Proteome analysis of organoleptic quality in tomato

C. Mihr, M. Faurobert, J.P. Bouchet, M. Causse

INRA Avignon, Unité de Génétique et Amélioration des Fruits et Légumes, France

T. Pawłowski

Institute of Dendrology PAN, Parkowa 5, 62-035 Kórnik, Poland

L. Negroni

INRA Gif-sur-Yvette, Plateforme de Protéomique du Moulon, France

N. Sommerer

M. Rossignol

INRA Montpellier, Unité de Protéomique, France

Keywords:, Lycopersicon esculentum, fruit quality, QTL-NILs, 2D-gel electrophoresis, mass

spectrometry

Abstract

Organoleptic quality of tomato fruits involves among others aroma, taste, colour and

texture. To study the genetic bases of these traits, QTL analyses of a recombinant inbred

line (RIL) population created from an intraspecific cross between a cherry tomato line

and a common tomato line were carried out.

QTLs with large effects on fruit weight, locule number and sugar content were detected

in the distal 50 cM segment of chromosome 2. Near isogenic lines (NILs) were generated

to characterise this particular chromosomal region.

A proteomic approach was initiated to investigate characteristics involved in fruit

quality at the protein level. Total protein extracts of the tomato pericarp were separated

by two-dimensional gel electrophoresis. Two NILs differing in the concerned segment of

chromosome 2 as well as the two parental lines of the RIL population were compared

during several fruit developmental stages. As expected there are more differences

between the parental lines than between the NILs. Several proteins of interest were

analysed by MALDI-TOF or ESI-MS/MS and candidate genes for the above mentioned

characteristics were obtained. Some of these genes are well known to be linked to fruit

development processes but many of them are related to stress and defence response.

INTRODUCTION

Since years consumers have complained about the flavour of tomato fruit. In order to

further improve its organoleptic quality involving colour, taste, aroma and texture, molecular

markers were used to map QTLs of important quality traits in a recombinant inbred line

population (RIL). This population was developed by intraspecific crossing between a cherry

tomato line (Cervil) with remarkable overall aroma intensity and a common tomato line

(Levovil) with ordinary tasty fruits (Causse et al., 2002).

A total of 131 QTLs representing 38 traits were mapped in different regions of the

chromosomes 2, 3, 4, 8, 9 and 12. Fine mapping of a particular region of chromosome 2 was

carried out to separate three linked QTLs, one for fruit weight (different from fw2.2, Frary et

al. 2000), one for locule number and one for sugar content, within about 20 cM). Near

isogenic lines (F8VC, F8VL) differing in this region (Fig.1) were obtained by marker-assisted

selection (Lecomte et al., 2004).

The two parental lines (Cervil, Levovil) as well as the two NILs were compared by

proteome analyses of the fruit pericarp at two different developmental stages (14 DAP and

red-ripe). Proteins of interest were identified by mass-spectrometry (MALDI-TOF, ESI-

MS/MS). The aim of our work is (a) to show the suitability of two-dimensional gel

electrophoresis to differentiate between closely related genotypes like NILs and (b) to find

candidate genes/proteins involved in fruit quality and development.

MATERIAL AND METHODS

Tomato plants were cultivated in a greenhouse during spring season (January-July) in

a full randomised trial.

Total proteins of tomato fruit pericarp were extracted by a phenol-based method

adapted from Mihr and Braun (2003).

Proteins were separated by two-dimensional gel electrophoresis (2DE). Isoelectric

focusing was carried out with precast IPG-gels pH 4-7. Each gel was loaded with 100 µg of

protein, except for the NILs at stage 14 DAP for which 110 µg were used. Second

dimensional separation was performed by Glycin-SDS-PAGE according to Laemmli (1970).

Analytical gels were silver stained (Heukeshoven and Dernick 1986) and compared visually.

Visual analyses were confirmed by quantification of spot volumes (percentage of total spot

volume) with the Image Master Platinum software (Version 5.0). For spot identification by

mass spectrometry 1 mg of protein sample was used and gels were stained with coomassie

blue colloidal (Neuhoff et al., 1985, 1990).

Proteins of interest were identified by MALDI-TOF and ESI-MS/MS. Mass spectra

were analysed with MASCOT (MALDI-TOF) or with SEQUEST (ESI-MS/MS),

respectively, whereas the following databases were used: Mass Spectrometry protein

Sequence DataBase (MSDB, version 2002), Tomato Consensus Database of TIGR (LeGI,

version 9, release April 2003) and Plant Genome Database (PlantGDB) Lycopersicon

esculentum.

RESULTS AND DISCUSSION

Proteome analyses by two-dimensional gel electrophoresis have been successfully

used to study different genetic and physiological processes in plants (see for review

Thiellement et al., 1999, Cánovas et al., 2004). Except the work of Sarry et al. (2004), who

examined the protein composition of different grape berry varieties only few data are

available about fleshy fruits examined by 2DE.

We here present results of our investigations of tomato fruit quality by 2DE and show

that this approach gives access to protein polymorphism between closely related genotypes.

This might be an interesting fact because in Lycopersicon esculentum polymorphism on

molecular level is known to be very low (Miller and Tanksley, 1990) though the degree of

phenotypic variation is high.

Between 1300 and 1900 reproducible spots among the different genotypes were

detected. About 1400 spots in Cervil / Levovil at stage 14 DAP and 1800 at stage red-ripe

were retained for the computer assisted analysis. For comparing the NILs 1900 spots at stage

14 DAP and 1300 at stage red-ripe, respectively, were taken into account.

As expected the number of variable spots was higher between the parental lines than

between the NILs. Comparing the parental lines 55 of 1400 spots (3,9 %) were variable at 14

DAP, among them 44 spots were more intense in Cervil than in Levovil. At red-ripe 46 of

1800 spots (2,4 %) showed significant differences, 27 of these were more intense in Cervil

(Fig. 2). However the comparison of NILs showed a higher degree of variation at stage red

ripe (18 of 1800 spots, i.e. 1,4 %; Fig. 3) than at 14 DAP (14 of 1900 i.e. 0,7 %). In F8VC the majority of the varying spots had a higher volume than in F8VL (9 of 14 at 14 DAP and 11 of 18 at red-ripe, respectively).

Moreover it is remarkable that the extent of variation is not only related to the

genotype but also to the developmental stage. Since the detected variations at 14 DAP and

red-ripe are not identical, the comparison of genotypes at several stages gives access to

variations based on differential gene expression during fruit development.

The tomato genome size is about 1000 Mb and 1000 cM, so the expected number of

genes in 20 cM might be roughly 300-600. With the proteomic approach we pointed out 32

differentially expressed proteins between the NILs among a total about 1600 spots. Such a

result seems quite correct with regard to the resolution power of 2DE.

Only a part of the varying protein spots is yet identified by MALDI-TOF and ESI-

MS/MS (Table 1). These spots were all picked from Cervil gels. This is the reason why they

were all, except one, more intense in Cervil than in Levovil or in F8VC than in F8VL, respectively. All the 20 identified proteins corresponded to tomato protein sequences or to

tomato consensus sequences (TIGR). They might be involved in the following metabolic

pathways:

-fruit ripening (DNA-binding protein E4),

-production of aroma compounds (Lipoxygenase B),

-stress response (e.g. HSP, peroxidase),

-sugar metabolism (acid invertase),

-amino acid synthesis (beta-alanine synthase).

Only for spot 26 the type of variation in the NILs is coherent with that in the parental

lines. The other spots differing between the NILs do not vary between Cervil and Levovil.

Spot 26 was identified as 1,3 beta glucanase, an enzyme which is known to be involved in

pathogen defence and to have anti-fungal activity, but its expression was also reported during

the ripening of banana (Medina-Suárez et al., 1997).

Nevertheless it is a striking result that most of the identified proteins are related to

stress and defence response. Among them there is a high number of heat shock proteins

(HSPs), in particular small heat shock proteins (sHSPs). In general HSPs are involved in

protein folding and stress tolerance (Miernyk, 1999 and Sun et al., 2002). But especially

sHSPs also seem to participate in embryogenesis, germination, pollen development and fruit

ripening (Sun et al., 2002).

Others are known to be implicated in fruit development i.e. E4 which is an ethylene

regulated gene (Lincoln et al., 1987), whose exact function is unknown.

Another one is lipoxygenase B, a fruit specific enzyme. Lipoxygenases are involved in

the oxidation of polyunsaturated fatty acids. The resulting hydroperoxides are broken down

by other enzymes to produce volatile aroma compounds (Robinson et al., 1995).

Moreover all proteins mentioned in table 1 show a high degree of variation during fruit

development (not shown).

If and in which way the variation of these proteins is involved in fruit quality and/or

fruit development has to be further evaluated. Additionally non-identified spots are currently

analysed by MALDI-TOF and/or ESI-MS/MS.

All our data will be compared with other tomato tissues before being included in a

fruit specific protein database. At the same time the here presented genotypes are investigated

on the trancriptomic level. The results will be compared and candidate genes will be tested for

genetic and physical colocalisation with QTLs of chromosome 2.

ACKNOWLEDGEMENT

We gratefully thank Esther Pelpoir and Philippe Duffé for technical assistance.

Literature cited

Cánovas, F.M., Dumas-Gaudot, E., Recorbet, G., Jorrin, J., Mock, H.P., Rossignol M. 2004.

Plant proteome analysis. Proteomics 4(2): 285-298.

Causse, M., Saliba-Columbani, V., Lecomte, L., Duffé P., Rousselle P. and Buret, M. 2002.

QTL analysis of fruit quality attributes in fresh marked tomato: a few chromosome

regions control the variation of sensory and instrumental traits. J. Exp. Bot. 53: 2090-

2098.

Frary, A., Nesbitt, T.C., Frary, A., Grandillo, S., van der Knaap, E., Cong, B., Liu, J., Meller,

J. Elber, R., Alpert, K.B and Tanksley, S. D. 2000. fw2.2: A quantitative trait locus key to

the evolution of tomato fruit size. Science 289: 85-88.

Heukeshoven, J. and Dernick R. 1986: Silver staining of proteins. p.22-27. In: B. J. Radiola

(ed), Electrophoresis Forum. Technische Universität München, Munich.

Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of

bacteriophage T4. Nature 227: 680–685.

Lecomte, L. Saliba-Colombani, V., Gautier, A., Gomez-Jimenez, M.C., Duffé, P., Buret, M.

and Causse, M. 2004. Fine mapping of QTLs of chromosome 2 affecting the fruit

architecture and composition of tomato. Molecular Breeding 13: 1-14.

Lincoln, J.E., Cordes, S., Read, E. and Fischer R.L. 1987: Regulation of gene expression

ethylene during Lycopersicon esculentum fruit development. Proc. Natl. Acad. Sci. 84:

2793-2797.

Medina-Suárez, R., Manning, K, Fletcher, J., Aked, J., Bird, C.R., and Seymour, G.B. 1997.

Gene expression in the pulp of ripening bananas. Plant Physiol 115: 453-461.

Miernyk, J. A.1999: Protein folding in the plant cell. Plant Physiology 121: 695-703.

Mihr, C. and Braun, H.P. 2003. Proteomics in plant biology. p.409-416. In: P. M. Conn (ed),

Handbook of Proteomics. Humana Press, Totowa.

Miller, J.C. and Tanksley, S.D. 1990: RFLP analysis of phylogenetic relationships and genetic

variation in the genus Lycopersicon. TAG 80: 437-448.

Neuhoff, V., Stamm, R. and Eibl, H. 1985: Clear background and highly sensitive protein

staining with Coomassie Blue dyes in polyacrylamide gels: a systematic analysis.

Electrophoresis 6: 427-448.

Neuhoff, V., Stamm, R., Pardowitz, I., Arold, N., Ehrhardt, W. and Taube, D. 1990: Essential

problems in quantification of proteins following colloidal staining with Coomassie

Brilliant Blue dyes in polyacrylamide gels, and their solution. Electrophoresis 11: 101-

117.

Robinson, D.S., Wu, Z. Domoney, C. and Casey, R. 1995: Lipoxygenases and the quality of

foods. Food Chemistry 54: 33-43.

Sarry, J.E., Sommerer, N., Sauvage, F.X., Bergoin, A., Rossignol, M., Albagnac, G. and

Romieu, C. 2004: Grape berry biochemistry revisited upon proteomic analysis of the

mesocarp. Proteomics. 4:201-15.

Sun, W., van Montagu, M. and Verbruggen, N. 2002: Small heat shock proteins and stress

tolerance in plants. Biochimica and Biophysica Acta 1577: 1-9.

Thiellement, H., Bahrman, N., and Damerval C., 1999: Proteomics for genetic and

physiological studies in plants. Electrophoresis 20: 2013-2026.

Tables

Table1. Varying proteins already identified by mass spectrometry. C = Cervil, L = Levovil,

VC = F8VC, VL = F8VL,

Variation Function Functional category Identification

C < L beta alanine synthase amino acid synthesis MALDI-TOF

C > L probable calcium-binding protein stress, defence ESI-MS/MS

C > L DNA-binding protein E4 development MALDI-TOF

C > L small heat shock protein stress, defence ESI-MS/MS

C > L heat shock protein stress, defence MALDI-TOF

C > L small heat shock protein stress defence MALDI-TOF

C > L outer membrane lipoprotein stress, defence ESI-MS/MS

C > L gluthation peroxidase stress, defence MALDI-TOF

C > L acid invertase sugar metabolism MALDI-TOF

C > L lipoxygenase B flavour compound synthesis MALDI-TOF

C > L leucoanthocyanidin dioxygenase anthocyan synthesis ESI-MS/MS

C > L mitochondrial small heat shock protein stress, defence MALDI-TOF

C > L DS2 protein stress, defence MALDI-TOF

C > L heat shock protein stress, defence MALDI-TOF

C > L small heat shock protein stress, defence MALDI-TOF

C > L small heat shock protein stress, defence MALDI-TOF

C > L TS-1 protein not known MALDI-TOF

C > L / VC > VL 1,3 beta glucanase stress, defence MALDI-TOF

VC > VL probable oxidoreductase development ESI-MS/MS

VC > VL isocitrate dehydrogenase energy / amino acid synthesis ESI-MS/MS

Figures

Fig. 1: Distal segment of chromosome 2 and characteristic differences between the QTL-NILs

F8VC and F8VL.

suc2.1 (3cM)

18 19 20

22

GC274 26 TG454, OPB08-

1.5C

TG462* 30

CD035 31

TG521, TG493 32

TG266

TG484, TG191, TG527, GC276 37 TG463 GC240 39

Rad23 43

OPAE4-0.9C 45

TG337-C, TG518-C 48

52 53

H35M47-143L, H35M47-162C

fw2.3 (3cM)

LoNb2.1 (2cM)

F8VL F8VC

CERVIL

LEVOVIL

Fig. 2. Spots varying between parental lines Cervil and Levovil. Numbers indicate spots

which were already identified by mass spectrometry (Black arrows: spot more intense in

Cervil than in Levovil; white arrows: spot more intense in Levovil than in Cervil)

Fig. 3. Spots varying between near isogenic lines F8VC and F8VL. Numbers indicate spots

which were already identified by mass spectrometry. (Black arrows: spot more intense in

F8VC than in F8VL; white arrows: spot more intense in F8VL than in F8VC)

pH 4 pH 7

2288

8899

2266

114444

6666 6688

6633

114488

22

5588

SDS-PAGE

2266 4477 110022

6655

pH 4 pH 7

SDS-PAGE