GDP-d-mannose 3,5-epimerase (GME) plays a key role at the intersection of ascorbate and...

GDP-D-mannose 3,5-epimerase (GME) plays a key role atthe intersection of ascorbate and non-cellulosic cell-wallbiosynthesis in tomato

Louise Gilbert1,†, Moftah Alhagdow1,†,‡, Adriano Nunes-Nesi2, Bernard Quemener3, Fabienne Guillon3, Brigitte Bouchet3,

Mireille Faurobert4, Barbara Gouble5, David Page5, Virginie Garcia1, Johann Petit1, Rebecca Stevens4, Mathilde Causse4,

Alisdair R. Fernie2, Marc Lahaye3, Christophe Rothan1 and Pierre Baldet1,*

1Institut National de la Recherche Agronomique (INRA), Universite de Bordeaux, Unite Mixte de Recherche 619 sur la Biologie

du Fruit, Institut Federatif de Recherche 103, BP 81, F-33883 Villenave d’Ornon Cedex, France,2Max-Planck-Institut fur Molekulare Pflanzenphysiologie, Am Muhlenberg 1, 14467 Potsdam-Golm, Germany,3Institut National de la Recherche Agronomique (INRA), Unite Biopolymeres Interactions Assemblage, UR1268, F-44316 Nantes,

France,4Institut National de la Recherche Agronomique (INRA), Unite Genetique et Amelioration des Fruits et Legumes, UR1052,

F-84143 Montfavet, France, and5Institut National de la Recherche Agronomique (INRA), Unite Securite et Qualite des Produits d’Origine Vegetale, UMR408,

F-84914 Avignon, France

Received 7 May 2009; revised 24 June 2009; accepted 26 June 2009.*For correspondence (fax +33 5 57122541; e-mail [email protected]).†These authors contributed equally to this work.‡Present address: Faculty of Agronomy, Al-Fateh University, PO Box 13040, Tripoli, Libya.

SUMMARY

The GDP-D-mannose 3,5-epimerase (GME, EC 5.1.3.18), which converts GDP-D-mannose to GDP-L-galactose, is

generally considered to be a central enzyme of the major ascorbate biosynthesis pathway in higher plants, but

experimental evidence for its role in planta is lacking. Using transgenic tomato lines that were RNAi-silenced

for GME, we confirmed that GME does indeed play a key role in the regulation of ascorbate biosynthesis in

plants. In addition, the transgenic tomato lines exhibited growth defects affecting both cell division and cell

expansion. A further remarkable feature of the transgenic plants was their fragility and loss of fruit firmness.

Analysis of the cell-wall composition of leaves and developing fruit revealed that the cell-wall monosaccharide

content was altered in the transgenic lines, especially those directly linked to GME activity, such as mannose

and galactose. In agreement with this, immunocytochemical analyses showed an increase of mannan labelling

in stem and fruit walls and of rhamnogalacturonan labelling in the stem alone. The results of MALDI-TOF

fingerprinting of mannanase cleavage products of the cell wall suggested synthesis of specific mannan

structures with modified degrees of substitution by acetate in the transgenic lines. When considered together,

these findings indicate an intimate linkage between ascorbate and non-cellulosic cell-wall polysaccharide

biosynthesis in plants, a fact that helps to explain the common factors in seemingly unrelated traits such as

fruit firmness and ascorbate content.

Keywords: GDP-D-mannose 3,5-epimerase, ascorbate, cell wall, tomato fruit, Solanum lycopersicum.

INTRODUCTION

Plants exhibit tightly regulated variations in vitamin C

(or ascorbate) content across plant species and between

tissues. No viable ascorbate-less mutant has ever been

reported, indicating that, as in animals, it is of crucial

importance in plants. Ascorbate is a major antioxidant that

ensures protection of plant cells against reactive oxygen

species (ROS) generated by physiological processes as well

as by biotic and abiotic stresses. In addition, ascorbate has

been implicated in the regulation of key developmental

processes involving cell division and cell expansion (Smir-

noff, 2000). A possible link between ascorbate and plant

growth control is the interconnection between the major

ascorbate biosynthetic pathway first described by Wheeler

et al. (1998) and cell-wall biosynthesis (Figure 1). Formation

ª 2009 The Authors 1Journal compilation ª 2009 Blackwell Publishing Ltd

The Plant Journal (2009) doi: 10.1111/j.1365-313X.2009.03972.x

of GDP-D-mannose is the initial step in the pathway of

ascorbate biosynthesis, and GDP-D-mannose is also a

known precursor for the synthesis of D-mannose, L-fucose

and L-galactose, and therefore for hemicelluloses such as

the (galacto)glucomannans and for the pectin rhamnoga-

lacturonan II (Reiter and Vauzin, 2001). It is worth noting that

the galactose residues in the (galacto)glucomannan are D-

galactose. However, the biosynthesis and function in plants

of these non-cellulosic cell-wall polysaccharides remain

poorly understood (Carpita and Gibeaut, 1993).

GDP-D-mannose 3,5-epimerase (GME, EC 5.1.3.18) pro-

duces GDP-L-galactose from GDP-D-mannose, and therefore

represents the intersection between L-ascorbate and cell-

wall polysaccharide biosynthesis. GME is the most highly

conserved protein involved in ascorbate biosynthesis (Wolu-

cka and Van Montagu, 2007), and has attracted considerable

interest in recent years. In addition to GDP-L-galactose, GME

releases another epimerization product, GDP-L-gulose,

which is considered to be a novel intermediate in an

alternative pathway of ascorbate biosynthesis in plants

(Wolucka and Van Montagu, 2003). GME may also regulate

ascorbate synthesis under stress conditions, and adjust the

balance between ascorbate and cell-wall monosaccharide

biosynthesis (Wolucka and Van Montagu, 2003). Thus, it has

been hypothesized that, in association with VTC2 (GDP-L-

galactose phosphorylase), the enzyme catalysing the sub-

sequent step of ascorbate biosynthesis, GME constitutes a

control point for regulation of the ascorbate pathway in

plants (Laing et al., 2007; Wolucka and Van Montagu, 2007).

However, experimental evidence in support of these hypoth-

eses is still lacking, possibly because knockout mutation of

the single GME gene identified so far in Arabidopsis is lethal.

Here we report that RNAi silencing of GME in tomato

effectively reduces ascorbate content in the plant, leading to

ROS accumulation, leaf bleaching and developmental

defects. We further show that transgenic plants display

strong alterations in cell-wall composition, with a consider-

able impact on plant and fruit mechanical properties. The

results reveal a crucial role for GME in the regulation of

ascorbate biosynthesis, give new insights into hemicellu-

lose biosynthesis in plants, and demonstrate the intimate

link between plant ascorbate and non-cellulosic cell-wall

polysaccharide metabolism.

RESULTS

Ascorbate content, regulation of ascorbate biosynthesis

and plant phenotype are altered in GME-silenced plants

Using a CaMV 35S promoter-controlled RNAi strategy tar-

geting a DNA fragment common to the two GME genes

found in tomato (SlGME1 and SlGME2), we generated 15

independent tomato primary transformants (P35S:SlgmeRNAi

lines). Of these, three lines showing moderate (L-70) to

severe (L-66 and L-108) phenotypic changes were selected

for further analyses. Corresponding T1 homozygous plants

displayed a significant reduction in both SlGME1 and

SlGME2 transcript and protein abundances (Figure 2a,b and

1 2

GDP-L-Galphosphorylase (GGP)

GDP-L-Gul

NAD

GMP

PPi

GTP

D-Glc

D-Man-1P

GDP-D-Man

GDP-L-Gal

GDP-L-Fuc

L-Gal 1-P

L-Gal

L-GalL

L-Ascorbate

GDP-D-Man PPase (GMP)

NADH

Cyt cox

Cyt cred

L-GulL

L-Gal 1-Pphosphatase (GP)

L-Gal DHase (GDH)

L-GalLDHase (GalLDH)

Cell wall & glycoproteinsGlucomannansGalactoglucomannansRhamnogalacturonan II

GDP-D-Man 3,5-epimerase (GME)

3

4

0

0,2

0,4

0,6

GME1

GME2

GMP1

GMP2

GMP3

GMP4GGP

GP1GP2

GDH

GalLDH

Leaf20 DPA fruit

Rel

ativ

e m

RN

A a

bu

nd

ance

(a)

(b)

Figure 1. L-ascorbic acid biosynthesis in plants and expression of ascorbate-

related genes in tomato.

(a) In the ascorbic acid pathway, the carbon skeleton (D-Man 1-P) is

synthesized from D-glucose via hexose phosphate intermediates. At the level

of the GDP-D-mannose 3,5-epimerase step, three GDP hexoses are precursors

of cell-wall and glycoprotein biogenesis. GDP-L-fucose is converted from

GDP-D-mannose via two reactions catalysed by GDP-D-mannose dehydratase

(1) and GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase (2). The

pathway that uses GDP-L-gulose as a precursor has not been elucidated (3);

activity of L-gulonolactone oxidase/dehydrogenase (4) has been found to exist

in plants but is not totally characterized.

(b) The relative abundances of ascorbic acid-related mRNAs were determined

in young leaves and fruit at 20 DPA in control plants. Data were obtained by

real-time RT-PCR normalized against Actin1, and are expressed as a percent-

age of the control.

2 Louise Gilbert et al.

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), doi: 10.1111/j.1365-313X.2009.03972.x

Table S1). The most severe reduction was observed in lines

L-108 and L-66. However, transcript abundance was not

significantly different between these two lines. Ascorbate

content was reduced by 40–60% in young fully expanded

leaves and by 20–40% in fruits at 20 days post-anthesis

(DPA) from P35S:SlgmeRNAi lines (Figure 2c). Leaf labelling

experiments with [U-13C6]-glucose highlighted the reduced

capacity of the transgenic plants to produce ascorbate, as

the flux of label into ascorbate was significantly decreased in

lines L-108 and L-66 (Figure 2d). Importantly, microarray

analysis of 20 DPA fruit identified GDP-L-galactose phos-

phorylase (VTC2) among the more than twofold up-regu-

lated genes in line L-108 (Table S2). The results of transcript

expression profiling using TOM1 tomato microarray in the

P35S:SlgmeRNAi transgenic and control plants are accessible

at http://terry.bordeaux.inra.fr:8080/files/. Quantitative RT-

PCR analysis further confirmed that regulation of ascorbate

biosynthesis was altered in both transformants. The tran-

script abundance of the genes of the upper part of the

pathway encoding GDP-L-galactose phosphorylase (SlGGP)

and GDP-D-mannose pyrophosphorylase (SlGMP) was

much increased (Figure 2e). In contrast, the last two steps

of the pathway, catalysed by L-galactose dehydrogenase

(SlGDH) and by L-galactono-1,4-lactone dehydrogenase

(SlGalLDH), were unaffected. The results were not as clear

for L-galactose-1-P phosphatase (SlGP).

Viable T0 plants with a stronger reduction in ascorbate

content of 75–85% were also obtained. These plants showed

extreme phenotypes, including very stunted growth and leaf

bleaching (Figure 3a), but were excluded from further anal-

yses as they did not produce flowers. The selected lines had

milder visual phenotypes. The most altered T1 homozygous

plants (lines L-108 and L-66) showed a 25% reduction in

chlorophyll content and a corresponding 40–60% decrease

in photosynthetic rate (Figure 3b). The leaves of transgenic

plants were also very sensitive to 100 lM methyl viologen,

indicating a lower ROS-scavenging activity (Figure 3b). As

perhaps expected, modification of the antioxidant status of

the plant also had an effect on the plant transcriptome. A

group of stress and redox signalling genes, including

thioredoxin and catalase, was up-regulated in developing

L-108 fruit (Table S2), and other up- or down-regulated

genes involved in hormonal signalling and signal transduc-

tion may have possible roles in stress responses and/or

plant growth control (Table S2).

Growth defects of GME-silenced lines are not rescued

by ascorbate supplementation

In the greenhouse, it soon became apparent that growth

of the aerial parts of the plant and fruit size were reduced in

RNAi-GME

Leaf Fruit

SlGME2

0

1

2

3

4

5

Rel

ativ

eS

lGM

E1

and

SlG

ME

2m

RN

A a

bund

ance

Leaf Fruit

SlGME1

0

1

2

3

4

5

ControlLine-70Line-66Line-108

GME1 GME2 GME1 GME2

GMP1GMP2

GMP3GMP4

GGPGP1

GP2GDH

GalLDH

Control Line-66 Line-108

0

1

2

3

Rel

ativ

e m

RN

A a

bund

ance

D-g

lc-6-

P

D-m

an-1

-P

GDP-D-m

anGDP-L

-gal

L-gal

L-

L-

gallac

tone

AsA

L-gal-

1-P

Fruit

Leaf

12.8 ± 1.7*12.4 ± 2.3*17.4 ± 2.8* 21.5 ± 2.3

23.6 ± 3.4*21.5 ± 4.5* 30.8 ± 3.4*48.9 ± 2.3

Line-108Line-66Line-70Control

0.014 ± 0.006*

0.047 ± 0.0080.015 ± 0.003* 0.532 ± 0.065*

1.013 ± 0.032

0.881 ± 0.033*L-66L-108Control

Label incorporation (µmol C1 equivalents.g DW-1.h-1)

Gal (Glc) AsA (Gal)

Total ascorbate content (µmol.g FW-1)

(a)

(b)

(c)

(d)

(e)

Figure 2. Gene expression, protein and ascorbate content in P35S:SlgmeRNAi

transgenic and control plants.

(a) The relative abundances of SlGME mRNAs were determined in young

leaves and fruit at 20 DPA in P35S:SlgmeRNAi plants (lines L-66, L-70 and L-108)

compared to control plants. Data were obtained by quantitative RT-PCR

normalized against Actin1.

(b) Comparison of specific regions of silver-stained two-dimensional gel

electrophoresis gels of the proteome of fruit pericarp at 20 DPA from

P35S:SlgmeRNAi line L-108 and control plants using 100 lg of proteins (see

Table S4).

(c) Ascorbate content in young leaves and fruit at 20 DPA from P35S:SlgmeRNAi

lines L-66, L-70 and L-108 and control (wild-type) plants.

(d) Labelling experiment with [U-13C6]-glucose in tomato leaflets. Data

(mean � SE, n = 3) are the calculated redistribution of label from glucose

(Glc) to galactose (Gal) and from galactose to ascorbate (AsA) after 4 h.

(e) Relative mRNA abundance for ascorbate-related genes in leaves of lines L-

66, L-108 and control plants. The ascorbic acid pathway is shown below the

graph. Data were obtained by real-time RT-PCR normalized against Actin1,

and are expressed as a percentage of the control.

Data represent means � SD of six individual plants per line, and asterisks

indicate values that are significantly different from those of the control (t test,

P < 0.05).

SlGME silencing, ascorbate and cell wall in tomato 3


the P35S:SlgmeRNAi L-66 and L-108 lines (Figure 4a). As

expected, ascorbate content was reduced in the seedlings,

although to a lesser extent than observed previously in

mature leaves (Figure 2c). This discrepancy can easily be

explained by the differences in environmental conditions

(greenhouse or growth chamber) and the tissues analysed

(mature leaves or young seedling). A detailed analysis of

fruit pericarp found a significant reduction in both cell

number and cell size in these lines (Figure 4a). In order to

investigate to what extent the growth defect observed in

the P35S:SlgmeRNAi plants was related to depletion of the

ascorbate pool, we germinated P35S:SlgmeRNAi (L-108) and

control seeds on L-ascorbate- or L-galactose-supplemented

media. With both compounds, the ascorbate content of

the transgenic seedlings was restored to values similar to

that of the control plants. However, supplementation failed

to enhance plant growth, and seedling height and fresh

weight remained significantly lower than that of the control

(Figure 4b), suggesting that plant growth defects cannot be

solely attributed to the reduced ascorbate biosynthetic

capacity of the P35S:SlgmeRNAi plants.

The GME-silenced lines have increased fragility

In addition to the visual phenotypes, P35S:SlgmeRNAi plants

appeared to be very fragile in the greenhouse. Ripe fruits

were found to be soft when picked, and stems were easily

broken when manipulated. Analysis of this trait in line L-108

using a straightforward system demonstrated that the force

necessary to break young stems (sucker shoots) was five

times lower in transgenic plants than in control plants (Fig-

ure 5). The fruit firmness of both young developing fruits (20

DPA) and orange (ripening) fruits was also considerably re-

duced (Figure 5). Interestingly, when the humidity of the

greenhouse decreased to below 60%, the fragility phenotype

of the plants was no longer detected. Because plant rigidity

is largely related to water potential (ww) and turgor pressure

(wp), this aspect was investigated in the leaf. The water

potential of transgenic leaves (L-108) was reduced by 50%,

but no significant changes were observed for osmotic

P35S:SlgmeRNAi line-108 P35S:SlgmeRNAi line-66

Line-108Line-66

Control 16.2 ± 2.39.8 ± 2.1 *6.3 ± 1.8 *

Chlorophyll(mg g FW–1)

Photosynthesis(nmolCO2 µmol photon–1)

1.43 ± 0.16 1.09 ± 0.13 * 1.05 ± 0.21 *

Paraquat effect(% chlorophyll loss)

46.5 ± 2.867.9 ± 4.6 *76.5 ± 6.5 *

(a)

(b)

Figure 3. Effects of oxidative stress on photosynthesis and chlorophyll

content in tomato leaf.

(a) Eight-week-old P35S:SlgmeRNAi plants that were severely affected, with a

strong bleaching phenotype.

(b) Photosynthesis was assayed in mature leaves from four P35S:SlgmeRNAi

plants of each line and the control at several light intensities, and chlorophyll

content was measured. Oxidative stress induced by methyl viologen (Para-

quat) was assayed on leaf discs incubated at 25�C for 18 h under continuous

light with 0.1 mM methyl viologen or without methyl viologen in MS medium.

Bleaching was quantified as the percentage chlorophyll loss after 18 h

treatment. Data are means � SE (n = 6). Asterisks indicate values that are

significantly different from those of wild-type plants (t test, P < 0.05).

Control L-66 L-108

Line-108Line-66ControlFruit diameter (mm) 22.2 ± 2.3 16.1 ± 2.1*15.7 ± 3.2*Pericarp (mm)Cell size (µm2)No. of cell layers

0.98 ± 0.18.8 ± 1.2

15.9 ± 1.1

0.69 ± 0.1*6.1 ± 1.0*

12.9 ± 0.9*

0.60 ± 0.1*4.9 ± 2.1*

12.7 ± 1.1*

Control Line-108

MS + GalM S + AsAMSMS

Height(cm)

6.8 ± 0.8

4.7 ± 1.0 *5.1 ± 1.0 *5.2 ± 1.1 *

Fresh weight(mg)

Total AsA(µmolg–1FW)

77 ± 4

64 ± 1 *60 ± 1 *

61 ± 2 *

1.30 ± 0.10

0.97 ± 0.14 *1.19 ± 0.18 1.25 ± 0 .19 *

Line-66

Line-108

Control

1.10 ± 0.03 *1.70 ± 0.13 *1.59 ± 0.19 *

53 ± 4 *57 ± 3 *

60 ± 2 *

4.0 ± 0.5 *3.9 ± 0.7 *2.9 ± 0.5 *

6.8 ± 0.36.0 ± 0.5

79 ± 674 ± 8

1.28 ± 0.112.06 ± 0.20

MSMS + GalMS + AsA

MSMS + GalMS + AsA

MSMS + GalMS + AsA

Control L-70 -108 -66

(a)

(b)

Figure 4. Growth of plantlets and fruits of P35S:SlgmeRNAi transgenic and

control plants.

(a) Plantlets at 30 days after sowing and sections of 20 DPA fruit pericarp from

P35S:SlgmeRNAi lines L-66, L-70 and L-108 and the control. Scale bars = 200 lm.

Several fruit traits were measured. Cell size values correspond to the mean

value for several calculations of cell number per surface unit (mm2) as

described by Cheniclet et al. (2005). All data are means � SD of a total of 10

fruits from six individual plants.

(b) Seedlings at 10 days after sowing of P35S:SlgmeRNAi line L-108 and the

control on MS medium, or MS supplemented with 1 mM L-galactose (Gal) or

0.25 mM L-ascorbic acid (AsA). Seedling height and weight at 10 days after

sowing and total ascorbate at 15 days after sowing were measured. Data are

means � SD of 20 individual plantlets. Asterisks indicate values that are

significantly different from those of the control (t test, P < 0.05).



potential (ws). Together with data from GC-MS metabolome

analyses of 20 DPA fruit indicating that the most significant

metabolic changes observed in P35S:SlgmeRNAi plants were

linked to central and cell-wall-related metabolism (Table S2),

these results suggested that the plant fragility and soft fruit

traits are possibly linked to modifications of cell-wall poly-

saccharides.

Modification of non-cellulosic cell-wall polysaccharides

Cell-wall composition analyses of the P35S:SlgmeRNAi lines L-

66 and L-108 performed on alcohol-insoluble residue (AIR)

indicated that wall preparations of both stem and 20 DPA

fruit were enriched in mannose and were depleted in

galactose relative to control plants (Figure 6 and Table S3).

The content of other monosaccharides was not altered, with

the exception of arabinose, which declined in the stem, and

rhamnose and uronic acids, which increased in the fruit. The

contect of other sugars did not change significantly. Fucose,

which is synthesized from GDP-D-mannose and is usually

absent in Solanaceae cell-wall polysaccharides, was present

at trace level in the L-108 stem. Interestingly, cellulose was

not affected, but hemicelluloses and starch were signifi-

cantly reduced by about 30% in the stem and fruit of GME-

silenced lines (Table S3). Given these changes in cell-wall

monosaccharide composition, transgenic plant cell walls

were most likely enriched in (galactogluco)mannans. Fluo-

rescence microscopy observations of L-108 using anti-b-1,4-

D-mannan antibodies (Handford et al., 2003) clearly showed

increased cell-wall labelling of both stem (Figure 7e,f) and

fruit (Figure 7a,b). The reduction of labelling of partly methyl

esterified homogalacturonan and rhamnogalacturonan I

domains of pectin by JIM7 and LM5 antibodies, respectively

(Jones et al., 1997; Clausen et al., 2003), in transgenic stems

(Figure 7g–j) also suggested alterations in both rhamnoga-

lacturonan I galactan side chains and methyl esterification of

pectins. However, no differences in LM5 labelling were ob-

served in the fruit (Figure 7c,d). Fine structural changes of

(galactogluco)mannans were further investigated by 1,4-b-

mannanase hydrolysis of the alcohol-insoluble residue and

by MALDI-TOF/MS fingerprinting (Lerouxel et al., 2002;

Quemener et al., 2007). The proportion of oligosaccharides

comprising three to five hexoses was significantly increased

in L-66 and L-108 plants, mostly in the fruit (Figure 6 and

Table S3). In addition, the acetyl esterification of these

(galactogluco)mannan oligosaccharides was substantially

modified. Taken together, these results indicate that GME

silencing induced an increase in cell-wall mannose content

linked to the synthesis of specific (galactogluco)mannan

structures with modified acetyl esterification. These new

structural features may confer different functional properties

to this hemicellulose and to the cell walls, and thus may

–40 –30 –20 –10 10 20 30 40

Stem(2.3 ± 0.2)

(2.8 ± 0.1)

(24.5 ± 0.7)

(0.1 ± 0.02)

(6.6 ± 0.9)(8.4 ± 1.1)

(4.5 ± 0.3)(30.1 ± 2.7)

(1.2 ± 0.1)

(26.0 ± 2.5)(4.0 ± 0.03)

(5.1 ± 0.9)(8.4 ± 0.9)(5.1 ± 0.9)

(5.7 ± 0.4)

Variation relative to the mean control value (%)

20 DPA fruit

–40 –20 20 40 60 80 100

GM4Ac2GM4Ac1GM3Ac2GM3Ac1

GM3

StarchHemicellulose

Cellulose

Uronic acidsGlucoseXylose

ArabinoseRhamnoseGalactoseMannose (1.4 ± 0.1)

(1.1 ± 0.1)

(18.3 ± 0.3)

(0.8 ± 0.2)

(8.4 ± 0.8)(9.1 ± 0.7)

(1.5 ± 0.1)(39.1 ± 2.8)

(0.3 ± 0.01)

(7.1 ± 1.4)(31.2 ± 2.4)

(3.8 ± 0.8)(8.8 ± 0.5)(5.7 ± 0.6)

(4.6 ± 0.2)

Figure 6. Analysis of cell-wall composition.

Change in the composition of the polysaccharide constituents of the cell wall in the stem and 20 DPA fruit from control plants and P35S:SlgmeRNAi lines L-66 (squares)

and L-108 (diamonds). The sugar content (weight %) of the AIR and fractions of glucomannan oligomers (GM) released by endo-mannanase were determined from

ions identified by MALDI-TOF/MS analysis. Their content is relative to the signal of the internal standard xyloglucan oligomer XXG (2 lg/0.5 mg of AIR). The

nomenclature used is GM3 for a glucomannan oligosaccharide with degree of polymerization 3 (three hexoses) with one (Ac1) or two (Ac2) acetyl ester groups. All

data are expressed as a percentage of the control according to the following formula (L)C)/C · 100, where L is the value for the transgenic tissue and C is that for the

control (Table S3). The vertical median axis represents the mean value of the control. Data represent means � SE (n = 6). Black symbols indicate values that are

significant different from those of the control (t test, P < 0.05).

–0.13 ± 0.1*

166 ± 61*

L-108–0.22 ± 0.1

849 ± 64

Control

ΨΨΨΨw

Breaking force (N)

–0.86 ± 0.1–0.91 ± 0.1

ΨΨΨΨs

Plants

ΨΨΨΨp (MPa)(MPa)(MPa)

0.73 ± 0.1 0.69 ± 0.1

Fruit 20 DPA

20.9 ± 5.6* 69.4 ± 6.5* L-10835.7 ± 10.980.1 ± 11.0Control

Stem

Leaf

Pressure (kPa)

Orange fruit

Figure 5. Fragility of the stem, fruit firmness, water (Ww) and osmotic (Ws)

potentials, and turgor pressure (Wp) of the leaf in P35S:SlgmeRNAi transgenic

line L-108 and control plants.

The fragility of young sucker shoots expressed as breaking force and the

firmness of 20 DPA and orange fruits (equivalent to the force necessary to

deform the fruit diameter by 3%) were measured as described in Experimental

procedures. Data represent means � SD (n = 30–40 fruits). Ww and Ws were

measured on leaves, and Wp was calculated according to the formula

Ww = Ws + Wp. Asterisks indicate values that are significantly different from

those of the control (t test, P < 0.05).



contribute to the alterations of plant and fruit mechanical

properties (fragility and firmness).

DISCUSSION

In this paper, we characterized the consequences of silenc-

ing expression of GME genes using the RNAi strategy. This

manipulation had a broad impact on plant and fruit devel-

opment, emphasizing both the important role of GME in

ascorbate biosynthesis as well as its pivotal function in plant

and fruit growth.

In tomato plants, GME is an important control point

between cell-wall and ascorbate metabolism (Figure 1). It is

noteworthy that repression of GME genes affects most

genes of ascorbate biosynthesis, especially that of

the downstream GGP tomato gene homologue of VTC2

(Figure 2 and Table S3). Indeed, GME and GGP transcripts

appear to be co-regulated in wild-type tomato (Figure 1b).

This tight transcriptional connection between ascorbate-

related genes encoding enzymes that share regulatory

properties is important. Therefore, our data confirm the

findings of Linster and Clarke (2008) that GGP (VTC2) plays a

major role in regulation of the L-ascorbate pathway. Sec-

ondly, while the existence of a VTC2 cycle involving GME

and GGP (Laing et al., 2007; Wolucka and Van Montagu,

2007) remains somewhat controversial at the catalytic level,

our results clearly support the suggestion that GME and

GGP share a crucial role in controlling L-ascorbate biosyn-

thesis in tomato. Further investigation is needed to fully

define the nature of this control, especially given the

proposed nuclear localization of VTC2 in Arabidopsis

(Muller-Moule, 2008).

In the P35S:SlgmeRNAi lines, the significant decrease in

GME expression and protein level reduced the capacity of

the plant to produce ascorbate (Figure 2) and impaired plant

development (Figure 4). Under normal growth conditions,

the size of the P35S:SlgmeRNAi plants and fruits correlated

perfectly with the ascorbate concentration (Figure 2), the

photosynthetic capacity of the plant, and its sensitivity to

oxidative stress (Figure 3). Given the accepted crucial role of

ascorbate in photoprotection (Foyer and Noctor, 2005),

these ascorbate-related phenotypes were perhaps expected.

However, there are also some differences between our

results and those previously reported. Dowdle et al. (2007)

concluded that, in vtc2 plants, growth is affected before

photoprotection as ascorbate concentration decreases, but

this sequence is not evident from our data. Furthermore,

in our previous work in tomato, in which we repressed

expression of GalLDH, the mitochondrially localized termi-

nal enzyme, the reduced organ growth resulted from neither

decreased ascorbate content nor impaired photosynthesis

but was most probably a consequence of the alteration of

mitochondrial respiration and function (Alhagdow et al.,

2007). Very little is known about the exact mechanisms by

which ascorbate regulates cell growth in plants (Smirnoff,

2000; Foyer and Noctor, 2005). Smirnoff hypothesized a

model whereby ascorbate acts as a redox buffer in the

cell-wall compartment with apoplastic ascorbate oxidase

activity and hydrogen peroxide signalling (Smirnoff, 2000).

Recently, Dumville and Fry (2003) postulated that, during

the first period of the tomato fruit ripening, ascorbate, in the

presence of H2O2 in the apoplast, could generate hydroxyl

radicals which contribute to fruit softening via solubilization

of polysaccharides such as pectins. More recently, Nunes-

Nesi et al. (2008) proposed that the interaction between

ascorbic acid and growth may involve ascorbate per se in

combination with photosynthesis and respiration processes.

(a) (b)

(c) (d)

(e) (f)

(g) (h)

(j)(i)

Figure 7. Scanning fluorescent micrographs of the stem and the pericarp of

20 DPA fruit from P35S:SlgmeRNAi transgenic line L-108 and control plants.

Fruit pericarp (a–d) and stem (e–j) tissue sections of control (a, c, e, g, i) and L-

108 (b, d, f, h, j) plants were embedded in resin and labelled with antibodies

against b-1,4-D-mannan (a, b, e, f), or LM5 (c, d, g, h) and JIM7 antibodies (i, j).

Observations were made under a fluorescent microscope after labelling with

a fluorescent-tagged secondary antibody. Scale bars = 100 lm.



Taken together, these data show that the link between

ascorbate and growth is far from well-established.

GME as a potential regulator of cell-wall composition

In the P35S:SlgmeRNAi plants, we have assumed that the

origin of the growth defects is most likely related to the

GME function. In plants, GDP-D-mannose and GDP-L-

galactose, the substrate and product, respectively, of GME,

are not only used for L-ascorbate synthesis but also for the

synthesis of cell-wall polysaccharides and protein glyco-

sylation (Smirnoff, 2000). In addition, GDP-D-mannose is

also precursor for GDP-L-fucose (Reiter and Vauzin, 2001).

GDP-D-mannose, GDP-L-galactose and GDP-L-fucose are

used to incorporate D-mannose, L-galactose and L-fucose

in cell-wall polymers. Supplementation of the tomato

P35S:SlgmeRNAi seedlings with L-ascorbate and L-galactose

rescued accumulation and ascorbate synthesis, but failed to

restore normal growth (Figure 4). This experiment demon-

strates that the growth delay of P35S:SlgmeRNAi seedlings is

independent of their reduced capacity to synthesize ascor-

bate, but is probably due to an alteration of cell-wall bio-

genesis (Baydoun and Fry, 1988). It is therefore tempting to

explain the growth deficiency of the P35S:SlgmeRNAi plants

by a key role for GME in cell-wall biogenesis. Analysis of

the cell-wall polysaccharides of P35S:SlgmeRNAi plants

revealed a significant increase in mannose and a decrease

in galactose content, but also changes for several other cell-

wall sugars such as arabinose, rhamnose, xylose and

uronic acids (Figure 6). Strong accumulation of (galactog-

luco)mannans in stem and fruit pericarp was also observed

(Figure 7). Many Arabidopsis mutants affected in cell-wall

biogenesis have been characterized (Farrokhi et al., 2006).

Among them, the embryo-lethal cyt1 mutant displayed

similar features to the P35S:SlgmeRNAi plants. The CYT1

gene encodes the GDP-D-mannose pyrophosphorylase en-

zyme. This mutation leads to arrest of the development of

the embryo at the heart stage, with reduced cellulose and

cell-wall mannose contents and lower ascorbate content

(Lukowitz et al., 2001). As with the tomato seedlings,

ascorbate supplementation of the cyt1 mutant does not

rescue the developmental phenotype. Lukowitz et al. (2001)

concluded that the growth arrest results from deficiency in

N-glycosylation, which affects cellulose biosynthesis.

Although no variation of the cellulose content occurred in

the P35S:SlgmeRNAi plants (Figure 6), slight changes in

protein glycosylation were observed in the most affected

lines (Figure S1). Hence, we cannot rule out a possible

alteration of N-glycosylation in the P35S:SlgmeRNAi plants,

which could affect essential mechanisms for the proper

folding, targeting and function of numerous cell-wall pro-

teins (Lerouge et al., 1998). In addition, as a vitamin,

ascorbate is an essential co-factor for numerous enzymes,

e.g. the prolylhydroxylase required for biosynthesis of cell-

wall hydroxyproline-rich glycoproteins (Sommer-Knudsen

et al., 1998). In addition to its role in N-glycosylation

(Lerouge et al., 1998) and in post-translational protein

modification for glycosylphosphatidylinositol membrane

anchoring (Fergusson, 1999), GDP-D-mannose is also the

precursor of glucomannan and galactoglucomannan, two

major hemicellulosic polymers of primary and secondary

cell walls. The increase in mannose resulting from blocking

of the GDP-D-mannose to GDP-L-galactose conversion, and

the decrease in galactose content, led to considerable

modification of the wall structure (Figures 6 and 7). GDP-D

3,5-epimerase produces GDP-L-galactose rather than the

D-galactose precursor used in synthesis of most cell-wall

polysaccharides including the pectin rhamnogalacturonan I

and the hemicelluloses xyloglucan and (galactogluco)

mannan. Cell-wall D-galactose is thought to originate from

epimerization of UDP-D-glucose (Seifert, 2004). Our results

(Figure 7) suggest that synthesis of the rhamnogalacturo-

nan I side chain in the stem could be down-regulated to

compensate for the increase in (galactogluco)mannan, as

a means of controlling cell-wall mechanical properties.

Indeed, pectic rhamnogalacturonan I galactan and arabinan

side chains can interact with cellulose, and appear to be

important in the control of the biophysical properties of

plants (Zykwinska et al., 2005). Moreover, the level of

acetylation of the (galactogluco)mannan-derived oligomers

is severely altered, particularly in fruits where the hemi-

cellulose content is higher (Figure 6). Mannans are mainly

known as major seed storage polysaccharides (Reid et al.,

2003; Edwards et al., 2004). They are also present in various

tissues such as siliques, flowers and stems in Arabidopsis

(Liepman et al., 2007), but their role is not clearly under-

stood. They are able to hydrogen bond to cellulose

(Whitney et al., 1998), and have been suggested to play a

structural role analogous to that of xyloglucans, introduc-

ing flexibility and forming growth-restraining networks with

cellulose (Schroder et al., 2004). Hence, in line with the

above proposition regarding the alteration of rhamnoga-

lacturonan I galactan side chains in the stem, an alteration

in the degree of O-acetylation of the (galactogluco)mannan

could also play a role in hemicellulose binding to cellulose

and thus cell-wall mechanical properties (Pauly et al., 1999).

Another important aspect is that reducing the synthesis of

L-galactose from GDP-L-galactose may impair synthesis of

rhamnogalacturonan II, which is a minor but essential

pectic component of the primary cell wall (Baydoun and

Fry, 1988). Although the rhamnogalacturonan II content and

composition were not investigated in our P35S:SlgmeRNAi

plants, it is noteworthy to draw a parallel with the fucose-

deficient Arabidopsis mutant mur1. The mur1 plants are

dwarfed and less flexible, as shown by the reduction of the

tensile strength required to break elongating inflorescence

stems (Reiter et al., 1993). Analysis of the cell-wall compo-

sition reveals that the two L-fucose molecules present in

rhamnogalacturonan II are replaced by L-galactose (O’Neill



et al., 2001). This simple substitution impairs the stability of

rhamnogalacturonan II, which partly contributes to reduc-

ing the overall wall strength (Ryden et al., 2003).

Clearly, silencing of GME expression has an impact on

cell-wall structure biogenesis. Nevertheless, it is not clear

whether this results from a direct effect or a structural

compensation of the GME repression on hemicellulose

biosynthesis. The striking examples of the mur1 and cyt1

mutants demonstrate how difficult it can be to define the

origin of the growth delay as well as the exacerbated fragility

of P35S:SlgmeRNAi plants (Figure 5). No cell-wall GME

mutant and ascorbate-deficient GME mutant have yet been

described in plants, presumably because such a mutation

would be lethal. In tomato plants, GME appears to play a

crucial role in both cell-wall and ascorbate metabolism.

Further investigation is required to define this control,

especially in fleshy fruit such as tomato, where it may

contribute to the taste (sugars and organic acids), texture

(cell wall) and nutritional value (vitamin C) of the fruit, which

are important in determining fruit quality.

EXPERIMENTAL PROCEDURES

Plant material

Cherry tomato plants [Solanum lycopersicum L. cv West Virginia106 (WVa106)] were grown in a greenhouse or in vitro culture asdescribed previously (Alhagdow et al., 2007). The RNAi-mediatedsilencing of tomato SlGME1 (SGN-U314898) and SlGME2 (SGN-U314369) genes was performed by stable transformation of tomato(Alhagdow et al., 2007) using a 510 bp DNA fragment correspond-ing to positions 89–598 of SlGME1 cDNA. Plants showing poly-ploidy or multiple insertion events were excluded from furtheranalyses. Cell number and size were measured in semi-thin sectionsof fruit pericarp at 20 days post-anthesis (DPA) (Alhagdow et al.,2007).

Measurement of plant fragility and fruit firmness

Sucker shoots of 3.5 � 0.5 mm diameter were rapidly cut to 7 cmlong (time < 30 sec) after harvest, and fixed on a flat support withadhesive tape (contact over 3 cm). To estimate the force necessaryto break the sucker, a tube was suspended 1 cm from the oppositeend of the sucker and filled with sand until rupture of the sucker. Thediameter of the sucker shoot was then precisely measured at thebreaking zone. Fruit firmness was measured using a Penelaup�

penetrometer (Serisud, Montpellier, France) as described by Chaıbet al. (2007).

Photosynthetic activity, water and osmotic potential

The photosynthetic activity of attached leaves was measuredusing a CO2 analyser with infrared detection (LCA3; AnalyticalDevelopment Corporation, http://www.analyticaldevelopment.com).Chlorophyll content was assayed as described by Wintermans andMots (1965). To analyse plant water status, a Scholander pressurechamber (PMS Instrument Company, Albany, USA, http://www.pmsinstrument.com) was used. Measurements were very rapidlycarried out (time < 1 min) on the 4th and 5th leaves from theapex at 25�C and 65–70% humidity. The osmotic potential wasmeasured on liquid recovered from the same squeezed leavesusing a freezing-point osmometer (model 13DR; Roebling).

Ascorbate content and oxidation stress tolerance

Leaf discs were incubated in Petri dishes with either water or 100 lM

methyl viologen (Paraquat; Sigma, http://www.sigmaaldrich.com/)under continuous light at room temperature (25�C). Leaf bleachinginduced by methyl viologen was quantified by measuring chloro-phyll content over the time course of incubation. Ascorbate contentwas measured as described previously (Stevens et al., 2006).

Labelling experiments with [U-13C6]-glucose

Leaflets (length 4–5 cm) were cut from fully photosynthesizingyoung leaves. The petiole was dipped in 1.5 ml Eppendorf tubescontaining 10 mM MES/KOH (pH 6.5) with 10 mM [U-13C6]-glucoseor unlabelled glucose. The incubation was carried out under con-tinuous light (400 lmol m)2 sec)1 photons) at 25�C. After the indi-cated time period, the leaflets were washed with water, the centralvein was removed, and the samples were frozen in liquid nitrogen.Extraction, analysis and calculation of the fractional enrichment ofmetabolite pools were performed as described by Roessner-Tunaliet al. (2004). The reaction rate from metabolic precursors throughintermediates to the end product was estimated by dividing theamount of label accumulating in the product by the calculated meanproportional labelling of the precursor pool (Tieman et al., 2006).

RNA, protein and metabolite analyses

RNA extraction and RT-PCR analyses using gene-specific primers(Table S4) were performed as described previously (Alhagdowet al., 2007). Expression analyses of ascorbate biosynthesis geneswere performed for four GDP-D-mannose pyrophosphorylase genes[SlGMP1 (SGN-U315592), SlGMP2 (SGN-U313112), SlGMP3(SGN-U313111) and SlGMP4 (SGN-U329408)], a GDP-L-galactosephosphorylase gene [SlGGP (SGN-U312646)], two L-galactose-1-phosphate phosphatase genes [SlGP1 (SGN-U345930), SlGP2(SGN-U317967)], an L-galactose dehydrogenase gene [SlGDH(SGN-U319047)] and an L-galactono-1,4-lactone dehydrogenasegene [SlGalLDH (SGN-U317657)]. Transcriptome analyses wereperformed on the 4th leaf and on 20 DPA fruit using TOM1 micro-arrays (Alhagdow et al., 2007). Protein profiling was performedusing total protein extracts from 20 DPA fruits by two-dimensionalgel electrophoresis, and protein identification by mass spectros-copy (MALDI-TOF, LC-MS/MS) as described by Faurobert et al.(2007). Metabolite extraction and GC-MS analyses of the samples ofleaflet and 20 DPA fruit were performed as described previously(Roessner-Tunali et al., 2003; Nunes-Nesi et al., 2005).

Cell-wall composition

Alcohol-insoluble residue (AIR) was produced from 50 mg dryweight of stem and 20 DPA fruit pericarp, and neutral sugars in theAIR were identified and quantified by GC as previously described(Quemener et al., 2007). The starch, hemicellulose and cellulosecontents refer to the weight percentage of glucose in AIR releasedafter amylase treatment (McCleary et al., 1997), acid hydrolysiswithout pre-hydrolysis, and acid hydrolysis after pre-hydrolysis,respectively. The starch glucose content was subtracted from theacid hydrolysates, whereas hemicellulose glucose was subtractedfrom the acid hydrolysate with pre-hydrolysis to obtain the yield ofglucose from cellulose. Enzymatic hydrolysis of (galactog-luco)mannans was performed on dried and re-ground AIRs using1,4-b-D-mannanase (0.2 U), and the hydrolysate was analysedby MALDI-TOF/MS fingerprinting (Lewandrowski et al., 2005;Quemener et al., 2007) on a M@LDI LR spectrometer (Waters,http://www.waters.com). For immunofluorescent labelling, tissueswere embedded in white acrylic resin (Leboeuf et al., 2005).



Semi-thin sections (1 lm thick) were cut on an RMC MT 7000ultramicrotome (Leica Microsystems GmbH, Wetzlar, Germany,http://www.leica-microsystems.com), and labelled with antibodiesagainst partly methyl esterified homogalacturonan (JIM7), pecticrhamnogalacturonan I galactan side chains (LM5) and b-1,4-D-mannan as described previously (Guillemin et al., 2005).

ACKNOWLEDGEMENTS

We thank C. Cheniclet and M. Dieuaide-Noubhani for technicalassistance, P. Lerouge (University of Rouen, France) for providingantibodies against b-1,2-xylose and a-1,3-fucose residues, andBenoıt Valot (INRA Le Moulon, France) for the MS/MS proteinidentification. Part of the work was performed using equipmentfrom the Biopolymers-Interactions-Structural Biology platform(INRA, Nantes, France). This work was supported by grants from theLibyan government (M.A.) and the Region Aquitaine (L.G.), and wasfunded by Region Aquitaine/Midi-Pyrenees cooperative program,the INRA AgroBi-VTC Fruit project and the France–Germany–Spaintrilateral project GENMETFRUQUAL, and was performed under theauspices of the EU SOL Integrated Project FOOD-CT-2006-016214.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. SDS–PAGE and affinoblot analysis of soluble proteinextracts of control and P35S:SlgmeRNAi transgenic lines.Table S1. Two-dimensional gel electrophoresis analysis of 20 DPAfruit from P35S:SlgmeRNAi and control plants.Table S2. Gene expression and metabolic profiles in 20 DPA fruit ofP35S:SlgmeRNAi versus control plants.Table S3. Polysaccharide constituents of the cell walls of stem and20 DPA fruit from P35S:SlgmeRNAi and control plants.Table S4. PCR primers used to amplify specific regions of genes ofinterest.Please note: Wiley-Blackwell are not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.

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