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Fluxes in central carbohydrate metabolism of source leaves in a fructan-storing C3 grass: rapid turnover and futile cycling of sucrose in continuous light under contrasted nitrogen nutrition status

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This work assessed the central carbohydrate metabolism of actively photosynthesizing leaf blades of a C3 grass (Lolium perenne L.). The study used dynamic (13)C labelling of plants growing in continuous light with contrasting supplies of nitrogen
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   Journal of Experimental Botany  , Vol. 63, No. 6, pp. 2363–2375, 2012doi:10.1093/jxb/ers020 Advance Access publication 27 February, 2012 RESEARCH PAPER Fluxes in central carbohydrate metabolism of source leavesin a fructan-storing C 3  grass: rapid turnover and futile cyclingof sucrose in continuous light under contrasted nitrogennutrition status Fernando A. Lattanzi 1, *, Ulrike Ostler 1, *, Melanie Wild 1, * , † , Annette Morvan-Bertrand 2 , Marie-Laure Decau 2 ,Christoph A. Lehmeier 1, ‡ , Fre´ de´ ric Meuriot 2 , Marie-Pascale Prud’homme 2 , Rudi Scha ¨ ufele 1 andHans Schnyder 1,# 1 Lehrstuhl fu ¨ r Gru ¨ nlandlehre, Technische Universita  ¨ t Mu ¨ nchen, Alte Akademie 12, 85350 Freising-Weihenstephan, Germany 2 UMR INRA-UCBN 950 EVA Ecophysiologie Ve´ ge´ tale, Agronomie and Nutritions NCS, Universite´  de Caen Basse-Normandie,Esplanade de la Paix, 14032 Caen Cedex, France* These authors contributed equally to this work. y Current address: Bavarian State Research Center for Agriculture, Vo¨ttinger Strasse 38, D-85354 Freising, Bavaria z Current address: Department for Ecology and Evolutionary Biology, Kansas Biological Survey, University of Kansas, 66047 Lawrence,KS, USA  #  To whom correspondence should be addressed. E-mail: schnyder@wzw.tum.de Received 28 October 2011; Revised 12 January 2012; Accepted 13 January 2012  Abstract This work assessed the central carbohydrate metabolism of actively photosynthesizing leaf blades of a C3 grass(   Lolium perenne  L.). The study used dynamic  13 C labelling of plants growing in continuous light with contrastingsupplies of nitrogen (‘low N’ and ‘high N’) and mathematical analysis of the tracer data with a four-poolcompartmental model to estimate rates of: (i) sucrose synthesis from current assimilation; (ii) sucrose export/use;(iii) sucrose hydrolysis (to glucose and fructose) and resynthesis; and (iv) fructan synthesis and sucrose resynthesisfrom fructan metabolism. The contents of sucrose, fructan, glucose, and fructose were almost constant in bothtreatments. Labelling demonstrated that all carbohydrate pools were turned over. This indicated a system inmetabolic steady state with equal rates of synthesis and degradation/consumption of the individual pools. Fructancontent was enhanced by nitrogen deficiency (55 and 26% of dry mass at low and high N, respectively). Sucrosecontent was lower in nitrogen-deficient leaves (2.7 versus 6.7%). Glucose and fructose contents were always low(  < 1.5%). Interconversions between sucrose, glucose, and fructose were rapid (with half-lives of individual poolsranging between 0.3 and 0.8 h). Futile cycling of sucrose through sucrose hydrolysis (67 and 56% of sucrose at lowand high N, respectively) and fructan metabolism (19 and 20%, respectively) was substantial but seemed to have nodetrimental effect on the relative growth rate and carbon-use efficiency of these plants. The main effect of nitrogendeficiency on carbohydrate metabolism was to increase the half-life of the fructan pool from 27 to 62 h and toeffectively double its size.Key words:  13 C, central carbohydrate metabolism, carbon fluxes, compartmental analysis, fructan, futile cycling, sucrose, store,tracer, turnover.  Abbreviations: FEH, fructan exohydrolase; FT, fructosyltransferase; SD, standard deviation. ª  The Author [2012]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: journals.permissions@oup.com   a  t  T  e  c h ni   s  c h  e  Uni   v e r  s i   t  Ã ¤  t  Mà ¼n c h  e n onM a r  c h 2  7  ,2  0 1 2 h  t   t   p :  /   /   j  x b  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om  Introduction This study was concerned with sucrose as a key branchpoint in the central carbohydrate metabolism of photosyn-thetically active, exporting leaf blades of perennial ryegrass( Lolium perenne  L.), a fructan-storing species. In particular,the fluxes of sucrose in distinct, interdependent processeswere assessed: fructan synthesis, sucrose hydrolysis, sucrosestorage in vacuoles, sucrose export/use by sinks, and sucrosesynthesis from current assimilation and from the productsof its hydrolysis or from fructan metabolism (sucrosecycling). This analysis was performed with plants growingin continuous light with either a high or a low nitrogenfertilizer supply rate.Fructans are the main long-term storage carbohydrate inthe vegetative parts of many important economic plantspecies, including wheat, barley, rye, oat, and C3 foragegrasses (Pollock and Cairns, 1991; Vijn and Smeekens, 1999). Fructans serve vital roles in seed filling and recovery frombiotic and abiotic stresses, providing carbohydrate sub-strates for growth and respiration when provision bycurrent assimilation is less than demand by sinks (Schnyder,1993; Morvan-Bertrand  et al. , 2001; Yang and Zhang, 2006; Valluru and Van den Ende, 2008). Fructans are stored invacuoles and are synthesized from sucrose by fructosyl-transferases (FTs) and degraded by fructan exohydrolases(FEHs) (Wagner  et al. , 1983; Frehner  et al. , 1984; Darwenand John, 1989). The transfer of the fructosyl residue of sucrose to acceptor sucrose or fructan molecules is exer-gonic and essentially irreversible (Wagner and Wiemken,1987; Duchateau  et al. , 1995). Fructans of   L. perenne  arestructurally diverse, with this diversity resulting from thecooperation of several FTs (Pavis  et al. , 2001 a , b ; Chalmers et al. , 2005; Lasseur  et al. , 2006, 2011). Degradation by FEHs involves the successive cleavage of terminal fructoseresidues of fructans. Most, if not all, of the released fructoseis used in resynthesis of sucrose for export (Pollock andCairns, 1991). However, resynthesized sucrose may beutilized again in fructan synthesis if fructan synthesis anddegradation occur simultaneously inside the same cell.Moreover, the glucose released during fructan synthesis isprobably used in resynthesis of sucrose for fructan synthe-sis. Evidence for such futile cycling of carbon through thefructan store has been put forward repeatedly (e.g. Pollock,1982; Wagner  et al. , 1986; Simpson and Bonnett, 1993; Morvan-Bertrand  et al. , 1999, Amiard  et al. , 2003), butits quantitative importance for sucrose partitioning (andcycling) has not been assessed. As fructan turnover in leaf blades can be high (Borland and Farrar, 1988; Farrar, 1989), sucrose cycling could be substantial.Carbohydrates can also be stored in the form of sucrose invacuoles (Kaiser  et al. , 1982). In  L. perenne  and otherfructan-storing grasses, vacuolar sucrose may even replacetransient starch as the diurnal storage compound in leaves orother photosynthetic organs/tissues (Wilson and Bailey, 1971, Riens  et al. , 1994; Isopp  et al. , 2000; Gebbing, 2003). This sucrose store serves night-time export of autotrophic tissue.Fructan and sucrose storage follow divergent distributionpatterns between organs and tissues: sucrose storage predom-inates in the photosynthetically active organs, and fructanstorage is most active in heterotrophic tissue (Volenec, 1986;Schnyder and Nelson, 1987; Pollock and Cairns, 1991; Riens et al. , 1994; Morvan-Bertrand  et al. , 2001; Gebbing, 2003). This contrast could result from a fundamental trade-off,a biochemical switch, between sucrose utilization in vacuolarsucrose storage and fructan synthesis in the same compart-ment. Yet, according to Martinoia  et al.  (2007), plants canhave several types of vacuoles with distinct functions, evenwithin one cell, theoretically providing a basis for the co-existence of vacuolar sucrose storage and fructan metabolismin different compartments in the same cell.Hydrolysis by invertase is another, potentially strong,sink for sucrose in source leaves (Huber, 1989). Guerrand et al.  (1996) found very high extractable soluble acidinvertase activity in mature photosynthesizing leaf blades of  L. perenne  induced to accumulate fructan. Similarly, maturephotosynthetically active leaves of the fructan-storing grass Lolium temulentum  contain intracellular invertase activitythat is sufficient to hydrolyse the total sucrose content of the leaf in 0.5 h (Kingston-Smith  et al. , 1999). Vargas  et al. (2007) found an upregulation of alkaline invertase in wheatleaves following exposure to cold or osmotic stress,conditions that are also conducive to fructan accumulation.Notably, some FEHs are also capable of hydrolysingsucrose (Kingston-Smith  et al. , 1999; Tamura  et al. , 2011).However, it is often not clear how much of the tissuesucrose is accessible by invertase in source leaves, asdifferent forms of invertase are distributed between cellularcompartments, including apoplast, cytoplasm, and vacuole(Kingston-Smith  et al. , 1999; Cairns and Gallagher, 2004; Vargas  et al. , 2007; Vargas and Salerno, 2010), and the activity may be regulated (Roitsch and Gonza´lez, 2004). Inparticular, Cairns and Gallagher (2004) found no sucrosehydrolysis in treatments blocking sucrose resynthesis inexcised, pre-illuminated and non-fructan-containing leavesof   L. temulentum . From this, they suggested that solubleacid invertase and sucrose did not share the same compart-ment in these leaves.Nitrogen nutrition is known to affect carbohydratemetabolism in fundamental ways (Stitt and Krapp, 1999). Nitrogen deficiency decreases sink strength and growth rate(Kavanova´  et al. , 2008), increases carbohydrate accumula-tion in vegetative tissue (Lehmeier  et al. , 2010 a ), andincreases the expression of carbohydrate storage-relatedgenes (Ruuska  et al. , 2008) and their activities (Wang andTillberg, 1996; Wang  et al. , 2000; Morcuende  et al. , 2004).From these relationships, one might expect that nitrogennutrition will also affect the turnover of leaf carbohydratepools and cycling of sucrose through fructan and otherstores, but, to the best of our knowledge, these effects havenot been explored.Accordingly, this work addressed the following question:how does the nitrogen fertilizer supply affect central carbo-hydrate metabolism in source leaves of a fructan-storing 2364  |  Lattanzi  et al.   a  t  T  e  c h ni   s  c h  e  Uni   v e r  s i   t  Ã ¤  t  Mà ¼n c h  e n onM a r  c h 2  7  ,2  0 1 2 h  t   t   p :  /   /   j  x b  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om  species,  L. perenne , in continuous light? In particular, howdoes it affect: (i) sucrose synthesis from current assimila-tion; (ii) sucrose export to heterotrophic tissue; (iii) sucrosehydrolysis and resynthesis; and (iv) fructan synthesis andsucrose resynthesis from fructan metabolism products?Lastly, is vacuolar sucrose storage disabled/suppressed bycontinuous light, as the requirement for buffering day/nightcycles in assimilation would not exist in continuous light? Inaddressing these questions, this study also investigated thepossible implication of vacuolar compartmentation onsucrose cycling, an issue recently raised and discussed byKruger  et al.  (2007). The present analysis employed dynamiclabelling (as described by Ratcliffe and Shachar-Hill, 2006)with  13 CO 2 / 12 CO 2  over intervals of 1 h to 8 d (Schnyder  et al. ,2003), measurement of the quantity and tracer time course insucrose, fructan, glucose, and fructose (Morvan-Bertrand et al. , 1999; Gebbing and Schnyder, 2001), and compartmen- tal modelling using similar principles to those used previouslyto characterize the substrate supply system of leaf growth andrespiration in the same species (Lattanzi  et al. , 2005; Lehmeier et al. , 2008, 2010 a , b ). Materials and methods Plant material and growth conditions Details of plant material and growth conditions for the presentexperiments have been described previously (Lehmeier  et al. ,2010 b ). Briefly, the experiment was performed in four plant growthchambers (Conviron E15, Winnipeg, Canada). Plants were grownsingly in pots (350 mm high, 50 mm diameter) filled with washedquartz sand and arranged at a density of 378 m  2 .All plants were grown in continuous light, with 275  l mol m  2 s  1 photosynthetic photon flux density at canopy height. Relativehumidity was controlled at 85% and temperature at 20   C at thelevel of the leaf growth zone. Every 3 h, the plants receiveda nutrient solution with either 1.0 mM nitrate (‘low N’) or 7.5 mMnitrate (‘high N’) as the sole nitrogen source. Control of CO  2  concentration and isotope composition The growth chambers formed part of the  13 CO 2 / 12 CO 2  labelling andgas exchange mesocosms described by Schnyder  et al.  (2003). Briefly,CO 2 -free air and CO 2  of known carbon isotope composition ( d 13 C, in & , with  d 13 C ¼ [( 13 C/ 12 C sample )/( 13 C/ 12 C VPDB standard )–1] 3 1000) weremixed and supplied to the growth chambers. Each chamber had itsown mixing system. The concentration of CO 2  (360  l l l  1 ) and its d 13 C ( d 13 C air ) in the chamber air was monitored with an infrared gasanalyser (Li-6262, Li-Cor, Lincoln, NE, USA) and a continuous-flowisotope-ratio mass spectrometer (CF-IRMS, Delta Plus, FinniganMAT, Bremen, Germany). At high N, one chamber was kept at d 13 C air  –28.8 &  [ 6 0.2 standard deviation (SD)] and the other at –1.7 & ( 6 0.2). At low N,  d 13 C air  was –3.6 & ( 6 0.2) in one chamberand –30.9 &  ( 6 0.3) in the other. The stability of the isotopiccomposition and concentration of CO 2  ( 6 3  l l l  1 SD) in chamberair was achieved by small periodic adjustments of air flow andCO 2  concentration in the mixing systems serving the chambers.All chambers were equipped with air locks, which permittedaccess to plants for sampling etc. with minimal disturbance of theconcentration and isotope composition of CO 2  (Lehmeier  et al. ,2008). Labelling and sampling When plants had three tillers (about 3 and 6 weeks after sowing athigh and low N), labelling was initiated by randomly swappingselected individual plants between chambers with different  d 13 C air (i.e.  13 C-enriched CO 2 / 13 C-depleted CO 2  and vice versa) withinthe same nitrogen fertilizer supply. At this stage, the plants in thetwo treatments had a very similar size, minimizing eventual effectsof nutrient levels on size-dependent carbon relations. In bothtreatments, plants were labelled for periods of 1, 2, 3, 4, 8, or 16 h,or for 1, 2, 4, 8, or 16 d. Four replicate plants were used for eachlabelling interval.At the end of the labelling period, plants were removed from thestands. In each plant, the blade (lamina) of the youngest fullyexpanded leaf was sampled from all mature tillers. Blade area wasmeasured on a subsample of 16–20 replicates. Thickness wasestimated as blade volume per area, with volume estimated fromfresh mass (Arredondo and Schnyder, 2003). Mature tillers weredefined as having at least three fully expanded leaves. Samplesfrom one plant were combined, weighed, frozen in liquid nitrogen,freeze dried for 72 h at –80   C, weighed again, ground to flourmesh quality with a ball mill, and stored at –30   C untilcarbohydrate extraction. Non-labelled control plants were sampledin the same way in both chambers of both treatments in parallelwith labelled plants. The controls were grown continuously in thesame chamber, i.e. in the presence of a constant  d 13 C air . Carbon and nitrogen elemental analysis Carbon and nitrogen elemental contents of bulk biomass sampleswere measured using the protocols described by Lehmeier  et al. (2008). Carbohydrate extraction, separation, quantification, and   13 C analysis Soluble carbohydrates were extracted from 100 mg of freeze-driedground tissue by addition of 2 ml of cold (4   C) 100 mM sodiumphosphate buffer (pH 7.4). Samples were mixed by vortexing threetimes for 30 s each at 4   C before centrifugation at 3200  g   for10 min at 4   C. The pellets were extracted once again with 2 ml of 100 mM sodium phosphate buffer using the same protocol. Thepellets were preserved for a later water extraction. The twosupernatants were combined, 6 ml of 96% acetone was addedfor protein precipitation, and the tubes were incubated overnightat –80   C. After warming, the tubes were centrifuged for 10 min at10 000  g   and the supernatants were preserved. The pellets from thesodium phosphate extraction were extracted twice with 2 ml of pure water at 60   C for 30 min and the samples centrifuged at3200  g   for 10 min at 4   C. The supernatants were pooled with theacetone supernatants and the extracts dried under reduced pressureand redissolved in 1 ml of water. Aliquots were passed througha column containing cation exchange resin (Dowex 50 W,H + -form, Sigma, St Louis, MO, USA) and anion exchange resin(Dowex 1X8, Formate form, Sigma, St Louis, MO, USA) toremove charged compounds (Smouter and Simpson, 1991). The columns were eluted with water, and the samples were againconcentrated under reduced pressure and redissolved in water.Glucose, fructose, sucrose, and fructan were separated by HPLCon a cation exchange column (Sugar-PAK, 300 mm 3 6.5 mm,Millipore Waters, Milford, MA, USA) as given in Guerrand  et al. (1996). After separation, the sucrose, fructan, glucose, and fructosewere collected separately and concentrated under reduced pressure.To make sure that the sucrose was not hydrolysed by residualinvertase activity during the sodium phosphate buffer extraction,relative sucrose, glucose, and fructose levels were compared withthe same samples (six independent replicates) extracted usinga classical ethanol/water extraction protocol. In this classicalprotocol, 20 mg of freeze-dried ground tissue were extracted with80% ethanol at 80   C for 15 min, followed by two extractions with Sucrose fluxes in  Lolium perenne  |  2365   a  t  T  e  c h ni   s  c h  e  Uni   v e r  s i   t  Ã ¤  t  Mà ¼n c h  e n onM a r  c h 2  7  ,2  0 1 2 h  t   t   p :  /   /   j  x b  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om  pure water at 60   C for 15 min. Samples were centrifuged betweeneach extraction at 10 000  g   for 10 min and the three supernatantswere pooled. The pooled supernatants were dried and desalted asdescribed above. For all samples, the two protocols (sodiumphosphate buffer and ethanol/water extraction) gave the samerelative proportions of sucrose, glucose, and fructose (data notshown), indicating that no sucrose hydrolysis occurred during thesodium phosphate buffer extraction.Aliquots of the aqueous solution of separated sugars containingapproximately 2 mg of carbohydrate were dried on Chromosorb,packed in tin cups and the  d 13 C of the samples was determined bycontinuous-flow isotope ratio mass spectrometry (Delta Plus,Finnigan MAT, Bremen, Germany) after combustion in anelemental analyser (NA 1110, Carlo Erba Instruments, Milan,Italy). Each sample was measured against a laboratory CO 2 working standard, which was calibrated previously against anIAEA secondary standard (IAEA-CH6, accuracy of calibration0.06 &  SD). Finely ground wheat flour served as an internallaboratory standard and was run regularly every tenth sample todetermine the precision of the isotope analysis; the SD in  d 13 C of sample repeats was 0.15 & .The extraction and purification scheme caused some loss of carbohydrates during the purification steps. To correct for theselosses, we used a procedure that eliminate the abovementionedrequirement of removing charged compounds and provided virtu-ally full recovery (Schnyder and de Visser, 1999). For this, 10 mg of  ground dry material was weighed into 2.2 ml capped Eppendorf tubes and 2 ml of water was added. The tubes were sealedimmediately and transferred to a 95   C water bath for 10 min,shaken for 30 min, and centrifuged at 10 000  g   for 10 min, and thesupernatants were removed with a pipette. Comparison with twoadditional extractions demonstrated that  > 98% of the total water-soluble carbohydrates were dissolved in the first extract. Water-soluble carbohydrates were analysed with a continuous-flow system:an aliquot of the extract was hydrolysed in 0.1 mM sulphuric acidfor 25 min at 92   C. The reducing power of the hydrolysedcarbohydrates was detected photometrically at 425 nm afterreduction of a potassium ferricyanide solution. Analysis of thereference sucrose, fructan, glucose, and fructose (all analytical gradefrom Merck, Darmstadt, Germany) yielded response factors for theindividual carbohydrates, which were proportional to the amount of hexose residues present in 1 g of substrate. The fraction of tracer in carbohydrates Tracer incorporation in carbohydrates was followed via the timecourse of the fraction of unlabelled carbon (  f  unlab ) in sucrose,fructan, glucose, and fructose of control plants and plantstransferred to and kept inside a labelling chamber for a distinctperiod of time (see above). The  f  unlab  fraction represented thefraction of carbon in carbohydrates that was derived fromassimilation in the chamber of origin, prior to transfer to thelabelling chamber. In the labelling chamber,  f  unlab  was graduallyreplaced by a labelled fraction (  f  lab ), which resulted fromassimilation after the transfer (  f  unlab +  f  lab ¼ 1). Note that, except forthe isotopic composition of CO 2  in the chambers, the conditions inthe chamber of srcin and in the labelling chamber were identical(see above). Details of the labelling data evaluation are given in deVisser  et al.  (1997) and Schnyder and de Visser (1999). The  f  unlab fraction was obtained from mass balance considerations as  f  unlab ¼ ( d 13 C s  –  d 13 C lab )/( d 13 C unlab  –  d 13 C lab ), with  d 13 C s  designatingthe isotopic composition of a certain carbohydrate sample(sucrose, glucose, fructose, or fructan, as appropriate), and d 13 C unlab  and  d 13 C lab  denoting the  d 13 C of the same carbohydrateextracted from control plants grown continuously in the chamberof srcin (unlabelled) and in the labelling chamber, respectively.Importantly, the direction of the transfer of labelled plants (from 13 C-enriched CO 2  to  13 C-depleted CO 2  or vice versa) had no effecton  f  unlab , as is expected for artefact-free labelling experiments andappropriate consideration of isotope discrimination effects duringlabelling. Compartmental analysis of tracer time courses The time course of   f  unlab  in the different carbohydrates is a reflectionof their turnover by labelled carbon. This time course is a functionof the metabolic pathways that include these carbohydrates, thecompartmentation of the carbohydrates, the size of the differentcarbohydrate pools, and the magnitude of the fluxes among them.These functional relationships can be represented in compartmentalmodels.In compartmental modelling terminology, a ‘pool’ is defined asa population of molecules that exhibit the same proportion of labelled carbon atoms; thus, a pool represents an entity withuniform isotopic composition (Lattanzi  et al. , 2005, Lehmeier et al. , 2008). This means that, for instance, compartmentalmodelling per se can not distinguish cytosolic and vacuolar sucrosepools if these pools exhibit identical tracer kinetics.The compartmental model chosen in the present study assumedthat: (i) the system is in a metabolic steady state; (ii) fluxes obeyfirst-order kinetics; and (iii) pools are homogeneous and well mixed,so that the flux(es) out of and into the pool, as well as the pool’s sizeand half-life, are constant. Growth of plants occurred in a constantenvironment with continuous light, which was conducive to steadyconditions of growth and related metabolism. Isotope discrimina-tion in carbohydrate interconversions was accounted for bycollection and analysis of control plants (see above). The fact thatlabelling with  13 C-depleted and  13 C-enriched CO 2  produced identi-cal results proved that this approach prevented artefacts related toisotope discrimination. For other assumptions of the compartmen-tal model, such as the constancy of carbohydrate pool sizes withlabelling duration and plant age, see Results and Discussion.The biologically realistic model that best reflected the tracer datain our experiments (‘constrained four-pool model’ in Fig. 1; see also Supplemental Table S2 in  JXB   online) is described by thefollowing set of differential equations (implemented in the softwareModelMaker; Cherwell Scientific, UK). The change in the amountof unlabelled carbon in pool  i   with time was given by the sum of  Fig. 1.  Four-pool compartmental model of central carbohydratemetabolism in source leaves of   L. perenne . Suc, sucrose; Glc,glucose; Fru, fructose;  F  In , tracer flux into the system.  Q i represents the size of the carbohydrate pool  I  , and  k  ij  is the rateconstant for the flux from pool  i   to pool  j  . Thus,  k  10  denotes theexport of sucrose from the system,  k  12  denotes the FT-catalysedtransfer of the fructosyl residue of sucrose to a fructan (or sucrose)acceptor molecule,  k  13  denotes glucose production by FT plusinvertase(-like) activities,  k  14  denotes fructose production byinvertase(-like) activity,  k  24  denotes cleavage of fructose fromfructan by FEH,  k  31  denotes glucose use in sucrose resynthesis,and  k  41  denotes fructose use in sucrose resynthesis. Formathematical details of the model, including the differentialequations describing the fluxes, see Materials and methods. 2366  |  Lattanzi  et al.   a  t  T  e  c h ni   s  c h  e  Uni   v e r  s i   t  Ã ¤  t  Mà ¼n c h  e n onM a r  c h 2  7  ,2  0 1 2 h  t   t   p :  /   /   j  x b  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om  fluxes of unlabelled carbon into that pool minus the fluxes of unlabelled carbon leaving the pool: df  unlab  Q 1  t  dt    Q 1  ¼  f  unlab In  t    F  In  þ  f  unlab  Q 3  t    k  31 Q 3 þ  f  unlab  Q 4  t    k  41 Q 4    f  unlab  Q 1  t    k  1 0  þ  k  1 2  þ  k  1 3  þ  k  1 4  Q 1 ð 1 Þ df  unlab  Q 2  t  dt    Q 2  ¼  f  unlab  Q 1  t    k  1 2 Q 1    f  unlab  Q 2  t    k  24 Q 2  ð 2 Þ df  unlab  Q 3  t  dt    Q 3  ¼  f  unlab  Q 1  t    k  1 3 Q 1    f  unlab  Q 3  t    k  31 Q 3  ð 3 Þ df  unlab  Q 4  t  dt    Q 4  ¼  f  unlab  Q 1  t    k  14 Q 1  þ  f  unlab  Q 2  t    k  24 Q 2   f  unlab  Q 4  t    k  41 Q 4 ð 4 Þ with  t  the time since the onset of labelling (labelling duration).  f  unlab_  Qi  ( t ) is the fraction of unlabelled carbon in pool  i   at time  t ,  F  In equals the flux into the system, and  f  unlab_In  the fraction of unlabelledcarbon in that flux [  f  unlab_In ( t ) ¼ 1 for  t  <0 and  f  unlab_In ( t ) ¼ 0 for  t > 0]. Q i   is the size of pool  i  , and  k  ij   the rate constant for the flux from pool i   to pool  j   (with  j  ¼ 0 denoting the flux leaving the system).For a system in metabolic steady state, pool sizes are constantwith time, and described by: F  In  ¼  k  1 0 Q 1  ð 5 Þ k  1 2 Q 1  ¼  k  24 Q 2  ð 6 Þ k  1 3 Q 1  ¼  k  31 Q 3  ð 7 Þ k  1 4 Q 1  þ  k  2 4 Q 2  ¼  k  41 Q 4  ð 8 Þ The half-lives reflect the turnover of pools. In a metabolic steadystate, they are determined by the rate constants for efflux alone.Thus, the half-life ( T  1/2 ) of the different pools were obtained as: T  1 = 2  Q 1   ¼  ln ð 2 Þ k  1 0  þ  k  1 2  þ  k  1 3  þ  k  1 4 ð 9 Þ T  1 = 2  Q 2   ¼  ln ð 2 Þ k  24 ð 10 Þ T  1 = 2  Q 3   ¼  ln ð 2 Þ k  31 ð 11 Þ T  1 = 2  Q 4   ¼  ln ð 2 Þ k  41 ð 12 Þ The rate constants were optimized such that  f  unlab_  Qi  ( t ) derivedfrom Equations (1)–(4) fitted the observed tracer kinetics of sucrose ( Q 1 ), fructan ( Q 2 ), glucose ( Q 3 ), and fructose ( Q 4 ) best.This was done under the constraints that: (i) the pool size ratios Q 1 / Q 2  were given by the observed ratios of carbohydrate content;and (ii) identical amounts of glucose and fructose were utilized forsucrose regeneration, conserving the stoichiometric ratio of 1:1.Interconversions between glucose and fructose were not considered(see Compartmental modelling of carbohydrate metabolism inResults). The mean residence time ( s ) of carbon in the system wasobtained as  s ¼ ( Q 1  +  Q 2  +  Q 3  +  Q 4 )/ F  10 .A number of biologically plausible, alternative variants of poolmodels were also implemented in ModelMaker using the sameprinciples and used to fit the observed tracer kinetics by optimizinghalf-lives of the pools. Results Leaf parameters Nitrogen fertilizer supply affected key leaf parameters intypical ways (Table 1). Plants were sampled at equal size,and thus total dry mass per leaf was similar for the low andhigh N supply. Nitrogen fertilizer greatly increased leaf areaand nitrogen content per unit dry mass, but it decreased leaf thickness and tissue density (i.e. dry mass per fresh mass).Nitrogen content per unit leaf area was unchanged becauseof the compensating effects of tissue density and leaf thickness on nitrogen content per unit dry mass. Carbohydrate concentration Nitrogen fertilizer supply also affected carbohydrate concen-tration: it decreased fructan concentration to approximately Table 1.  Leaf parameters: area, thickness, fresh and dry mass,and nitrogen content per unit biomass and per unit leaf areaPlants were grown with 1 mM nitrate (low N) or 7.5 mM nitrate(high N) in the nutrient solution. Results are shown as means 6 SEof 16–20 replications. Numbers in a row with different superscriptletters indicate a statistically significant difference at  P   < 0.05. Parameter Low N High NMean SE Mean SE  Area per leaf (cm 2 ) 2.9 a 6 0.3 7.8 b 6 0.7Leaf thickness (mm) 0.57 a 6 0.04 0.30 b 6 0.01Fresh mass per leaf (mg) 150 a 6 13 226 b 6 19Dry mass per leaf (mg) 54 a 6 5 64 a 6 6Nitrogen per dry mass (mg g  1 ) 9.3 a 6 0.6 24.5 b 6 1.7Nitrogen per area (g m  2 ) 2.0 a 6 0.2 2.0 a 6 0.1 Table 2.  Mean concentration of glucose, fructose, sucrose andfructan in the blade (lamina) of the youngest fully expanded leaf of perennial ryegrass.Plants were grown with 1 mM nitrate (low N) or 7.5 mM nitrate(high N) in the nutrient solution. Results are shown as mg g  1 freshmass (means 6 SE) of two to seven replicates. Numbers in a rowwith different superscript letters indicate a statistically significantdifference at  P   < 0.05. Low N High NMean SE Mean SE Sucrose 10.6 a 6 1.8 19.9 b 6 3.2Fructan 217 a 6 36 78 b 6 13Glucose 4.8 a 6 1.3 4.5 a 6 0.7Fructose 5.5 a 6 1.7 3.6 a 6 0.6 Sucrose fluxes in  Lolium perenne  |  2367   a  t  T  e  c h ni   s  c h  e  Uni   v e r  s i   t  Ã ¤  t  Mà ¼n c h  e n onM a r  c h 2  7  ,2  0 1 2 h  t   t   p :  /   /   j  x b  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om
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