Original Article

Natural 13C distribution in oil palm (Elaeis guineensis Jacq.) andconsequences for allocation pattern

Emmanuelle Lamade1, Guillaume Tcherkez2, Nuzul Hijri Darlan3, Rosario Lobato Rodrigues4, Chantal Fresneau5, Caroline Mauve6,Marlène Lamothe-Sibold6, Diana Sketriené5 & Jaleh Ghashghaie5

1UPR34 Performance of Perennial Cropping Systems, CIRAD-PERSYST, Montpellier 34398, France, 2Research School of Biology,College ofMedicine, Biology andEnvironment, AustralianNationalUniversity, Canberra, AustralianCapital Territory 2601, Australia,3Indonesian Oil Palm Research Institute, IOPRI, Jl. Brigjen Katamso 51, Medan, North Sumatra, Indonesia, 4Embrapa AmazoniaOcidental, Rodovia AM-010, km 29, Manaus 319, Brazil, 5ESE, Université Paris-Sud, CNRS UMR 8079, Orsay cedex 91405, Franceand 6Plateforme Métabolisme-Métabolome, Université Paris-Sud

ABSTRACT

Oil palm has now become one of the most important crops,palm oil representing nearly 25% of global plant oil consump-tion. Many studies have thus addressed oil palm ecophysiologyand photosynthesis-based models of carbon allocation havebeen used. However, there is a lack of experimental data oncarbon fixation and redistributionwithin palm trees, and impor-tant C-sinks have not been fully characterized yet. Here, wecarried out extensive measurement of natural 13C-abundance(δ13C) in oil palm tissues, including fruits at different matura-tion stages. We find a 13C-enrichment in heterotrophic organscompared to mature leaves, with roots being the most 13C-enriched. The δ13C in fruits decreased during maturation,reflecting the accumulation in 13C-depleted lipids. We furtherused observed δ13C values to compute plausible carbon fluxesusing a steady-state model of 13C-distribution including meta-bolic isotope effects (12v/13v). The results suggest that fruitsrepresent a major respiratory loss (≈39% of total tree respira-tion) and that sink organs such as fruits are fed by sucrose fromleaves. That is, glucose appears to be a quantitatively importantcompound in palm tissues, but computations indicate that it isinvolved in dynamic starch metabolism rather that C-exchange between organs.

Key-words: carbon allocation; isotope composition;oleosynthesis cost; respiratory losses; 13C/12C fractionation.

INTRODUCTION

Oil palm (Elaeis guineensis Jacq., Arecaceae) is presently oneof the most important crops, with more than 17 million ha cul-tivated worldwide and an annual production of 300 � 106 t offresh fruit bunches (in 2012, FAOSTAT, www.fao.org). It is be-lieved to be the most efficient oil-producing plant in the world,with a maximal oil yield of ≈12.2 t ha�1 year�1 (world averageabout 4 t ha�1 year�1) far beyond rapeseed (Brassica napus)or sunflower (Helianthus annuus) (2.3 and 1.5 t ha�1 year�1, re-spectively). The oil content in palm fruits is very high, up to 80%

on a deciwatt basis. Unsurprisingly therefore, there are cur-rently intense efforts to better understand carbon fixation andallocation and metabolism of oil production (oleosynthesis) inthis species. Oil palm photosynthesis has been studied sincethe 1970s (Corley 1973; Dufrêne & Saugier 1993; Henson1992; Lamade & Setiyo 1996), and so key parameters such asmaximal photosynthesis rate, photosynthetic quantum effi-ciency, and light compensation point as well as saturating lightlevels have been documented. Carbon allocation to buncheshas been mostly described by photosynthesis-driven models(Gerritsma 1988; Dufrêne 1989; Kraalingen et al. 1989), butoleosynthesis velocity and carbonmetabolism itself are believedto play a key role in determining fruit bunches developmentand fruit sink-strength. The genome sequence of the genusElaeis has been described (Singh et al. 2013), and a compar-ative transcriptomics analysis with date palm has shown therole of genes involved in carbohydrate metabolism (such asinvertase) in addition to lipid production during fruit matu-ration (Bourgis et al. 2011). Recent studies (Bourgis et al.2011; Tranbarger et al.; 2011; Dussert et al. 2013) of lipidbiosynthetic chain and regulations along fruit maturationfurther emphasized the role of certain precursors (such assugars) of oleosynthesis.

On the one hand, oleosynthesis is a high energy-demandingmetabolic process, associated with a substantial carbon (respi-ratory) cost. At the fruit scale, the production of oil-containingtissue is associated with a metabolic yield amongst the lowestknown values in crops, of 0.54 (total biomass production) and0.34 gCg�1C (lipid production), that is, with a requirement ofmore than 2.5 kg of starch to produce 1kg of fruit dry matter(De Vries & van Laar 1983). At the whole palm tree scale, itis believed that about 62% of fixed CO2 is lost by respiration(Dufrêne, 1989). Corley and Tinker (2003) quote respiratorylosses between 60 and 80% (cited from Breure 1988; Henson1992; Lamade & Setiyo 1996). The ratio of fresh fruit bunchesmass to total dry matter (i.e. bunch index) is quite high, around0.46 (Wahid et al. 2004), but the carbon use efficiency (CUE, theratio of net fixed carbon – including all respiratory losses – togross carbon fixation) is ≈0.38 (Dufrêne 1989, Henson & Chan2000; Lamade & Bouillet 2005). Furthermore, fruit yield is sub-ject to considerable variation along the year, due to changes inCorrespondence: E. Lamade. e-mail: emmanuelle.lamade@cirad.fr

© 2015 JohnWiley & Sons Ltd 1

doi: 10.1111/pce.12606Plant, Cell and Environment (2015)

the rate of inflorescence production (Legros et al. 2009a),variations in the male/female inflorescence cycle (Henson& Harun 2004) or inflorescence abortions (Breure &Menendez 1990). It is well known that in addition tocropping practice and unfavourable environmental condi-tions such as drought, altered source/sink relationships maybe the cause of abortions (Combres et al. 2012; Cros et al.2013). For example, there is an increase in abortion ratewhen the carbon supply is limited by severe pruning (Legroset al. 2009a) or defoliation.

On the other hand, oil palm exhibits large leaf assimila-tion rates (up to 30 μmolm�2 s�1; Lamade & Setiyo 1996)and high leaf surface area (from 6 to 12m2, depending onvarieties), and large carbon storage mostly in the form ofstarch and glucose in the trunk (Legros et al. 2006; 2009a).Other organs like petioles or leaves may also play a roleas a transitory storage. Recently, Zahari et al. (2012) ob-served substantial carbon reserve in the form of glucosein petiole.

The relatively high yield of fruit production thus proba-bly arises from a high photosynthetic capacity and an effi-cient remobilization of stored carbohydrates to sustainfruit development. In other words, carbohydrate storagein the trunk may serve as a ‘buffer’ when total photosyn-thesis is limited by dry conditions or severe pruning ordefoliation (Henson et al. 1999; Legros et al. 2009a,b). Car-bon allocation and storage pattern are thus a central con-cern for both agronomists and oil palm breeders.Ecophysiological modelling and biomass measurementshave suggested that in mature trees, fruit bunches androots are responsible for a respiratory loss representing 18and 14% of gross primary production (GPP), respectively,and that carbon partitioning to fruits and roots stands for30 and 10% of gross carbon fixation, respectively (Dufrêne1989, Lamade et al. 1996, Melling et al. 2008). Recently,using phenological modelling, it has been shown that tro-phic competition amongst organs drives sex determinationand inflorescence abortion rate (Combres et al. 2012) andtherefore contributes to determining fruit production, withlow/high fruit production during strong/poor competitiveperiods (ratio of gross assimilation to total organ demandof 0.5/1.5, respectively). Nevertheless, to our knowledge,there are only few experimental data on carbon fluxes inoil palm, such as direct respiration measurements on all or-gans, or isotopic labelling with 13C.

It should be recognized that 13C-labelling of trees ishighly demanding and expensive, with necessary precau-tions to avoid 13C-losses and complicated instrumentationin the field (Epron et al. 2012). Rather than 13C-labelling,advantage can be taken from 13C natural abundance(δ13C). Recently, δ13C values in sugars have been predictedwith a very good accuracy with a model that took into ac-count known metabolic partitioning patterns and isotope ef-fects in crop plants such as sugar beet (Beta vulgaris) andwheat (Triticum aestivum) (Gilbert et al. 2012). C3 plantssuch as oil palm fractionate against 13CO2 during photosyn-thesis (mainly during CO2 diffusion through stomata intothe leaf and carboxylation by Rubisco, Farquhar et al.

1982). As a result, δ13C in organic matter of C3 plants ison average almost equal to �28‰, that is, about 20‰13C-depleted relative to atmospheric CO2 (δ13C=�8‰).Nevertheless, such an average value encompasses substan-tial disparities amongst plant tissues or metabolites (seeHayes 2001 for a review) caused by post-C fixation pro-cesses, the so-called ‘post-photosynthetic fractionations’.These lead to extensive changes in the isotopic signal ofnet fixed carbon and also cause differences in the isotopecomposition between plant organs and compounds(Tcherkez et al. 2011a). In general, sink heterotrophic plantorgans are 13C-enriched compared to leaves (for a review,see Badeck et al. 2005), and within leaves, the lamina is13C-depleted compared to main veins (Badeck et al.2009). Several explanations have been hypothesized, suchas a fractionation against 13CO2 in respiration (Cernusaket al. 2009; Ghashghaie & Badeck 2014) and isotope effectsin sugar metabolism (Tcherkez et al. 2011a). In particular,the fractionation against 13C during sucrose cleavage by in-vertase (≈1‰) and isomerization of glucose into fructose(≈1‰) has been shown to be of fundamental importancein determining δ13C values in sucrose and starch in storageorgans (Gilbert et al. 2012), because it leads to a progres-sive 13C-enrichment in sucrose imported by sink organs.Within plant organs, elaborated compounds such as ligninor lipids tend to be 13C-depleted compared with primaryassimilates (sugars) and cellulose (Gleixner et al. 1993,Schmidt & Gleixner 1998), and leaf-respired CO2 appearsto be 13C-enriched relative to sucrose in the dark (for a re-view, see Ghashghaie et al. 2003) and 13C-depleted in thelight (Tcherkez et al. 2011b). Such isotopic differences arethe consequence of isotope effects associated with enzymesinvolved in C–C bond modifications (formation or cleav-age). Typically, irreversible reactions usually fractionateagainst 13C, thereby depleting their products in 13C (e.g. py-ruvate dehydrogenase, PDH), while reversible reactions mayfavour 13C, particularly when equilibria lead to the formationof C–C bonds (e.g. aldolase, which evolves fructose-1,6-bisphosphate from triose phosphates). That lipids are depletedcan be related to both the intramolecular heterogeneity in 13C-abundance within glucose (Rossmann et al. 1991) and the oxi-dation of pyruvate to acetyl-CoA by the PDH. This enzymefractionates against 13C, leading to 13C-depleted acetyl-CoA,which is in turn consumed to synthesize fatty acids (Melzer &Schmidt 1987).

Here, we carried out measurements of photosynthesisand sugar content, took advantage of natural 13C abun-dance (δ13C values) in palm tree bulk material (total or-ganic matter, TOM) of different tissues as well as inpurified metabolites (soluble sugars, starch and lipids) andfollowed δ13C values during fruit development and matura-tion. We further exploited δ13C values as input data into amodel to generate plausible carbon allocation values to or-gans and respiratory losses. Our results show that sink or-gans are all 13C-enriched (up to 1.8‰) compared toleaves, with the exception of mature fruits due to theirhigh content in 13C-depleted lipids. Carbon fluxes com-puted from the 13C-distribution suggest that carbon

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© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment

allocation to fruits represents about 30% of photosyntheticC fixation and that fruit respiration is the largest CO2 lossof oil palm (≈39% of whole tree respiration).

MATERIAL AND METHODS

Plant material and growth conditions

The study took place at Aek Pancur Research Station (3°30’N,98°48’E, 25m above sea level, North Sumatra, Indonesia) be-tween April 2003 and April 2004 on 10 oil palm adult treesplanted in 1995. Trees belonged to a clonal material (Deli×LaMé type;MK60: LM007T×DA128D) planted in a genetic trialby Indonesian Oil Palm Research Institute. Monthly local me-teorological data (years 2003 and 2004) are plotted in Fig. S1.Average daily values were: temperature 27 °C and vapour pres-sure deficit 0.62kPa. Annual rainfall was 1999mm. Treescontinuously produced inflorescences (and thus fruits), with apeak at 2.9± 0.2 (monthly frequency of inflorescence appear-ance) in January 2004 (Fig. S2) likely induced by wet condi-tions in November and December 2003 (Fig. S1).

Gas exchange measurements

Gas exchangemeasurements were performed on eight trees onleaf rank 7–9 and on leaflets sampled around point B (upperthird section of the leaf, see figure 2 in Lamade et al. 2009) withan open system, using a portable infrared analyser [IRGA,LCA4 type, Analytical Development Company (ADC),Hoddesdon, Herts, UK] and an adapted assimilation chamber(PLCN4 type, ADC). Light response curves were carried outin the morning from 7:00 to 10:00 h (time at which photo-synthesis is already induced), with VPD< 1kPa and airtemperature< 36 °C. In order to avoid variability because oflocation (surface area chosen) within each leaflet, measure-ments were always performed on the upper third of the lamina.A time course (diel variations) of gas exchange was occasion-ally performed, with leaf gas exchange measured (at least onfour different trees and several measurement per hour) on leafrank 17 from 7:00 to 18:00 h. Leaf respiration at night was mea-sured using a semi-closed system connected to a LCA2 infraredgas analyser (ADC) following themethod already described byLamade et al. (2009).

Biomass measurement

Standing biomass was evaluated on an individual basis andwas performed concurrently with sampling. For all treesstudied, leaf biomass (leaf rank 17) was estimated usingleaf surface area (Tailliez & Ballo, 1992) and specific leafarea completed by the length of rachis and petiole andtheir respective density (in kgDWm�3). Trunk volumewas determined by measuring the diameter at three differ-ent levels (base: at 50 cm from top soil; middle: at halfheight and top: under petiole base of leaf rank 33) andthe height (from base to top). Trunk density has been de-termined by sampling bulk material along the trunk. Rootbiomass was estimated as in Lamade and Setiyo (2002).

Fruit growth was followed (measured and weighed) bysampling fruits on a monthly basis, from the stage just be-fore pollination to full fruit maturation stage (6monthslater).

Sampling

Leaf sampling was performed as in the work of Lamade et al.(2009). Briefly, each leaf is made of a petiole (around 1m)and a rachis (around 4 to 6m) with more than 100 leaflets oneach side. Leaf sampling was carried out on leaf ranks 1 (theyoungest autotrophic leaf on the canopy), 9, 17, 25, 33 and soon (with some additional leaves including, for each rank, thepetiole, rachis and leaflets) following the spiral pattern of leafemission. Leaf numbering used positive numbers for autotro-phic leaves and negative numbers for heterotrophic, emergingleaves. Trunk organic matter was collected at five different lo-cations: (i) basal section (denoted as TBa in Fig. 3); (ii) middlesection (TMi); (iii) apical section (TAp); (iv) meristem (TMe)and (v) terminal bud (TBu, which is formed by all of thegrowing leaves before rank �6, that is, heterotrophic whiteleaves). Sampling was performed using a hand-made auger(8mm diameter× 45 cm length). Particular care was taken tosample the whole trunk radially. Roots were collected at15 cm depth with a dutch auger at five different locationsaround the tree base as in the work of Lamade et al. (1996).Fruit sampling was performed at seven different developmen-tal stages (30d between them) numbered from 0 (before pol-lination) to 6 (maturity) so as to follow fruit formation andmaturation. At least one bunch per tree was selected (just af-ter pollination) and sampled every month, and fruit samplingwas performed at three different levels (at the base of thebunch, in the middle and at the top). All samples were col-lected before 11:00 for both years, disinfected and quenchedwith ozone, frozen with liquid nitrogen, stored at �80 °Cand oven-dried for at least 2 d at 70 °C. They were then finelyground (Type MM200, Retsch, Haan, Germany). Fruitsamples were made of the whole fruit (mesocarp+ seed andkernel) ground into powder.

Sugars and lipid extraction and quantification

Sugars were extracted as in the work of Tcherkez et al.(2003) from fine powder samples. Briefly, after extractionwith MilliQ water and centrifugation, soluble sugars wereanalysed (quantified and separated) by liquid chromatogra-phy (high-performance liquid chromatography), which wasassociated with no isotope fractionation (Duranceau et al.1999). Sugars were manually collected and freeze-dried.Starch was extracted using solubilization with HCl and floc-culation with cold methanol after Duranceau et al. (1999).Lipids were extracted using the method of Deléens et al.(1984) modified by Tcherkez et al. (2003), with hot ethanoland chloroform. The lipidic chloroform phase was evapo-rated under N2 at 60 °C. Glass tubes were weighed to de-termine the lipid content. For isotopic analysis, lipidswere redissolved in chloroform and transferred into thicktin capsules, and chloroform was evaporated.

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© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment

Elemental and isotopic analyses

Organic matter and individual, purified compounds were allplaced in tin capsules for isotope analyses. Isotope analyseswere carried out with an elemental analyser (FlashEA1112, Thermo-Scientific, Waltham, MA, USA) coupled withan isotope-ratio mass spectrometer (Elementar, Villeurbanne,France). The elemental carbon content was given in percent(mass fraction). Isotope composition was expressed in deltanotation (in percent), that is, as deviation of the carbon isotoperatio (13C/12C, denoted as R) from the international standardVienna Pee Dee Belemnite: δ13C= (Rsample�RV-PDB)/RV-

PDB. A lab standard (glutamic acid) as well as the InternationalAtomic Energy Agency standard USGS-40 (glutamic acid,δ13C=�26.39%) was measured every five samples to assessmeasurement accuracy and correct for any drift of the massspectrometer.

Calculations

Modelling C fluxes from natural 13C abundance requires theknowledge of the isotopic input, that is, the δ13C value of car-bon fixed by leaf photosynthesis, as well as the δ13C value ofCO2 lost by respiration. Due to technical difficulties to carryout online isotope measurements in the field (oil palm planta-tion in Sumatra), the δ13C of both the photosynthetic influxand respiratory efflux was calculated using gas exchange pa-rameters and isotopic mass balance, respectively. The calcula-tion of the Δ value (photosynthetic carbon isotopefractionation) using the equation of Farquhar et al. (1982) re-quired the knowledge of internal conductance (gm) so as to cal-culate cc/ca, the chloroplastic-to-external ratio of CO2 molefraction.

Internal conductance

Internal conductance was estimated using the relationshipbetween net assimilation (A) and stomatal conductance(gc). Net CO2 assimilation is given by von Caemmererand Farquhar (1981):

A ¼ gc� ca � cið Þ ¼ gm� ci � ccð Þ;where gc is stomatal conductance and ci is intercellular CO2

mole fraction. In this equation, ca accounts for boundary layerresistance (and thus could also be denoted as cbl). The equationcan be rearranged to

A ¼ 11gcþ 1

gm

ca � ccð Þ

that gives

1gc

¼ �1gm

þ ca � ccA

: (1)

In Eqn 1, it can be seen that plotting the reciprocal of gc as afunction of the reciprocal ofA gives �1/gm as the intercept. Ofcourse, in this relationship, the slope is not constant because(ca–cc) varies with photosynthesis. Therefore, the convergence

to the intercept is only visible for large values of stomatal con-ductance and assimilation. In practice, regression analysis toobtain the intercept was carried out with data for which1/A< 0.1 (A> 10μmolm�2 s�1). Such a relationship is shownin Fig. S3. It should be noted that we use the intercept of the re-lationship shown in Eqn 1, and thus, this method does not re-quire the knowledge of the slope (i.e. the knowledge of cc) toestimate gm.

Isotopic input

The isotopic photosynthetic input was calculated using theassimilation-weighted average (denoted as Δcalc) of the photo-synthetic fractionation Δ over the time course of the light pe-riod of the day:

Δcalc ¼∑iAi�Δcalc i

∑iAi

; (2)

where Ai is the net assimilation value and Δcalc i is the isotopefractionation at time i. Δcalc was calculated as in the work ofFarquhar et al. (1982):

Δcalc ¼ aca � cica

þ es þ alð Þ ci � ccca

þ bccca

� d; (3)

where a (4.4‰), es (0.7‰), al (1.1‰) and b (29‰) are the iso-tope fractionation associated with diffusion in air, dissolutionand diffusion in the liquid phase and carboxylation, respec-tively. d is the isotopic correction caused by (photo)respira-tion. The day respiratory component is given by eRd/kca,where e is the respiratory fractionation, Rd is the day respira-tion rate, and k is ‘carboxylation efficiency’. With standardvalues of e=10‰ (Tcherkez et al. 2011b), k=0.1molm�2 s�1

and Rd=0.5μmolm�2 s�1 at ca=380μmolmol�1, it gives0.1%. Even with substantial variations in k or Rd, the day re-spiratory component is unlikely to be larger than 0.2% (vonCaemmerer & Evans 1996). The photorespiratory componentis given by fΓ*/ca, where f is the photorespiratory fractionation(11%, Tcherkez 2006; Lanigan et al. 2008) and Γ* the compen-sation point ‘in the absence of Rd’ (≈40μmolmol�1, vonCaemmerer & Evans 1996), giving about 1.1‰. Thus, here,the value of d used was fixed at 1.2‰. The use of Eqn 3 re-quires the knowledge of cc, which was calculated using leaf in-ternal conductance determined as explained previously (here,0.68molm�2 s�1).

A typical daily time course of cc/ca, A and Δcalc is shown inFig. S4. Under our conditions, the average Δcalc was estimatedto be 20.8‰ (Table 1 and Fig. S4). The δ13C value of atmo-spheric CO2 used here was that recorded at the Indonesianstation Bukit Koto Tabang, that is, �7.9% in 2003–2004(www.esrl.noaa.gov). As a result, the average isotopic inputwas estimated to be �28.7‰.

Isotopic mass balance

Because we did not had access to experimental δ13C valueof evolved CO2, we took advantage of isotopic mass bal-ance, assuming that the whole oil palm tree is in the

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isotopic steady state as has been shown in other plants(Klumpp et al. 2005; Bathellier et al. 2008). That is, the car-bon isotope composition of the photosynthetic influx wasassumed to match the isotope composition of formed bio-mass plus CO2 efflux:

0 ¼ δp � ∑ibiδbi þ∑

iriδri

� �: (4)

Where δp is the isotope composition of the photosyntheticinput (δ13C of GPP), δi

b and δjr are the isotope compositions

in biomass and CO2 evolved by compartment (plant organ) i,respectively. bi and ri stand for biomass synthesis and respira-tory loss expressed relative to GPP (and thus ∑bi+ ri=1). Inpractice, the respiratory component was simply decomposedinto two terms, one for leaf respiration and the other one forall of the heterotrophic organs. The ri value for leaves (de-noted as ri leaves) obtained here with gas exchange (leaf CO2

respiratory loss during the night periods) was 12% (Table 1).The ri value for heterotrophic organs was thus computed as1–CUE–ri leaves=1–0.38–0.12= 0.50 (for references on CUE,see Introduction). bi values were obtained from biomass (de-scribed previously) corrected for elemental carbon content(%C). Eqn 4 was solved for δi

r of heterotrophic organs withan imposed value of δi

r for leaves. Two possibilities were ex-plored here: Leaf respired CO2 was 13C-enriched (fraction-ation of �1‰) or 13C-depleted (+1‰). The minimal valueused here (�1‰) might appear rather low. However, it shouldbe noted that in oil palm, leaves are not only made of autotro-phic tissues but also include a substantial amount of heterotro-phic tissues (thick petiole and rachis), and thus CO2 evolvedby palms is unlikely to be considerably 13C-enriched as in com-mon herbaceous species. In addition, the fractionation usedhere for foliar respiration in darkness integrates full night pe-riods (and not only the first hours following dawn), at theend of which evolved CO2 becomes much less 13C-enriched(Tcherkez et al. 2003). Nevertheless, the assumption on δi

r

for leaves had a modest impact only on eventually computedfluxes (Fig. S5).

Isotopic models

The 13C-distribution in compounds was modelled in thesteady state using the approach of Tcherkez et al. (2004)and Gessler et al. (2009), that is, using isotopic mass-balance with known isotope effects. Here, by ‘isotope ef-fect’, we mean the 12C-to-13C ratio of reaction rate(12v/13v). Quite generally, the model assumes that the isoto-pic influx equals the isotopic efflux. For example, if a certaincompound i (1) is consumed by n reactions associated withisotope effects (denoted as αk, k ∈ [1,…,n]) and (2) comesfrom m reactions associated with isotope effects (denotedas βj, j ∈ [1,…,m]), we have

Ri ∑n

k¼1

Fk

αk¼ ∑

m

j¼1

GjRj

βj;

where Fk and Gj are the fluxes associated with reactions con-suming and producing compound i, respectively.Rjs are the iso-tope ratios in substrates of reactions producing i. The steadystate applied to concentrations is so that

∑n

k¼1Fk ¼ ∑

m

j¼1Gj:

Such equations are written for sugars (glucose, fructoseand sucrose) and starch, as well as TOM (immature leavesand roots), and lipids and non-lipidic biomass in fruits. Thereactions considered were as follows: respiration, starchsynthesis, starch degradation, sucrose phosphate synthase(SPS), invertase (sucrose hydrolysis), glucose/fructose inter-conversion and in heterotrophic organs, biomass produc-tion. Biomass production was assumed to be zero inmature leaves. In fruits, two further fluxes were considered:lipid synthesis and non-lipidic biomass production. Knownisotope effects were that of (i) invertase, which fractionatesagainst 13C during sucrose cleavage in the fructose moiety,by 1‰ (Mauve et al. 2009; Gilbert et al. 2012); (ii) glucoseisomerization, which fractionates against 13C by 1‰ duringconversion of glucose into fructose (Gilbert et al. 2011);(iii) respiration (see previous text) and (iv) lipid synthesis,which fractionates on average by 5‰ in terrestrial plants(Park & Epstein 1961). There is no isotope fractionationduring starch degradation and resulting sugar export(Badeck et al. 2005, Maunoury-Danger et al. 2009). We as-sumed no isotope effect in SPS and starch synthesis, as inthe work of Gilbert et al. (2012). For example, the equa-tions for model A in leaf fructose (Fru) are as follows:

RFru� rαr

þ P� r � σαi

þ σαFs

� �¼ P�Rp; (5)

where Rp stands for the isotope ratio in net fixed carbon and P,r and σ are the net photosynthetic input (fixed at 100 so as toexpress other fluxes in %), leaf respiratory efflux and sucrose

Table 1. Photosynthetic and respiratory properties of oil palm leavesforDeli x La Mé clonal material, Aek Pancur Estate (North Sumatra).Mean± SE (n= 4 trees)

Parameter Value Units

Average daily CO2 assimilation 11.3 ± 0.9 μmolCM�2 s�1

Daily CO2 fixation 469± 30 mmolCM�2 d�1

Average night respiration 1.42 ± 0.09 μmolCM�2 d�1

Daily respiratory CO2 loss(night period)

59.5 ± 2.1 mmolCM�2 d�1

CO2 loss (night period), inpercent of CO2 fixation

12.7 ± 0.5 ‰

Leaf apparent CUE 87.2 ± 0.5 ‰Assimilation-weighted dailyaverage of Δcalc

20.8 ± 0.4 ‰

CUE, carbon use efficiency.

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production (SPS flux), respectively. αr, αi and αsF are the iso-

tope effects associated with respiration, fructose-to-glucoseisomerisation and SPS (fructose moiety), respectively (thus,here, αs

F=1). Note that we assumed fructose to be the sub-strate of respiration because in glycolysis, fructose-6-phosphateis indeed downstream to glucose-6-phosphate. Similarly in glu-cose (Glc), we have the following:

RGlc� S1αa

þ σαGs

� �¼ RFru�P� r � σ

αiþ Rstarch�S�1

α�a; (6)

where S1 and S� 1 stand for fluxes of starch synthesis and deg-radation, respectively, and associated isotope effects are αa andα� a, respectively (here, αa= α� a=1). αs

G is the isotope effectassociated with SPS (glucose moiety, here, αs

G=1). In fruits,we have the following:

RGlc� S1αa

þ σαGs

þ Bαb

� �¼ RFru� j � r � σ � L

αi

� �

þRstarch� S�1

α�aþ RSuc�G

jαGj

;

(7)

where B, L and j are fluxes of non-lipidic biomass production,lipid synthesis and invertase, respectively, and αb, αl and αj

G

are the associated isotope effects. RSuc�G is the isotope ratioin the glucosyl moiety of sucrose. Steady-state requirementsare so that in leaves (Eq. 7), S1 =P� r� 2σ +S� 1. In fruits(Eqn 7), we have S1 = 2j� 2σ� r�B+S� 1. Similar mass-balance equations are used for sucrose in leaves andsugars in other organs. It should be noted that here, weassumed there was no isotope effect in starch synthesisfrom leaf glucose, despite the fact that starch has provedto be generally 13C-enriched compared to net-fixed CO2

(Tcherkez et al. 2004). Nevertheless, such a 13C-enrich-ment depends upon metabolic partitioning between su-crose and starch during photosynthesis, which is notknown in oil palm. In addition, the synthesis of 13C-enriched starch in the light is compensated for by the pro-duction of 13C-depleted sucrose and thus in the steadystate, considering isotope effects in starch and sucrosesynthesis would yield the same results of C-allocation inleaves.

To avoid under-determination of the mathematical sys-tem, certain variables had to be fixed: Total respiratory flux(all organs) was fixed at 62% of the photosynthetic carboninput (CUE of 0.38); the isotope composition of CO2

evolved by all of heterotrophic organs was fixed (explainedpreviously) and the flux of SPS in roots was assumed to bezero. Three models were explored here: model A, in whichsucrose produced by leaves directly fed all organs; modelB, in which sucrose produced by leaves was first exportedto the trunk, which redistributed it to other organs andmodel C, which is similar to A except that glucose is the

circulating carbohydrate instead of sucrose. Because mostequations were non-linear, flux values were solved numeri-cally using the Excel solver (Microsoft). The accuracy ofthe model was estimated calculating the average Euclideandistance between observed and predicted δ13C values, inpermil. Note that in model C, there are more variablesto be determined by numerical resolution. We thereforeimposed as further constraints that there was no net su-crose synthesis in roots and new leaves. In addition, wecarried out several resolutions with fixed glucose importflux in roots and eventually chose the set of values thatgave the minimal Euclidean distance (stepwise numericalresolution).

RESULTS

Natural 13C-distribution

The δ13C values in palm organs are shown in Fig. 1. Theaverage δ13C value in leaf material (TOM) was about�28.6‰ (leaflets), such that the apparent photosyntheticisotope fractionation Δ was about �7.9–(�28.6) = 20.7‰.Leaf soluble sugars were significantly (P< 0.05) 13C-enriched compared to TOM, and their δ13C value wasnot significantly different from that of leaf starch, therebysuggesting that sugar metabolism (sucrose and starch pro-duction and degradation) was intense so that no isotopicfractionation occurred. That is, there was apparently littlemetabolic partitioning between starch and sucrose, meaningthat glucose was easily interconverted between starch andsucrose. TOM was 13C-enriched in heterotrophic organscompared to leaves, by 1.6‰ in trunk and up to 1.7‰ inroots. By contrast, there was no visible 13C-enrichment insugars (both soluble sugars and starch) compared to leaves,suggesting that sugars in sink organs were inherited from

Figure 1. Carbon isotope composition in palm tree organs, in totalorganic matter (TOM), starch and soluble sugars (weighted average ofSuc, Glc and Fru). Data shown for fruits are time average over fruitdevelopment from initiation to maturation. Data shown are mean± SE(n= 10). TBu, trunk apical bud; TMe, trunk apical meristem; TAp,trunk apical section; TMi, trunk middle section; TBa, trunk basalsection; nd, not determined.

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that produced in leaves with no fractionation along phloemtransport. Therefore, the apparent isotope fractionation be-tween sugars (potential carbon source) and TOM (biomassproduced) was larger in leaves (up to 3.3‰) than in sinkorgans (0.5 to 2.3‰). That said, there was substantial isoto-pic heterogeneity amongst biomass fractions, typically infruits (see below).Oil palm leaves are very large (3–5kgDWleaf�1) due to

multiple leaflets, very thick petioles and a very long rachis.The δ13C values of leaf parts are plotted in Fig. 2A. Thecomparison between leaflets and petiole or rachis of thesame leaf shows a slight isotopic enrichment in petioleand rachis of about 0.4‰. In leaflets, starch was onaverage 13C-enriched by 2‰ (Fig. 2B) and thus 13C-enriched compared to TOM of whole leaf (including peti-oles and rachis).

Leaf gas exchange

Daily photosynthesis reached a maximal value of30μmolm�2 s�1 (Fig. S4C) and was on average of11.3μmolm�2 s�1 (Table 1). Respiration in darkness wasabout 1.4μmolm�2 s�1 so that integrated C loss repre-sented 12.7% of total photosynthetic CO2 fixation in thelight. The relationship between reciprocals of net CO2 as-similation and stomatal conductance indicated that internalconductance was relatively high, at 0.68molm�2 s�1 (Fig.S3) under natural light levels, and much larger at high light(2.35molm�2 s�1). With such an internal conductance, theCO2 drawdown between the atmosphere and carboxylationsite (cc/ca) was near 0.7 (Fig. S4B), and therefore, the aver-age photosynthetic fractionation (assimilation-weighted)against 13C was 20.8‰ (Table 1, Fig. S4A).

Sugar content

The tissue content in starch, glucose, fructose and sucrosewas determined by purification (starch) and high-performance liquid chromatography (soluble sugars).

Original values in mgg�1DW were normalized (mean-centred) so as to draw a heat-map and thus compare sugarcontents between organs and sugar profiles between sugars(Fig. 3). It appears that the trunk (TMe and TAp) had thehighest relative content in sucrose and petioles, rachis andtrunk base (TBa) were enriched in glucose. Fructose wasmost concentrated in leaflets, petiole and rachis in matureleaves. By contrast, fruits were relatively depleted in fruc-tose, likely reflecting intense fructose utilization by catabo-lism (respiration) and anabolism (syntheses). Starch wasmost abundant at the top of the trunk (TAp) and tran-siently increased in fruits after 90d. Theglucose � fructose/sucrose ratio, which is a relative action ra-tio of sucrose cleavage versus sucrose production, appearedto be the highest in mature leaves thereby suggesting thatsucrose synthesis is relatively low compared with hexoseproduction by photosynthesis. The sucrose-to-starch ratiowas the highest in petiole and rachis suggesting that su-crose was poorly committed to starch synthesis in these or-gans, that is, petiole and rachis were likely to accumulatestarch at a low rate.

Fruit development

While there was no significant change in the isotope com-position in vegetative parts along the year considered(2003–2004; Lamade et al. 2009), the δ13C value in fruitschanged a lot during fruit development and maturation(Fig. 4). In fact, fruit TOM started at nearly �27‰ anddeclined down to �29‰ at maturity (Fig. 4A). Meanwhile,there was only modest changes in the δ13C value in sugars(starch and soluble sugars), with a progressive 13C-deple-tion. By contrast, there was a clear 13C-enrichment in lipidsof about 1.5‰ from fruit initiation to maturity (from �31to �29.5‰). The largest change in the metabolic composi-tion of the fruit was the considerable increase (15-fold in-crease) in lipid content (Fig. 4B), which eventuallyrepresented 600mgg�1DW at maturity (thus, 80‰ of fruit

(a) (b)

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Figure 2. Carbon isotope composition in palm leaves: δ13C in petioles (black) or rachis (white) as a function of δ13C in total organic matter of leafletblade (of the same leaf) (A) and relationship between total organic matter and starch in leaflets (B). Data shown aremeans (n= 5). SE (<0.4‰) is notshown for clarity.

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carbon content). As a result, there was a relationship be-tween the δ13C value in fruit TOM and the carbon elemen-tal content (Fig. 4C): typically, at the two last stages (5 and6) of fruit development (after 150 and 180d, respectively),fruits were 13C-depleted and carbon-enriched due to theprevalence of lipids (which contain about 75% carbon).The progressive 13C-enrichment and C-content increase inlipids reflected enhanced oleosynthesis and thus the de-crease in the fractionation against 13C during lipid produc-tion, due to a higher metabolic commitment. In fact, theapparent fractionation between soluble sugars (potentialcarbon source) and lipids declined from 5 to 3.5‰ fromstage 0 (fruit initiation) to stage 6 (maturity) (Fig. 4A). Itshould be noted that such a progressive 13C-depletion infruit organic matter due to lipid accumulation was probablycompensated for by an accumulation of 13C-enriched com-pounds (e.g. cell wall material, fibres and 13C-enrichmentof lipids) to satisfy isotope mass balance. Using simple con-servation equations (mass balance), the residual organicmatter (not made of lipids nor sugars) could be calculatedto be between ≈400 (stage 0) and ≈80mgCg�1DW (stage6), with a δ13C value between �27.3 and �24.4‰,respectively, that is, eventually 3‰ 13C-enriched comparedwith TOM.

Carbon allocation pattern

The δ13C values were used as input data into a model that tookinto account isotope fractionations, so as to calculate probableallocation fluxes at two fruit developmental stages (0 and 6).Three models were used here: carbon redistribution directlyfrom leaves in the form of sucrose (model A) or via palm trunk,which redistributed sucrose to other organs (model B), and car-bon redistribution directly from leaves in the form of glucoserather than sucrose (model C). Model C was explored here be-cause due to the lack of labelling studies in oil palm, the natureof the molecular species transported in the tree is not knownwith certainty. The involvement of sucrose seems likely be-cause sink organs such as fruits express genes encoding inver-tase (Dussert et al. 2013). However, there are substantialamounts of free glucose in oil palm and thus, the possibility ofglucose being the translocated molecule cannot be presentlyexcluded. In eachmodel, two scenarios were tested: respiratoryfractionation in leaves (denoted as e) of +1 and �1‰. The de-tailed output of the models is shown in Figs S5 and S6, and theresulting allocation schemes are represented by Fig. 5 (modelsA and B) and Fig. S6 (model C).With the calculated fluxes, theaverage Euclidean distance between observed and predictedδ13C values was 0.42‰ (model A) and 0.56‰ (model B). The

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-22TOMLipidsSol. sugarsStarch

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Figure 4. Metabolic and isotope composition in palm fruits: δ13C of different fractions (A) andmetabolic content (B) during fruit development (fromstage 0, initiation, to stage 6, mature fruit); relationship between δ13C in total organic matter and C elemental content (C). In (C), fruit developmentstage is indicated with numbers. Data shown are mean ± SE [n= 4 (A and B) to 25 (C)]. There is no δ13C value for soluble sugars at stage 6 due to thevery low content that did not allow isotope measurement. TOM, total organic matter.

Figure 3. Heat map of sugar content (Suc, Glc and Fru) or sugar ratios (Glc � Fru/Suc and Suc/starch) in palm tree organs. Original data inmg g�1DW were mean-centred for each sugar or sugar ratio of interest. The average content in sucrose, glucose and fructose is 4.6, 19.6 and10.3mg g�1DW, respectively. Relative intensity is indicated with green and red colours (smaller and higher than average, respectively). Numbersindicate leaf rank (leaflets L, petiole P and rachis R) or developmental stage (fruits F). L, leaflets; P, petioles; R, rachis; TBu, trunk apical bud; TMe,trunk apical meristem; TAp, trunk apical section; TMi, trunk middle section; TBa, trunk basal section; Ro, roots; F, fruits.

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use of positive or negative respiratory fractionation e did not af-fect much the values (Fig. S5) – because under our conditions,the change in e was compensated for by isotopic steady state(thus adjusting respiratory fractionation in sink organs

accordingly). Regardless of the model used, net starch synthe-sis in leaves was very small or even negative (model A at stage0 and model B), reflecting the fact that transitory starch wasremobilised and did not accumulate in leaves. In model A,

Figure 5. Allocation pattern in oil palm computed from δ13C values (seeMaterial and methods or Supporting Information for further details), usingtwomodels based on sucrose redistribution: carbon redistribution directly from leaves (A) or via palm trunk that redistributes to other organs (B). In(A) and (B), numbers are in percent, that is, expressed relative to photosynthetic C fixation fixed at 100. Values in black and grey were obtained atstages 0 and 6 of fruitmaturation, respectively. δ13C values shown for respiredCO2were obtained bymass balance with e= +1‰ (dark) or�1‰ (grey).Asterisks indicate that the values were fixed as a model constraint: leaf respiration (12% of fixedCO2), total respiration (fixed CUE of 0.38) and trunkgrowth [in (B)]. (C) Distribution of palm respiration efflux, in percent of total CO2 loss. Mature leaf respiration remains constant (fixed modelconstraint). The different values shown correspond to stage 0 (A0 and B0) and 6 (A6, B6) of fruit development, with model A (direct export fromleaves, A0 and A6) or B (redistribution through palm trunk, B0 and B6). The dashed vertical line represents the average of fruit CO2 loss (39.9%).Resp, respiration; St.s., starch synthesis.

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carbon allocation was well balanced between sink organs, ex-cept for juvenile heterotrophic leaves that captured a minimalpart of gross C fixation (11–15%, Fig. 5A). In model B, carbonwas also minimally allocated to juvenile heterotrophic leaves(≈13%). In both models A and B, the largest respiratory fluxwas that of fruits. Respiratory losses were re-calculated as per-centage of total palm tree respiration (Fig. 5C). Fruit respira-tion represented between 35 and 45% of total CO2 loss, withan average value of 39.9%. The CUEof sink organs, computedas 1 minus the ratio of respiration to carbon import, tended tobe ≈0.6 in vegetative organs and ≈0.1 in fruits (Fig. S5)., Inother words, the respiratory loss by respiration in fruits wasthe largest when compared to carbon consumption. Fruit de-velopmental stage did not change much the allocation pattern(Fig. 5A, B and C). Regardless of the model A or B used andthe developmental stage, lipid synthesis represented a smallfraction of gross carbon fixation (<1%). This result is not sur-prising: Should lipid synthesis be high, soluble sugars wouldbe considerably 13C-enriched in the steady state (due to thefractionation associated with lipid synthesis), contrary to whatis observed. Outputs of model C are rather different from thatobtained with models A and B (Fig. S6)., In particular, unreal-istic values were obtained for root respiratory efflux (≈0) and ahigh starch remobilization in leaves (≈50% of net-fixed C).Fruit respiration was also very low, which contradicts the ener-getic requirements of lipid synthesis. These results are not sur-prising, because the conversion of glucose to fructosefractionates against 12C, thereby depleting in 13C the glucosepool; the numerical resolution thus yielded a high (naturally13C-enriched) starch remobilization inmost organs, to compen-sate for such a depletion. Under our conditions, glucose thusappeared to be unlikely to represent the major circulatingsugar in oil palm because calculated fluxeswere not satisfactory(but see also Discussion).

DISCUSSION

Internal conductance

Due to the lack of isotope fractionation data obtained onlineor fluorescence data, we estimated internal (mesophyll) con-ductance for CO2 with a regression between reciprocals ofstomatal conductance and net assimilation (Fig. S3). We esti-mated a value of 0.68molm�2 s�1, which cannot be com-pared because to our knowledge, there is no gmdetermination in oil palm in the literature. In the hybrid E.guineensis×oleifera, which has much lower photosynthesisrates than the present palm tree species, a value of0.3molm�2 s�1 has been obtained (Rivera-Méndez et al.2012). Values up to 0.45molm�2 s�1 are reported in mostmonocots (Flexas et al. 2012), and a regression analysis usingreciprocals from assimilation/light and assimilation/VapourPressure Deficit (VPD) curves obtained in oil palm (cultivatedin Ivory Coast) and reported byDufrêne (1989) suggests valuesof 0.28 and 1.1molm�2 s�1, respectively. Fluorescence mea-surements on similar trees cultivated in Indonesia also suggestan average internal conductance of about 0.60molm�2 s�1

(Lamade, unpublished). The use of the gm value estimated heregave a calculated average of the 12C/13C fractionation of 20.8%(Table 1), not far from that back-calculated (ignoring respira-tory C losses) using the δ13C value in atmospheric CO2 and thatin leaves (Fig. 1) [(�7.9 to �28.6)/(1–28.6/1000)= 21.3‰] or inTOM of the whole palm tree [(�7.9 to �27.5)/(1–28.6/1000)= 20.1‰]. However, it should be noted that if internalconductance was much lower, the allocation values computedwould not be that different and in particular, the prevalenceof fruits in total tree respiration would remain valid (Fig. S7).

Natural 13C-distribution

There was a clear isotopic difference between autotrophicleaves and heterotrophic organs, with a 13C-enrichment ofup to 1.6‰ (Fig. 1). δ13C in TOM showed a 13C-enrich-ment gradient from autotrophic (leaflets being the most13C-depleted) to heterotrophic tissues (roots being the most13C-enriched). Trunk base was slightly (insignificantly) 13C-enriched (�26.9‰) compared to trunk top (�27.08‰) andmiddle (�27.6‰). The tendency of heterotrophic organs to be13C-enriched is in agreement with data in the literature ob-tained in many other species (Badeck et al. 2005; Cernusaket al. 2009; Werner & Gessler 2011; Ghashghaie & Badeck2014). Furthermore, in oil palm, examination of the transitionfrom heterotrophic to autotrophic metabolism in leaves hasshown that heterotrophy is associated with a 13C-enrichmentin organic matter (Lamade et al. 2009).

It should also be noted that sugars are 13C-enriched, with arather similar δ13C value in starch and soluble sugars (Fig. 1),especially in leaves. This finding is unexpected, because leaveswere sampled in the light (Material and Methods) and there-fore, the isotope composition in soluble sugars was expectedto reflect that in ‘day sucrose’, which has been shown to be13C-depleted as compared with transitory starch or ‘night su-crose’ (Tcherkez et al. 2004; Gessler et al. 2008). This suggeststhat a substantial fraction of the soluble sugar pool (maybestored in vacuoles) originated from starch degradation. Infact, leaves appeared to be relatively starch-depleted com-pared to other organs (Fig. 3), with a starch content of about10–30mgg�1DW, that is, 1 to 3% only, thereby suggestingthat oil palm leaf metabolism did not involve a progressivestarch build-up but rather, quantitative remobilization of tran-sitory starch. Accordingly, our calculations suggest a verysmall or negative net starch synthesis in leaves (Fig. 5). How-ever, leaf sucrose content was also low, and the sucrose-to-starch ratio was relatively low (Fig. 3), maybe reflecting fasttranslocation to sink organs. A similar observation has beenreported by Mialet-Serra et al. (2005) in coconut leaves(Cocos nucifera L.). Our data and that of Legros et al.(2006) in oil palm differ from those obtained in coconut palm(Mialet-Serra et al. 2005, Mialet-Serra et al. 2008), which haslarge amounts of sucrose in petioles (125mgg�1DW) andalso in trunk heart (75–300mgg�1DW).

Despite these differences between these two species, theinvolvement of glucose rather than sucrose as the central

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circulating sugar in oil palm is unlikely, considering the un-realistic nature of numerical results obtained with model C(Fig. S6). This agrees with the fact that invertase has beenfound to be amongst the highest expressed enzymes duringfruit maturation (Dussert et al. 2013), suggesting indeedthat sucrose was the molecular species imported by fruitsand then cleaved to glucose and fructose, subsequentlyabsorbed via a transporter (such as GPT2, identified byBourgis et al. 2011). Metabolomics analyses carried out onmesocarp tissue further showed that glucose and glucose-6-phosphate were quantitatively important at all stages offruit development (Neoh et al. 2013). Because glucose is,quite often, more abundant than sucrose (especially in pet-ioles and rachis, Fig. 3), it is plausible that glucose plays animportant role, either as a transitory storage or as an inter-mediate in sucrose or starch futile cycles (synthesis-degra-dation). Legros et al. (2006) identified glucose as theprevalent reserve carbohydrate in oil palm (53% of totalcarbohydrates), while starch was identified as the mainform of reserve coming from photo-assimilate accumulation(Legros et al. 2009a). In addition, there is an oppositetrend in starch and glucose content along seasons at thetop of the trunk (Legros et al. 2009b). A dynamic equilib-rium between starch synthesis and degradation into glucosethus appears presently more likely than the involvement ofa sucrose futile cycle.

Allocation pattern

Our allocation pattern calculated from δ13C values withmodels A and B suggests that fruits and roots representthe largest carbon sink (Fig. 5), while heterotrophic leavesrepresent a visible fraction of fixed carbon, of 11–15%.Using biomass distribution, Dufrêne (1989) suggested anapparent allocation pattern to roots and leaves of about37% of net primary production, while trunk and fruits rep-resented 7 and 17%, respectively. Under our conditions, or-gan biomass distribution was rather (in percent of wholetree C-assimilated) as follows: leaves 25%, roots 10%,fruits 23% and trunk 42%. It should be noted that the al-location pattern calculated here did not originate frompresent biomass distribution but rather took into accountactual carbon sinks (growth demand and respiration cost)using isotopic signatures. In other words, mature leavesare not considered as sink (i.e. growing) organs, and theallocation pattern depicted in Fig. 5 reflects carbon usageof photosynthetically fixed carbon and not a simplebiomass-integration.Our two allocation scenarios based on sucrose redistribu-

tion (direct carbon distribution from leaves or via thetrunk, A and B) yielded comparable results, and therefore,we are presently not able to validate or invalidate the as-sumption that palm trunk acts as a transit compartmentfor all sink organs. Such a role played by the trunk couldbe interpreted as an alternative pathway of fruit fillingand new leaves development, aside direct sugar transloca-tion from mature leaves. As stated previously, the involve-ment of the trunk sugar reserve may be involved in

compensating for a deficit in photosynthetic activity underunfavourable environmental conditions (Introduction). Thatis, the trunk may play the role of a ‘buffer’ (where carbonassimilated not used for growth is transiently accumulatedas starch or glucose). In fact, palm trunk has been previ-ously suggested to be the main transit and reserve com-partment for carbohydrates, fuelling developing sinkorgans like fruits and new leaves (Scheideker et al. 1958;Gray 1969; Mansor & Ahmad 1991; Henson et al. 1999).This mechanism might be the underlying cause of the ob-servation that, with the same environment and genetic ma-terial, oil palm trees with low yield have a bigger trunkwith little sugar remobilization (Corley & Tinker, 2003).

In fruits, the carbon allocation to (non-lipidic) biomass,starch and lipid synthesis represented up to 6% of gross assim-ilated C (Fig. 5B), with a flux to lipid synthesis of 0.1%. Thesevalues were probably underestimated, though. The total fruitlipidic biomass represented≈13% of total tree carbon biomass(600mgCg�1DW Fig. 4B, for a total fruit biomass of47 kg tree�1). However, this amount is progressively synthe-sized during fruit development (180d), representing an aver-age lipid synthesis of 4.6molC tree�1 d�1, that is, 3% of theaverage daily photosynthetic fixation. The allocation valuescomputed here are modestly sensitive to assumptions madefor modelling. In particular, using a lower value of CUE tendsto increase both the allocation and the respiratory loss of fruits(Fig. S8).

Respiratory losses

Amongst all oil palm organs, fruits represented the largestCO2 loss calculated here (39%, Fig. 5C), regardless ofthe model (A or B) used for computations. As such, fruitsappeared to be the least metabolically efficient organs, withan apparent (computed) CUE of about 0.1. A low CUEvalue matches the metabolic requirement of lipid synthesis(fatty acid synthesis is associated with a theoretical maxi-mal CUE of 0.33). That is, the biomass production-to-respiration ratio was about 10 here, while calculations fromrespiratory requirements for maintenance and growth ledto an estimate of about 1.2 (Dufrêne 1989). However, itshould be noted that with a lipid content of ≈13% of treecarbon biomass and an optimal metabolic conversion rateof source carbon into lipids of one third, this represents acarbon loss of 26 units (=2× 13), while whole-tree respira-tory loss is of 100× (1/0.38–1) (where 0.38 is CUE)=163units, and thus lipid fruit synthesis itself already standsfor 16% of whole-tree respiratory loss. Apart from fruits,the rest of CO2 loss is well distributed amongst other or-gans, representing 10–15% of total CO2 loss (Fig. 5C). Thisrange is also consistent with previous estimations of rootCO2 efflux: root respiration was estimated by soil CO2 effluxmeasurements as 27% of assimilated C (Lamade et al. 1996)in Benin (under dry conditions and palm trees with large rootbiomass) and about 8% (Lamade, unpublished) in NorthSumatra (under humid conditions and palm trees with smallroot biomass, as observed here).

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© 2015 John Wiley & Sons Ltd, Plant, Cell and Environment

Perspectives

Here, we generated an allocation pattern in oil palm based onobserved δ13C values. Such a pattern does not rely on biomassdistribution (except for the estimate of respiratory fraction-ation) and therefore is not biomass-based but rather reflects ef-fective carbon fluxes and utilization. The present allocationvalues obtained here are consistent with known carbon re-quirements and suggest a particularly high CO2 loss by fruitrespiration. Oleosynthesis per se is commonly considered as ahigh energy-consuming process.While the carbon source (pho-tosynthesis) is usually believed to be sufficient for bunchorganic-matter production (Corley & Tinker, 2003), our esti-mate of the respiratory cost associated with fruit maturationmay indicate the involvement of carbon remobilization fromtrunk reserve. Natural 13C abundance thus provides a usefultool to examine C (re)distribution in oil palm, not requiring ex-pensive pulse-chase experiments with 13CO2 at the tree level.We nevertheless recognize that our studywas limited by techni-cal difficulties in the field. For example, it might be improvedby directmeasurement of the δ13C value in respiredCO2 or iso-tope exchange of the palm plantation with Eddy covarianceflux determination, and this will be addressed in a subsequentstudy. Furthermore, future studies are warranted to examinevariation in13C-distribution between genotypes and growthconditions so as to give insights into C-allocation parametersthat should be targeted for yield improvement.

ACKNOWLEDGMENTS

The authors warmly thank the field staff of IOPRI (IndonesianOil Palm Research Institute) and especially that from theMarihat Research Station for financial, technical and logisticsupport throughout this work. The authors also thank the re-search unit UMR Eco&Sol (Cirad, Inra, IRD, MontpellierSupAgro) for financial support and Dr. Jean Dauzat fromUMR AMAP for the 3D simulated palm figure. TheLaboratoire d’Ecologie, Systématique et Evolution (CNRS-UMR 8079) as well as the Platform Métabolisme-Métabolomeare also acknowledged for facilitating metabolite and organicmatter preparation and isotopic analyses, respectively.

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Received 18 May 2015; accepted for publication 26 June 2015

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Figure S1. Climatic parameters of Aek Pancur Research Sta-tion in 2003 and 2004:A, monthly average of daily global radi-ation (blac symbols, in 106 Jm�2 d�1) and vapour pressuredeficit (VPD, white symbols, in mbars); B, monthly rainfall(grey bars), maximal temperature (Tmax), average temperature(Tmean) and minimal temperature (Tmin).Figure S2. Phenological evolution of oil palm trees sampled inthe present study, with the rate of leaf emission (dashed line,open symbols) and inflorescence appearance rate (solid line,closed symbols). Mean±SE (n=10).Figure S3. Relationship between the reciprocal of ‘total’ (sto-matal + boundary layer) conductance for CO2 and the recipro-cal of photosynthesis, under usual conditions (closed symbols)

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and under high light (open symbols; 1000 µmolm�2 s�1 photonflux density) at 380 µmol mol�1 CO2: photosynthesis obtainedon individual leaves (leaflets) at high light (PAR within1400�1600 µmol/m�2/s�1, open discs) and photosynthesis ob-tained at sub-saturating light (<900 µmol/m�2/s�1) inmornings(9:00 to 11:00 am) along 20 days (closed discs). The linear re-gression was only carried out with data associatedwith large as-similation values (1/A < 0.1 sm2 µmol�1). The regressioncorresponds to the theoretical relationship: 1/gc = �1/gm +(ca�cc)/A in which the intercept (�1/gm) is used to compute in-ternal (mesophyll) conductance. Intercepts are �1.47 (closed)and �0.35 (open) s m2 mol�1, i.e., gm=0.679 and 2.35 molm�2 s�1.Figure S4. Typical diel course of 12C/13C fractionation calcu-lated from ci/ca and internal conductance gm (Δcalc, A), cc/ca(B) and net CO2 assimilation (C) leaf photosynthesis in thefield during the light period. Time is counted from sunrise tosunset. InA, the horizontal dashed line stands for the assimila-tion-weighted average of the fractionation (mean daily frac-tionation, 20.8‰). cc/ca was calculated using internalconductance (gm) graphically estimated in Fig. S3.Figure S5.Output of the model (natural isotope distribution inthe steady-state) at stage 0 (A, B, E, F) and 6 (C, D, G, H) offruit maturation, with models A (A, C) and B (E, G). A, C,E, G, carbon flux relative to the photosynthetic input (fixedat 100). Dark grey bars represent the export in leaves and theimport (influx) in other organs. B, D, F, H, carbon use effi-ciency i.e. 1 � R/I where I is the photosynthetic input (leaves)or the carbon input into the organ considered (other organs),and R is respiration. In these computations, leaf carbon use ef-ficiency is fixed at 88% since leaf respiratory loss is fixed at12% of assimilated carbon (see the description of the model).

The error shown for each bar represents the extreme valuesobtained under different computation conditions: leaf respira-tory fractionation (e) equal to +1 or �1‰.Figure S6. Allocation pattern in oil palm computed from δ13Cvalues: output of model C based on glucose redistributionrather than sucrose. InA, numbers are in%, i.e., expressed rel-ative to photosynthetic C fixation fixed at 100. Values in blackand grey were obtained at stage 0 and stage 6 of fruit matura-tion, respectively. δ13C values shown for respired CO2 were ob-tained by mass balance with e = +1‰ (dark) or �1‰ (grey).Asterisks indicate that the values were fixed as a model con-straint. B, distribution of palm respiration efflux, in % of totalCO2 loss. Mature leaf respiration loss remains constant (fixedmodel constraint). The different values shown correspond tostage 0 (S0, black bars) and 6 (S6, grey bars) of fruit develop-ment. C, carbon use efficiency. Abbreviations: resp, respira-tion; St.s., starch synthesis; Suc s., sucrose synthesis. Note theunrealistic values of root respiration (at zero) and thus themaximal CUE of 1.Figure S7. Sensibility of calculated allocation and respirationvalues to new leaves (NL), roots, trunk and fruits with respectto internal conductance: relative changes (in % of valuesshown in Fig. 5) when internal conductance gm is decreasedfrom 0.68 (value used in Fig. 5) to 0.34molm�2 s�1.Figure S8. Sensibility of calculated allocation values to newleaves (NL), roots, trunk and fruits with respect to the imposedvalues of tree respiratory loss (50%, 62%and 80%), i.e., the ra-tio of total respiratory loss to carbon assimilation (= 1 � CUE,where CUE is carbon use efficiency). Allocation values (in %)are shown (A) as well as the contribution of fruits to total res-piration (B). The calculations carried out in this figure utilizethe outputs of model A at stage 0 as an example.

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