determine in vivo transport capac-ities at the tonoplast and the chloroplast envelope. To overcomethis limitation and to minimize the number of unknown parame-ters in simulations, transport processes were modeled by themass-action law – that is, the transport rate was assumed to beproportional to the concentration of the transported metabolite,disregarding possible effects of saturation, inhibition or activa-tion. Even with this simplification, simulations yielded a multi-tude of solutions for the system of differential equations, because

cyclic transport events cannot be restricted without exact knowl-edge of maximum transport activities. Therefore, we decided toidentify the minimum rate constants that were necessary to allowsimulation of a metabolic steady state. This identification wasperformed by increasing upper and lower bounds stepwise for therate constants until every metabolite concentration could be sim-ulated within its experimentally determined standard deviation.The limiting rate constants identified this way were then fixed,and only kinetic parameters of enzymatic metabolite interconver-sions were subject to further runs of parameter identification.Kinetic parameters and transport rate constants, which resultedfrom parameter identification, are summarized in Tables S3–S5.

Simulation of limiting metabolite transport rates indicated dif-ferences between cold-sensitive and cold-tolerant accessions(Fig. 7). While C24 did not show significant modulation oftransport rates during cold exposure (Fig. 7a), rates of sucrosetransport into chloroplasts and hexose transport into the vacuolewere elevated in Rsch (Fig. 7b) and, to an even larger extend, inTe (Fig. 7c). Additionally, rates of sucrose transport betweencytosol and vacuole were significantly elevated in Te as comparedto Rsch and C24. In contrast to the elevated rates of metabolitetransport in cold-tolerant accessions, rates of metabolite intercon-version were found to be either lower than or similar to those inC24 (Fig. 8a–c). Most significant differences between C24 andRsch occurred in rates of cytosolic sucrose cleavage and hexosephosphorylation, which were higher in cold exposed C24, whilenonacclimated plants showed higher rates in Rsch (Fig. 8a,b). InTe, rates of metabolite interconversions were intermediate andshowed a slight decrease during cold exposure (Fig. 8c). In allaccessions, plastidial as well as vacuolar rates of sucrose cleavagewere significantly lower than in the cytosol.

Discussion

Arabidopsis accessions differ significantly in carbohydratecompartmentation during cold exposure

Many studies have emphasized the importance of primary carbo-hydrate metabolism during cold acclimation, and various regula-tory instances significantly affecting this process have beenidentified (Strand et al., 1997, 1999; Klotke et al., 2004; Zutheret al., 2004; Lundmark et al., 2006; N€agele et al., 2011). Yet, dueto the complexity of the cellular network of metabolic interac-tions and compartmentalization, still many aspects of metabolismand its regulation are not well understood. As already stated,compartmentalization of metabolism in eukaryotic cells signifi-cantly affects activity and function of enzymes as well as concen-trations and regulatory impact of metabolites (Lunn, 2007; Klieet al., 2011). While compartmentalization of enzymes can be pre-dicted computationally based on the presence of sequence motifs(Emanuelsson et al., 2000), the subcellular allocation of metabo-lites is difficult to assess because of redundant biochemical path-ways, as for example the cytosolic and plastidic pathways ofcarbohydrate oxidation (Masakapalli et al., 2010), and varioustransport processes enabling rapid exchange of small compounds(Linka & Weber, 2010; Wingenter et al., 2010; Schneider et al.,

µmol

C6

gDW

–1

Fig. 5 Starch contents at the mid of the light phase during cold exposureto 4°C in Arabidopsis thaliana, C24 (blue bars), Rsch (red bars) and Te(green bars). Bars, means of measurements� SD (n = 5).



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2012). In the present study, compartmentalization of the centralcarbohydrate metabolism was studied to gain further insight intoregulatory instances involved in the cold acclimation process inArabidopsis thaliana.

Cold tolerance, expressed as LT50 values, showed significantdifferences before and during a 1-wk period of cold acclimationin three accessions, C24, Rsch and Te. Dynamics of cold accli-mation revealed a fast increase in tolerance during the first 3 d,which is in accordance with previous findings (N€agele et al.,2011) and showed that Te from Scandinavia is the most tolerantaccession at any time point of cold acclimation. Substantial redis-tribution of metabolites during the first 3 d in the cold occurredfor sucrose and hexoses, while cytosolic increase of the raffinoseconcentration was delayed. In vitro experiments performed byHincha et al. (2003) showed that sucrose and raffinose can pro-tect large unilamellar liposomes from damage during drying,indicating a possible role in protecting the plasma membraneduring freeze-induced dehydration. However, a lack of raffinosein a raffinose synthase mutant isolated from the cold-tolerantaccession Col-0 did not alter cold tolerance determined as freeze–thaw stability of the plasma membrane (Zuther et al., 2004). Inagreement with this, cytosolic raffinose content did not correlatewith cold tolerance in the present study, again implying that the

plasma membrane is not a target for protection by raffinose, ashas recently been demonstrated (Knaupp et al., 2011).

Besides raffinose, cytosolic sucrose concentration was alsonot correlated with cold tolerance at day 7 of the cold expo-sure. However, the sucrose concentration had already rapidlyincreased during the first day of cold exposure, thus accompa-nying the significant decrease of the LT50 in all accessions. Ithas been hypothesized that cold acclimation is the result of asequential accumulation and disappearance of several metabo-lites (Alberdi et al., 1993). In this sense, sucrose could serve asan early protectant of cellular membranes, but is later substi-tuted by other compounds. In line with this, cytosolic sucrosewas highest at day 1 in Rsch, when the increase in cold toler-ance was maximal in this accession, while it peaked at day 3in Te that displayed a larger gain in tolerance between day 1and 3 than did Rsch. The very high concentrations of cyto-solic sucrose at day 1 in Rsch and day 3 in Te, reaching con-centrations of 45–50 mM, may also indicate saturation duringcold acclimation. It might thus be speculated that cytosolicsucrose accumulates rapidly after cold exposure, serving as atransient cryoprotectant for cellular membranes at early stagesof cold exposure, while later it becomes replaced by a meta-bolically less critical compound, for example, raffinose. At that

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Fig. 6 Compartment specific metabolite concentrations of sucrose (a–c), hexoses (d–f) and raffinose (g–i) during cold exposure in Arabidopsis thaliana,C24 (blue bars), Rsch (red bars) and Te (green bars). Bars, means� SD (n = 5). Concentrations were calculated based on compartment specific volumes(see Materials and Methods).





stage, sucrose in the cytosol would serve as a substrate for thesynthesis of other cryoprotectants or has a regulatory role incold acclimation. In this context, it is interesting that raffinoseaccumulation follows the rise in cytosolic sucrose. As sucroseand galactinol are the substrates for raffinose synthesis takingplace in the cytosol (Peterbauer & Richter, 2001), theincrease of sucrose in the cytosol might be considered a trig-ger for raffinose production. Raffinose could then be trans-ported across the chloroplast envelope, probably by an activetransport mechanism as proposed by Schneider & Keller(2009), serving in the plastids as a cryoprotectant of the thy-lakoids (Knaupp et al., 2011). However, both cytosolic andplastidial sucrose content increased significantly faster than raf-finose content, suggesting an additional transitory protectiveeffect of sucrose for the thylakoids. This is difficult to testbecause of the simultaneous role of sucrose as substrate forraffinose synthesis. A likely reason for a substitution of sucroseby raffinose might be the reduced metabolic reactivity of raffi-nose and its marginal regulatory influence on primary carbonmetabolism.

While an increase of plastidial sucrose and raffinose wasalso found in cold-acclimated cabbage (Santarius & Milde,1977) and Arabidopsis, accession Columbia (Col-0) (Knauppet al., 2011), a decrease of the cytosolic content has onlybeen shown for Arabidopsis, accession Col-0 (Knaupp et al.,2011). The obvious difference in behaviour of Col-0 and theaccessions investigated in the present study might be relatedto the longer time of cold exposure, that is, 14 vs 7 d, usedby Knaupp and co-workers. This would imply that coldacclimation is either not saturated after 7 d, or that during alonger cold exposure, leaves develop that are in a metabolicstate different from those that were shifted from warm tocold. The latter has clearly been demonstrated by Strandet al. (1999). However, it is difficult to judge whether 14 din the cold would be long enough to bring about such aneffect, because the rosette of the plants is a mixture of shiftedand newly developed leaves. Also, a deviation of the labora-tory strain Col-0 from the accessions used in our study can-not be excluded.

(a)

(b)

(c)

Fig. 7 Surface plots of simulated limiting rates of metabolite transport forArabidopsis thaliana, C24 (a), Rsch (b) and Te (c). Values of transportrates, which are indicated by the color bar, were calculated to be minimalfor the successful simulation of a metabolic steady state after 0, 1, 3 and7 d of cold exposure. Nomenclature of transport rates refers to Fig. 1.

(a)

(b)

(c)

Fig. 8 Surface plots of simulated rates of metabolite interconversion forArabidopsis thaliana, C24 (a), Rsch (b) and Te (c). The surface representsmean values of metabolite interconversions calculated in steady-statesimulations (n = 50). Nomenclature of reaction rates refers to Fig. 1.



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NewPhytologist784

Steady-state simulations indicate differences in metabolicreprogramming

The rapid accumulation of sucrose and other metabolitesobserved in all accessions can be considered an immediate conse-quence of the cold-induced thermodynamic effects on enzymeactivities and/or transport rates, which can be deduced from theexperimentally determined maximum turnover rates. For exam-ple, vacuolar and cytosolic invertase activity dropped approxi-mately five-fold in the cold in all accessions, while the reductionin hexokinase activity was even stronger directly after the temper-ature shift, and activity came back to only c. 20% of the initialvalue after 7 d (Fig. 4). However, sugar accumulation would alsoresult from reduced consumption of assimilates in the cold whengrowth is stalled, or from an at least transiently reduced export ofcarbohydrates to sink organs, as predicted by modeling theimpact of cold acclimation on photosynthesis and assimilateexport to sink organs (N€agele et al., 2011). Although thermody-namic and growth effects of low temperature should be similarfor all accessions, simulated reaction rates of steady-state modelsdiscriminated the cold-sensitive from the cold-tolerant accessions,the latter showing a more pronounced reduction predominantlyof cytosolic sucrose cleavage and hexose phosphorylation. Reac-tion rates in the sensitive accession C24 were also reduced at 4°C,yet the reduction was not as strong as in Rsch and Te. While thisproves that the cold response of the tolerant accessions is moredistinct, simulations of reaction rates at the whole-cell level didnot reveal whether differences in turnover rates between tolerantaccessions and C24 would result in substantial differences at themetabolite level that could explain the differential cold tolerance.

This led us to implement intracellular redistribution of metab-olites during cold acclimation in our model of steady-state simu-lations of carbohydrate metabolism in order to investigatewhether metabolite dynamics at the compartment level could bemore significant than at the whole-cell level. To this end we firstidentified the minimal rates of metabolite exchange between cel-lular compartments that would allow establishing a metabolichomeostasis, that is, a steady state, which would be compatiblewith the reduced enzyme activities as well as the modified metab-olite concentrations observed in cold exposed plants. Certainly itwould have been preferable to measure transport activities. How-ever, the insufficient characterization of many of the transportersprecluded this concept. We thus applied a modeling approachusing stepwise increments of upper and lower bounds for thetransport rate constants. This was done until a steady state ofmetabolite concentrations could be simulated within the experi-mentally determined ranges of enzyme activities given as the stan-dard deviations of the measurements. We then compared thecalculated limiting transport rates obtained for cold-sensitive andcold-tolerant accessions.

While simulated rates of metabolite transport did not changesignificantly during cold exposure in C24, modeling suggestedsubstantially elevated rates of sucrose transport across thechloroplast envelope, as well as hexose transport across the tono-plast, in Rsch and even more so in Te. Additionally, sucrosetransport across the tonoplast was modeled to be strongly

elevated in cold-acclimated Te. As mentioned above, becauseknowledge on intracellular sugar transporters is still incomplete,especially regarding sucrose and raffinose import into plastids(Schneider & Keller, 2009), the results of the simulations dependon the assumption that bidirectional transport of hexoses, sucroseand raffinose is possible across the plastid envelope as well as thetonoplast. This provided, our findings indicate that intracellularmetabolite transport could make a substantial contribution tosustaining metabolic homeostasis during cold acclimation. Giventhat only few metabolites, at the whole-cell level, could becorrelated to cold tolerance of various accessions of Arabidopsis(Hannah et al., 2006), intracellular re-distribution might in addi-tion also be important to achieve protection of cellular organellessuch as plastids against damage during freeze–thaw cycles.

As stated above, raffinose synthesis is delayed when comparedwith the time course of cold acclimation, and thus sucrose trans-port into the plastids might be necessary to protect the thylakoidsuntil raffinose accumulation catches up. A very interesting resultof the simulations was that, after 3 d of cold exposure, the rate ofsucrose transport across the chloroplast envelope and the tono-plast could even discriminate the two cold-tolerant accessionsRsch and Te. The latter, which at day 3 was c. 1.7°C more cold-tolerant than Rsch, not only displayed higher transport rates, butalso had significantly higher sucrose concentrations in the cytosoland vacuole, while hexoses predominantly accumulated in Rsch.Our data indicate that an increased transport of sucrose fromcytosol into the vacuole allowed for more sucrose to accumulatein mesophyll cells of Te, probably because of the low vacuolarinvertase activity. In contrast, high cytosolic hexose concentra-tions were observed in Rsch at day 3 of the cold exposure, whichwere again reduced at day 7, when the cold tolerance of Rschdrew near that of Te. It is tempting to speculate that the highcytosolic hexose concentration in Rsch at day 3 could be relatedto the low gain in cold tolerance between days 1 and 3 in thisaccession. High hexose concentrations may cause feedback-inhi-bition of cytosolic invertase, thus inhibiting sucrose cyling, whichis important for buffering primary carbohydrate metabolismagainst environmental disturbances (N€agele et al., 2010). In fact,cytoslic sucrose cleavage was calculated to be higher in Te than inRsch and, thus, higher transport rates for hexoses into the vacuolemust be claimed for Te. In this respect, it is interesting that thetonoplast monosaccharide transporter, TMT1, which re-directsglucose and fructose from the cytsol into the vacuole, was dem-onstrated to be strongly induced at low temperature in the cold-tolerant accession Col-0 (Wormit et al., 2006). This may, in fact,point to a major difference in the cold-acclimation strategies ofRsch and Te: by shuffling sucrose into the vacuole, Te reducesaccumulation of hexoses in the cytosol which in turn allows for asustained cytosolic sucrose cycling and a stabilization of primarycarbohydrate metabolism against environmental changes (N€ageleet al., 2010). An exchange of sucrose between cytosol and vacuolemay thus be important for supporting regulation of cytosolicreactions of primary carbon fixation.

Of course, a final proof of the concept of limiting transportrates as applied in this work would rely on measuring transportactivities in planta. However, under conditions of insufficient





knowledge of transporters involved, the approach of mathemati-cally simulating transport processes at limiting transporter activi-ties may help to understand how cold-induced processes takingplace at the intracellular membranes could be involved in coldacclimation. The simulations plausibly demonstrated the impor-tance of intracellular re-distribution of carbohydrates and indi-cated that, besides raffinose transport into the plastids, which hasalready been demonstrated experimentally (Schneider & Keller,2009), transport of sucrose into the plastids and the vacuole aswell as hexose transport across the tonoplast might become limit-ing for cold acclimation in Arabidopsis thaliana. Taking intoaccount that the volumes of compartments may change duringthe process of cold acclimation, particularly the cytosolic and vac-uolar volume (Strand et al., 1999), predicted minimum rates oftransport, as well as metabolite interconversion for steady-statesimulations, would vary to a corresponding extent. Yet, as long asthe effect on compartment volumes can be assumed to be system-atic across all considered accessions, the inference derived fromour model simulations would hold although absolute values ofthe minimal transport rates may deviate. This would not be thecase if volume changes were different for the different accessions.However, investigations by us (Hannah et al., 2006) and others(Cook et al., 2004) have shown that global metabolite changesduring cold acclimation, which would result from changes in cel-lular water content, are very similar for cold-sensitive as well ascold-tolerant accessions.

Taken together, the present study reveals a significant elevationof cytosolic, plastidial and vacuolar sucrose concentrations as anearly response to cold exposure, being stronger in the cold-toler-ant accessions Rsch and Te as compared to the sensitive accessionC24. While C24 did not show any further alterations in subcellu-lar carbohydrate allocation, the tolerant accessions displayed anexchange of raffinose for sucrose especially in the plastids. Thesubstitution of sucrose by raffinose is in agreement with the pro-tective role of raffinose for the thylakoids and may indicate theneed for a replacement of sucrose by a metabolically less reactivecompound in tolerant accessions. Steady-state simulations of car-bohydrate metabolism suggested that intracellular carbohydratetransport processes are indispensable in establishing a low tem-perature compatible carbohydrate homeostasis in tolerant acces-sions, while in the cold-sensitive accession C24 this adjustmentappears less pronounced.

Acknowledgements

We would like to thank Annika Allinger for excellent plant culti-vation. We also thank the members of the Department of PlantBiotechnology at the University of Stuttgart for fruitful discus-sions and constructive advices, and the referees for their construc-tive advice during the reviewing process for this article.

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Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Table S1 Relative sugar concentrations in subcellular compart-ments

Table S2 Total sugar concentrations during exposure to 4°C

Table S3 Identified values of KM and Ki for cytosolic/plastidialand vacuolar invertase

Table S4 Summary of calculated limiting rate constants formembrane transport reactions

Table S5 Summary of calculated reaction rate

Notes S1 Procedure for calculation of subcellular metabolite dis-tribution by a system of linear equations

Notes S2Model structure for simulation of steady-state carbohy-drate compartmentation

Please note: Wiley-Blackwell are not responsible for the contentor functionality of any supporting information supplied by theauthors. Any queries (other than missing material) should bedirected to the New Phytologist Central Office.





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Approximating subcellular organisation of carbohydrate metabolism during cold acclimation in different natural accessions of Arabidopsis thaliana