Effects of exogenous glucose on carotenoid accumulation in tomato leaves

Physiologia Plantarum 134: 246–256. 2008 Copyright ª Physiologia Plantarum 2008, ISSN 0031-9317

Effects of exogenous glucose on carotenoid accumulationin tomato leavesAnne Mortain-Bertranda,b,c, Linda Stammittia,b,c, Nadege Telefa,b,c, Patrice Colardellea,b,c,Renaud Brouquissed, Dominique Rolina,b,c and Philippe Galluscia,b,c,*

aINRA, UMR 619, 71 Avenue Edouard Bourleaux, BP 8, F-33883 Bordeaux, FrancebUniversite de Bordeaux 1, UMR 619, 71 Avenue Edouard Bourleaux, BP 81F-33883 Bordeaux, FrancecUniversite de Bordeaux 2, UMR 619, 71 Avenue Edouard Bourleaux, BP 81F-33883 Bordeaux, FrancedUMR Interactions Biotiques et Sante Vegetale, INRA, Agrobiotech, 400 route des Chappes, BP 167, F-06903 Sophia Antipolis, France

Correspondence

*Corresponding author,

e-mail: [email protected]

Received 4 February 2008; revised 23 April

2008

doi: 10.1111/j.1399-3054.2008.01130.x

To investigate the effect of carbohydrate on carotenoid accumulation in leaves,excised plants of tomato (Lycopersicum esculentum var. cerasiformae, wva

106) were supplied with glucose through the transpiration stream for 48 h.We

report here that sugar accumulation in leaves led to a decrease of carotenoid

content, which was related to the reduction of Chl. The decrease in carotenoid

amount correlated with a sugar-induced repression of genes encoding

enzymes of the carotenoid and of the Rohmer pathways. The lower 1-deoxy-

D-xylulose-5-phosphate synthase transcript level probably leads to a decreased

metabolic flux through the methylerythritol pathway and subsequently toa lower amount of substrate available for plastidic isoprenoid synthesis.

Differences between responses of young (sink) and mature (source) leaves to

carbohydrate accumulation are discussed.

Introduction

Carotenoids (carotenes and xanthophylls) are C40 iso-

prenoids consisting of a polyene chain with conjugated

double bonds, responsible for their absorption spectrum

and color (yellow, orange or red). Carotenoids can eitherbe acyclic (lycopene), cyclized (b-carotene) and oxygen-

ated (xanthophylls). They are synthesized in plastids

where they accumulate and bind specific hydrophobic

proteins. Carotenoids are essential for all photosynthetic

organisms. In chloroplasts, they participate in light har-

vesting and in energy transfer to Chl, as well as in pho-

toprotection by quenching the excited Chl (reviewed

in Demmig-Adams and Adams 1996). They also havestructural functions in the photosynthetic pigment–protein

complexes of the reaction center and the light-harvesting

antenna. In flowers and fruits, carotenoids accumulate

in chromoplasts, conferring characteristic yellow, red or

orange colors to these organs and facilitating pollination

and seed dispersion by insects and animals.

A simplified depiction of the carotenoid pathway isshown in Fig. 1. Carotenoid biosynthesis necessitates the

synthesis of the universal isoprenoid precursor isopen-

tenyl diphosphate (IPP). IPP is a five-carbon building block,

which is condensed to its isomer dimethylallyl diphos-

phate (DMAPP) to give a C10 compound, the geranyl

pyrophosphate (GPP). Further IPP condensations by

prenyltransferases lead to the formation of a C15 com-

pound, the farnesyl pyrophosphate and to geranylgeranylpyrophosphate (GGPP), a linear C20 molecule, which is

the direct precursor of carotenoid biosynthesis. In the

Abbreviations – DW, dry weight; DMAPP, dimethylallyl diphosphate; DXP, 1-deoxy-D-xylulose- 5-phosphate; DXS, 1-deoxy-D-

xylulose-5-phosphate synthase; G-6-P, glucose-6-phosphate; GGPP, geranylgeranyl pyrophosphate; GPP, geranyl pyrophosphate;

IPP, isopentenyl diphosphate; LCY-B, lycopene b-cyclase; LCY-E, lycopene e-cyclase; MEP, methylerythritol; MS, Murashige and

Skoog medium; NPQ, non-photochemical quenching; PDS, phytoene desaturase; Pi, inorganic phosphate; PSY2, phytoene

synthase.

246 Physiol. Plant. 134, 2008

plastid compartment, IPP and DMAPP are produced

through the methylerythritol (MEP) pathway, also desig-

nated 1-deoxy-D-xylulose- 5-phosphate (DXP) pathway

(Lichtenthaler et al. 1997). The MEP pathway is initiatedby the transketolation of pyruvate and glyceraldehyde

3-phosphate, which leads to DXP then to MEP, and pro-

vides the precursors necessary for carotenoid biosyn-

thesis (Fig. 1).

The condensation of two molecules of GGPP by the

enzyme phytoene synthase (Psy2) produces phytoene,

which is subsequently dehydrogenated by the phytoene

desaturase (PDS) and the z-carotene desaturase to yield z-carotene and lycopene, respectively. Lycopene is con-

verted into b-carotene by the action of b-cyclase (LCY-B),

an enzyme that introduces b-ionone rings at both ends

of themolecule.A competing e-cyclase (LCY-E)may intro-

duce a single e-ionone ring in lycopene giving d-carotene.Genetic and molecular studies have established that

nuclear genes encode all the enzymes of the pathway and

have led to the cloning ofmost of the corresponding genes

in many higher plants (Hirschberg 2001).

At that time, the carotenoid biosynthesis pathway is

well established (Bramley 2002), but the regulatory

mechanisms that control this pathway are still poorly

understood. In chromoplasts of fruits and flowers,carotenoid accumulation is developmentally regulated,

in part by changes in the expression of genes encoding

enzymes of the pathway (Bramley 2002, Fraser et al.

1994, Lois et al. 2000, Ronen et al. 1999). However, other

mechanisms have been suggested including hormones

(Giovannoni 2004), light (Alba et al. 2000, Liu et al.

2004), but also the availability of substrates produced

through the MEP pathway (Cunningham 2002) and seq-uestration mechanisms (Vishnevetsky et al. 1999). In

green tissues, carotenogenesis is also controlled by light:

although expression of genes does take place in etiolated

plants, light has a greater than four-fold stimulatory effect

on carotenoid accumulation (Giuliano et al. 1993), and

PsymRNA levels have been shown to increase in the light

(Welsch et al. 2000) through a phytochrome-mediated

regulation (Alba et al. 2000, von Lintig et al. 1997). Inchloroplasts, contrary to chromoplasts, carotenoids are

part of pigment–protein complexes localized in thyla-

koids. Carotenogenesis in green tissues must, therefore,

be coordinated with the formation of others photosystem

components, including Chl. Indeed, Frosch and Mohr

(1980) provided evidence that the formation of Chl is

required to allow the accumulation of large amount of

carotenoids in the plastids of Sinapis alba. Their datasuggest that carotenogenesis is controlled by light in

a phytochrome-dependentway and also by the amount of

Chl that drains the pool of free carotenoids by complex

formation. However, accumulation of carotenoids has

been reported to be inhibited in the mutant of barley

(Nielsen and Gough 1974) and in Chlamydomonas

(Plumley and Schmidt 1995) arrested in Chl synthesis.

In addition, Corona et al. (1996) showed a decrease incarotenoid content of tomato seedlings treated with

gabaculine, an inhibitor of Chl synthesis. Therefore,

factors controlling Chl synthesis are also likely to be

involved in the control of carotenogenesis. Among them,

soluble sugars have been shown to be negative regulators

of photosynthesis (for review see Gibson 2005). Sugar-

mediated repression of photosynthesis is part of general

mechanisms aimed to maintain photosynthesis homeo-stasis over a wide range of growing conditions. Yet, when

sink demand is restricted [i.e. during water or low

temperature stress or under inorganic phosphate (Pi)

deficiency], sugars accumulate in leaves and have direct

effect on photosynthetic genes and Chl abundance (Paul

and Foyer 2001). Thus, soluble sugars may also play

essential function in the control of carotenoid abundance

Fig. 1. Pathway of isoprenoid synthesis in plant plastids. Modified from

Lois et al. (2000) and Cunningham (2002). GA3P, glyceraldehyde 3-

phosphate; GGPP; GGPS, geranylgeranyl diphosphate synthase; PEP,

phosphoenolpyruvate; ZDS, z-carotene desaturase.

Physiol. Plant. 134, 2008 247

because these pigments are part of the photosynthetic

apparatus in chloroplasts of leaves.

In contrast to the large amount of work that has focused

on the repression of photosynthesis by sugars (Araya et al.

2006, Gibson 2005, Krapp et al. 1991, Pego et al. 2000),

few works have been performed on the effects of sugarson carotenoids. Sucrose has been shown to promote color

change of Citrus fruit epicarp (Huff 1984) and to stimulate

carotenoid accumulation in pericarp disks of tomato

fruits (Telef et al. 2006). In leaves, however, if carotenoid

and Chl synthesis are coordinated, sugars, whose accu-

mulation leads to a loss of Chl (Krapp et al. 1991), are

expected to repress carotenogenesis as well.

In this study, we investigated the function of solublesugar accumulation on carotenoid amount in tomato

leaves using excised plants. We demonstrate that when

excised plants were incubated in glucose supplemented

medium, soluble sugars accumulated in young and

mature leaves. This accumulation resulted in a coordi-

nated decrease of Chl and carotenoid content probably

mediated through the repression of 1-deoxy-D-xylulose-

5-phosphate synthase (DXS), PSY2, PDS and LCY-B geneexpression.

Materials and methods

Culture conditions

Tomato plants (Lycopersicum esculentum var. cerasifor-mae, wva 106)were grown in a 16-h light/8-h dark photo-

period (300 mmol photons m22 s21 irradiance, 25�C/18�C). The stem of plants with five leaves was cut just

above cotyledons and surface sterilized by a rapid soak in

50% ethanol and then rinsed in sterile water. The stem

was cut a second time while submersed in water to main-

tain continuous water column in the xylem. Excised plants

were then placed for 48 h in 50% diluted Murashige andSkoog (MS) medium, without sugar (G0) or supplemented

with 55 mM (G1) or 110 mM (G2) glucose, under the same

conditions of light and temperature as intact plants.

After 2 days, at the end of the light period, young (the

two youngest) and mature leaves were separately col-

lected without petiole and frozen in liquid nitrogen.

Leaves from fully intact plants were used as a control.

Chl fluorescence

Chl fluorescencemeasurementswere performed on young

and mature leaves with a PAM-200 system (Eurosep,

Cergy-Pontoise, France). Conventional fluorescence

nomenclature was used (Van Kooten and Snel 1990). The

ratio of variable to maximal fluorescence was determined

at the end of the 8 h darkness period to be sure that leaves

were dark adapted.Non-photochemical quenching (NPQ)

was calculated as ðFm2Fm#Þ=Fm#, where Fm is the

maximum fluorescence in the dark-adapted state, and

Fm#is the maximum fluorescence after exposure to actinic

light (PAR of 1850 mmol photons m22 s21).

Pigment and sugar determination

Frozen tissues were ground in liquid nitrogen into a fine

powder. The same powder was used for all experiments,

except it was lyophilized before pigment determination.

Soluble sugars were extracted with boiling ethanol/

water and analyzed enzymatically according to Kunst

et al. (1984). Determination of NADH was made at340 nmwithMR5000 (Dynatech Laboratories, St. Cloud,

France). Starch contained in the insoluble pellet was

hydrolyzed into glucose using amyloglucosidase (EC

3.2.1.3; Merck, Lyon, France) after heating at 135�C for

1 h (Moing et al. 1994). The glucose was then measured

as mentioned above. Sugars were quantified from

standard solutions of glucose, fructose and sucrose.

Pigments were extracted from 10 mg of lyophilizedpowder as described by Fraser et al. (2000). The pooled

chloroform extracts were dried upon a stream of nitrogen

and stored at 220�C prior to HPLC. Samples were pre-

pared by dissolving the dried residues in 200 ml of ethyl

acetate. Pigments were separated using a reverse phase

C30 (250 � 46 mm) column (YMC Inc., Cluzeau, Sainte-

Foy-La-Grande, France) and eluted with a 1 ml min21

gradient of (A) methanol, (B) water/methanol (20/80)containing 0.2% ammonium acetate and (C) tert-methyl

butyl ether (Fraser et al. 2000). Chromatography was

performed on a Spectra system (Thermo Finnigan,

Villebon Sur Yvette, France) with diode array monitoring

between 200 and 800 nm. Data were collected and

analyzed using the PC1000 (Thermo Finnigan) software.

Pigments were identified and quantified by using stand-

ards: b-carotene, lutein, Chl a and Chl bwere purchasedfrom Sigma Chemical Co. (Lille-Lezennes, France)

Violaxanthin and neoxanthin were isolated from tomato

leaf tissue.

31P-NMR spectroscopy

A 500 MHz NMR spectrometer (Brucker, Wissenbourg,

France) was used to analyze the most abundantphosphorylated compounds. Samples were prepared

from 4 g fresh powder as described by Brouquisse et al.

(2001). Spectra were acquired as in Brouquisse et al.

(2001). The resonance assignments were made using

published data (Brouquisse et al. 2001, Roby et al. 1987)

and comparison with spectra of the pure suspected

compounds.


Extraction of total RNA and RT-PCR

Total RNA was extracted from 100 mg of frozen groundmaterial as described (Telef et al. 2006). Reverse

transcription was carried out using 2 mg of total RNA

and oligo-dT as a primer. The reaction mixture included

10 mM dNTP, 1.5 mM oligo-dT, 20 mM DTT, 24 U

RNAsin, 400 U M-MLV reverse transcriptase in a total

volume of 32.7 ml. The reaction was allowed to proceed

for 1 h at 42�C and stopped after 5 min at 80�C.Typically, PCR reactions contained 0.1 mM dNTPs,

1.25 U Taq polymerase, a volume of RT reaction mixture

corresponding to 60 ng RNA, 1 mM of couples of primers

in a total volume of 50 ml of PCR buffer (Promega,

Charbonnieres, France). The amplification reactions con-

sisted of 30 s at 95�C, 40 s at 54�C and 30 s at 72�C. Theminimum number of cycles needed to visualize the

transcripts on an agarose gel was first determined by

analyzing PCRafter 20, 25 and30 cycles. Inmost cases, 25cycles were appropriate. PCR products were analyzed in

1.2% agarose gel and visualized using ethidium bromide

staining. Signal intensity was quantified using the quantity

one software (Bio-Rad, Marnes La Coquette, France). Each

value represents the signal intensity normalized to control

and elongation Factor factor 1 alpha signals.

Results

Glucose supply enhanced carbohydrate andphosphorylated compound accumulation in leaves

In a first set of experiments, the aerial parts of 4-week-old

plants were cut off and placed in MS medium supple-

mented with 55 mM (G1), 110 mM (G2) or without (G0)

glucose (see Materials and methods). Glucose was

preferred to sucrose because of its stronger effects on

sugar accumulation as observed in preliminary exper-

iments (data not shown). Measurement of hexose

(glucose and fructose), sucrose and starch demonstratedthat soluble carbohydrates content increased inG0 leaves

compared with intact plant leaves (C) (Fig. 2). The

increase in soluble sugars was stronger in young (sink)

leaves (3.5-fold increase) than in mature (source) leaves

(1.2-fold increase) (Table 1). As a consequence, youngG0

leaves contained twice as much soluble carbohydrates as

mature leaves. Starch amount was similar in young and

mature leaves [174 and 163 nmol mg21 dry weight(DW), respectively] and decreased to approximately the

same level (44 and 30 nmol mg21 DW) in young and

mature G0 leaves. Feeding the plants with 55 mM (G1)

glucose led to amarked increase of the total carbohydrate

content when compared with G0 leaves incubated in MS

medium only (Table 1). As shown in Fig. 2, this increase

wasmainly because of a very high accumulation of starch

(12- and 15-fold in young and mature leaves, respec-tively). Increasing external glucose concentration to

110 mM had no additional effect on starch amount but

resulted in increased sucrose content in mature leaves.

The main effect was observed on fructose and glucose,

which were 1.6–2.3 higher in leaves of G2 plants

compared with G1. As a conclusion, the total carbohy-

drate amount in both young and mature leaves was

positively correlated to the glucose concentration in themedium (Table 1). This indicates that part of the

supplemented glucose was translocated to the leaves.

Sucrose

250300350

(nm

ol m

g–1 D

W)

50100150200

0

Young leaves Mature leaves

Fructose

250300350400

50100150200

(nm

ol m

g–1 D

W)

0CG2G1G0 G2G1G0C

G2G1G0C G2G1G0C G2G1G0C G2G1G0C

CG2G1G0 G2G1G0C

Mature leavesYoung leaves

(nm

ol m

g–1 D

W) Starch

400500600700

0100200300


(nm

ol m

g–1 D

W) Glucose

150

200

250

0

50

100


Fig. 2. Carbohydrate content [nmol mg21 DW]of tomato leaves. Excised plantswere placed inMSmediumwithout (G0) orwith 55 mM (G1) or 110 mM

(G2) glucose. After 48 h, fructose, glucose, starch and sucrose were determined in young and mature leaves as well as in intact plant leaves (C). Data are

means � SE of three independent assays.


In vitro 31P-NMRwas used to assess themetabolic state

of the leaves through the analysis of mature leaf extracts,including hexose phosphates, Pi, ATP and phosphoryl-

choline. Fig. 3 displays 31P spectra obtained with leaves

from intact plants or from excised plants incubated with

0, 55 or 110 mM glucose. Excision led to a marked

accumulation of Pi and, to a lesser extent, of P-choline,

whereas the amount of glucose-6-phosphate (G-6-P)

decreased. ATP and ADP contents remained unchanged.

In glucose-fed leaves, Pi declined considerably and the

sugar phosphates increased slightly.

Glucose supply limits Chl accumulation andrepresses Rubisco gene expression

Soluble sugars are known to repress Chl synthesis and

Rubisco accumulation and to reduce photosynthesis

efficiency (Krapp et al. 1991). Changes in Chl abundance

and Rubisco small-subunit mRNAamountwere therefore

analyzed in young and mature leaves of control and G0,

G1 and G2 plants. As shown in Fig. 4A, excision led to

a significant decrease (30%) in total Chl level of youngleaves but not of mature ones. In addition, Chl amount

decreased with increasing glucose concentration:221

and236% in G1 and G2 (mature and young) leaves

compared with G0. Chl a and Chl b were similarly

affected, leading to a constant Chl a/b ratio (Fig. 4A). This

suggests that the decrease in Chl affected both the Chl a

containing core complexes and the peripheral light

harvesting Chl a/b complexes. Similarly RT-PCR anal-ysis (Fig. 4B) showed that the rbcS transcript level

Table 1. Influence of feeding glucose on carbohydrate content of leaves.

Excised plants were placed in MS medium without (G0) or with 55 mM

(G1) or 110 mM (G2) glucose. After 48 h, fructose, glucose, starch and

sucrose were determined in young and mature leaves as well as in intact

plant leaves (control). Soluble carbohydrates, glucose, fructose and

sucrose; total carbohydrates, soluble carbohydrates 1 starch. Carbohy-

drates are expressed in nmol mg21 DW.

Soluble carbohydrates Total carbohydrates

Young leaves Mature leaves Young leaves Mature leaves

Control 87 139 261 302

G0 307 175 351 206

G1 357 432 877 908

G2 561 824 1066 1192

Fig. 3. 31P-NMR spectra of mature leaves. Excised plants were incubated for 48 h in MS medium without (G0) or with 55 mM (G1) or 110 mM (G2)

glucose. Leaf extracts were prepared as described in Materials and methods. Spectra were acquired at 400 MHz with an free induction decay (FID)

resolution of 0526 Hz, a 60� radiofrequency pulse and 1024 transient repeated every 2 s. F-6-P, fructose-6-phosphate; GPC, glycero phosphatidylcholine;

3-PGA, 3-phosphoglyceric acid; P-chol, phosphorylcholine; P-eth, phosphorylethanolamine; PEP, phosphoenolpyruvate; Pi, inorganic phosphate.


observed inG0 leaveswasmore reducedwhen plantswere

fed with glucose. Yet, these results are in agreement

with previously described effects of glucose on photosyn-

thesis in other plant species (Araya et al. 2006, Jang andSheen 1994, Krapp et al. 1993). It is noteworthy that the

decrease in rbcS expression was higher in mature leaves.

Glucose supply does not change Chl fluorescenceparameters

To control whether the decrease in Chl amount disrupts

PSII functioning, fluorescence measurements were per-formed on leaves before harvest. Measuring Chl fluores-

cence at room temperature is a non-invasive approach

that provides information about the organization and

functionality of PSII reaction center (Schreiber et al.

1986). The Fv/Fm ratio, that reflects the photochemical

quantum efficiency of PSII and photo-inhibitory damages

(Krause and Weis 1991), was determined after illumina-

tion of dark-adapted leaves with a saturating pulse (seeMaterials and methods). As shown in Table 2, the Fv/Fmratios were not significantly different whether the leaves

were excised (G0) or not (C), and whether the excised

plants were fed with 55 mM (G1) or 110 mM (G2)

glucose. Araya et al. (2006) also reported that Fv/Fm did

not change with sugar treatment. The magnitude and

timing of NPQ of Chl fluorescence, a protective process

allowing the dissipation of excessive excitation energy

in the antenna as heat, was also investigated during

exposure of leaves to high light. In mature leaves (Fig. 5),as in young leaves (data not shown), the NPQ response

reached a plateau after 9 min of illumination with high

light. The induction of NPQwas not different whether the

measurements were performed on leaves from intact

plants or from excised plants fed with or without glucose.

Glucose supply limits carotenoid accumulation

The carotenoid composition of intact or excised leaves

was determined (Fig. 6A). As shown for Chl, the amount

Fig. 4. Response of tomato leaves to carbohydrate accumulation. (A) Total Chl and Chl a/b ratio (d). (B) Expression of rbcS. Chl amount (in pmol mg21

DW)was determined by HPLC. SE bars are shown (n ¼ 3). The expression of rbcSwas studied by semiquantitative RT-PCR from the same samples used for

carbohydrates analysis. Each value represents the signal intensity normalized to C and EF1a signals.

Table 2. Fv/Fm values of young andmature tomato leaves. Excised plants

were placed in MS medium without (G0) or with 55 mM (G1) or 110 mM

(G2) glucose. After 48 h, the ratio Fv/Fm was determined in young and

mature leaves as well as in control plants (C). Data are means � SE of 10–

15 measurements on different leaves.


C 0.790 � 0.010 0.782 � 0.007

G0 0.786 � 0.004 0.777 � 0.010

G1 0.786 � 0.003 0.774 � 0.007

G2 0.780 � 0.008 0.763 � 0.011


of lutein and b-carotene decreased, in young leaves only,

when excised plants were cultured in MS medium (220and 230%, respectively compared with control leaves).

The decrease in carotenoids was amplified when plants

were incubated in glucose supplemented medium: in

young leaves of G2 plants, lutein and b-carotene were 45

and 60% lower than in young leaves of intact plants,

respectively. A similar decrease was observed in mature

leaves, although the effect was not as strong with only

a 30% decrease for both lutein and b-carotene. The b-carotene-derived pigments violaxanthin, zeaxanthin and

antheraxanthin, which were four- to six-fold less abun-

dant than lutein in intact leaves, also decreased with

increasing glucose concentration (data not shown). Thus,

glucose supplementation induced a coordinated reduc-

tion of all detectable carotenoids resulting in unchanged

carotenoid pattern in leaves. It is noteworthy that,

whatever the conditions and the leaves (youngormature),the ratio Chl/lutein 1 b-carotene was not significantly

different (comprised between 3.1 and3.4), indicating that

Chl and carotenoids were coordinately affected by

excision and glucose supply.

To unravel the mechanisms responsible for the

glucose-dependent reduction of carotenoid amount,

the expression of the genesDXS, PSY2, PDS, LCY-B and

LCY-E (Fig. 1) was investigated using semiquantitativeRT-PCR. DXS, PSY2, PDS, LCY-B gene expression was

negatively correlated with carbohydrate content of

leaves (Fig. 6B). However, although all these genes

were repressed by sugars, slight differences were

observed. The excision- and glucose-induced reduction

0.9

0.7

0.8

0.5

0.6

0.3

0.4NP

Q

0.1

0.2 C

G0

G2

0151413121110987654321

Time (min)

Fig. 5. Induction of NPQ in glucose-accumulating mature leaves. Excised plants were placed for 48 h in MS medium supplemented with 110 mM

glucose (G2) or not (G0) and illuminated with a 16 h light (300mmol photonm22 s21)/8 h dark cycle. At the end of the second dark period (dark-adapted

leaves) fluorescence was measured before and during exposure to actinic light (1850 mmol photons m22 s21). NPQ was calculated as ðFm2Fm#Þ=Fm#.

Fig. 6. Effect of glucose accumulation on carotenoid synthesis in leaves. (A)

Lutein and b-carotene amounts. (B) Expression of DXS, Psy2, PDS and b-

cyclase. Pigment amount (in pmol mg21 DW) was determined by HPLC. SE

bars are shown (n ¼ 3). The expression of genes was studied by semi-

quantitative RT-PCR from the same samples used for Chl and rbcS analysis.

Each value represents the signal intensity normalized to C and EF1a signals.


ofDXS transcript level was higher in mature leaves than

in young leaves. The abundance of PSY2, PDS and LCY-

B mRNA also decreased after excision and glucose

supply, with a stronger effect in mature leaves; however,

the excision-induced decrease was not as strong as for

DXS. Expression of LCY-E was only detectable in leavesharvested from intact plants. The signal was below

detection in all other conditions making difficult the

observation of glucose real impact on LCY-E expression

(data not shown).

Discussion

In plants as in many other organisms, soluble sugars areessential primary metabolites. They also play major roles

as regulatory molecules controlling gene expression,

plant physiology, metabolism, cell cycle and plant

development (Gibson 2005, Koch 1996, Paul and Foyer

2001). Soluble sugars limit photosynthesis efficiency,

reduce Chl accumulation and repress the expression of

genes involved in carbon assimilation including the RbC

genes (Pego et al. 2000). Despite the accurate descriptionof sugar-induced feedback repression of photosynthesis,

little attention has been devoted to the effect of sugar on

carotenoid abundance in leaves. However, carotenoids

are essential components of the photosynthetic appara-

tus, and their synthesis is expected to be correlated to Chl

accumulation and photosystem organization. To analyze

the effects of sugars on carotenoid accumulation in

mature and developing tomato leaves, glucose wassupplied to leaves of excised plants through the transpi-

ration stream.

The glucose-induced increase of leavescarbohydrate content limits RBCS gene expressionand Chl accumulation

Incubation of detached leaves was already proved to bean appropriate experimental system to study sugar effects

on photosynthesis regulation (Krapp et al. 1991) or

carbohydrate metabolism (Cairns et al. 2002, Wei et al.

2002). Similarly, our results show that using plant with

excised roots allowsmodifying the carbon equilibrium of

the plant even when no glucose was provided. In G0

plants, soluble carbohydrates accumulated both in young

and, to a lesser extent, in mature leaves (Fig. 2, Table 1).This accumulation was probably because of the inhibi-

tion of carbohydrate export to roots, whereas at the same

time, photosynthesis was still functioning efficiently

(Fig. 5, Table 2). It is unclear, however, to which extend

soluble sugars were efficiently metabolized in G0 plants

as they accumulated neither G-6-P nor starch and were

characterized by high Pi content (Fig. 3).

Feeding excised plants with glucose led to an even

greater accumulation of soluble carbohydrates and to

amassive synthesis of starch in young aswell as inmature

leaves (Fig. 2). Analysis of phosphorylated compounds in

mature G1 and G2 leaves showed that G-6-P abundance

was similar to that found in control non-excised plantsindicating that glucose was efficiently absorbed and me-

tabolized (Fig. 3). This, together with the sugar-induced

upregulation of genes involved in starch synthesis (Koch

1996), may explain the significant increase in starch con-

tent observed in G1 and G2 plants (Fig. 2).

Additionally, the increase of endogenous soluble sugar

content of excised leaves resulted in a decrease of both

rbcS mRNA level and Chl amount (Fig. 4), characteristicof sugar inhibition of photosynthesis (Krapp et al. 1991,

Pego et al. 2000). Although we cannot completely rule

out that wounding also affected the expression of genes

involved in photosynthesis and Chl abundance (Ehness

et al. 1997, Krapp et al. 1993), the reduction of rbcS tran-

scripts and of Chl amounts was correlated to the increase

of endogenous soluble sugars. This suggests that the effect

observedwas primarily because of the increase of sugars.Taken together, these results demonstrate that the

manipulation of endogenous soluble sugar amounts using

excised plants causes the repression of photosynthetic

parameters and therefore allows the study of their effects

on carotenoid accumulation.

Carbohydrate accumulation correlates witha decrease in carotenoid content

The sugar-induced loss of Chl was accompanied by

a progressive decrease in all carotenoids. The effect of

soluble sugars seemed stronger in young leaves than in

mature ones with a 50 and 30% decrease of total caro-

tenoids in G2 plants, respectively, compared with control

plants (Fig. 6). Similarly, sugar-induced reduction for Chl

amount was more pronounced in young than in matureleaves (55 and 38%, respectively; Fig. 4). Yet, in both

cases, the decreases of Chl and carotenoid amounts were

coordinated in response to soluble sugar increase, sug-

gesting that the integrity of the photosynthetic apparatus

was maintained. This conclusion is consistent with the

lack of variation of the Fv/Fm ratios, even in G2 conditions

(Table 2), which indicates that the decrease in Chl and

carotenoids does not disturb the efficiency of the absor-bed light energy transfer to the PSII reaction centers.

Additionally, the lower amount of carotenoids detected

in sugar-accumulating leaves did not result in a reduction

of NPQ of Chl fluorescence (Fig. 5). NPQ measures the

de-excitation of singlet Chl (1Chl) that accumulates in the

light-harvesting complexes under conditions of excessive

illumination (Demmig-Adams 1990) and is one of the


mechanisms allowing carotenoids to protect plants

against photodamage. NPQ correlates with the synthesis

of zeaxanthin and antheraxanthin from violaxanthin

through the xanthophyll cycle (Demmig-Adams 1990).

However, in addition to the xanthophyll-cycle pigments,

which are derived from b-carotene, b-carotene itself(Sharma and Hall 1992) as well as a-carotene-derived

xanthophylls such as lutein (Niyogi et al. 1997) may

contribute to the dissipation of excess absorbed light

energy. Thus, the reduction of all carotenoids observed in

excised plants should have led to a reduced ability to de-

excite 1Chl, illustrated by a decrease in NPQ and then to

an increased sensitivity to high light. This was not the

case, demonstrating that the concomitant reduction ofcarotenoid and Chl allows maintaining the photoprotec-

tion capacity.

As a conclusion, the coordinate regulation of Chl and

carotenoid abundance in response to sugar allows the

leaves to reduce the amount of absorbed light, therefore

their photosynthesis rate, while maintaining both energy

transfer efficiency and photoprotection capacity.

Soluble sugars affect the expression of genes ofthe Rohmer pathway and of the carotenoidpathway

Carotenoid accumulation in leaves is determined by the

steady-state amount resulting from continuous biosyn-

thesis and simultaneous degradation because of photo-

oxidation. To evaluate the effect of sugars on carotenoidbiosynthesis, the expression of genes involved on carot-

enoid biosynthesis was analyzed. Our results show that

the expression of Psy2, PDS and LCY-B genes was

reduced in leaves accumulating soluble sugars in

a concentration-dependent manner (Fig. 6). In mature

G0 leaves, reduction of the expression of genes involved

in carotenoid biosynthesis was observed although no

detectable change in Chl content had occurred. Thismakes it unlikely an indirect effect of sugars on the

expression of genes involved in carotenoid accumulation

mediated by variations in Chl content. Taken together,

these results suggest a coordinated negative regulation

of the carotenoid pathway genes in response to sugar,

which in turn, may limit carotenoid renewal and ac-

count for the sugar-dependent reduction of pigment

accumulation.Additionally, because both Chl and carotenoid amount

decreased, sugars are also expected to act before

geranylgeranyl diphosphate synthase that represents a

branching point in plastid isoprenoids (Fig. 1). As a matter

of fact, the expression ofDXS gene,which encodes the first

enzyme of the Rohmer pathway, was significantly reduced

in mature and young leaves. DXS has been reported to

control the carbon flux entering the Rohmer pathway,

which provides precursors for carotenoid and ubiquinone

biosynthesis inEscherichia coli (HarkerandBramley1999,

Matthews and Wurtzel 2000) as well as for plant

isoprenoids (Estevez et al. 2001, Lois et al. 2000). Thus,

limitation of DXS gene expression may result in a globalreduction of plastidic isoprenoid biosynthesis (Estevez

et al. 2001), which is consistent with our observation that

the sugar-induced decrease in Chl and carotenoid

occurred in a coordinate manner. It is, however, unclear

whether, in our conditions, this loss of pigments was

accompanied by a decrease of other isoprenoid-derived

molecules such as ABA, gibberellins and tocopherol.

It is noteworthy that the sugar-induced reduction ofPSY2, PDS, LCY-B andDXSmRNA, as that of rbcSmRNA,

was more pronounced in mature than in young leaves,

particularly at high glucose concentration in the incuba-

tion medium (Fig. 6). This is consistent with the results of

Araya et al. (2006),which indicate that glucose repression

of photosynthetic genes is significant in source leaves but

not in young leaves. This may be partly explained by the

highest concentrations of soluble sugars accumulated inmature leaves compared with young leaves, at least in G1

and G2 plants (Table 2). This observation, however, is

contradictory with the demonstration that pigment

accumulation was more sensitive to sugar in young than

in mature leaves. It suggests that in addition to gene

expression, other mechanisms are responsible for the

rapid decrease of pigment abundance in young leaves.

In conclusion we demonstrate that, in addition to itseffect on Chl and photosynthetic proteins, soluble sugars

limit carotenoid amounts. This effect can be correlated

with the limitation of the expression of DXS, a gene that

controls the synthesis of plastidic IPP, the general

precursor of isoprenoid biosynthesis. Additionally, genes

from the carotenoid/isoprenoid pathway were also

repressed suggesting a coordinate direct or indirect

regulation by sugars of this pathway in leaves.

Acknowledgements – This work was supported by the

Aquitaine region and by the ‘Pole Aquitain de Nutrition et

Sante’. N. T.was a recipient of a grant from the FrenchMinistry

of Research and Technology. We would like to thank M.

Maucourt for his valuable technical help.

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Effects of exogenous glucose on carotenoid accumulation in tomato leaves

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