Proteomic analysis of chicory root identifies proteins typically involved in cold acclimation

DATASET BRIEF

Proteomic analysis of chicory root identifies proteins

typically involved in cold acclimation

Herve Degand1, Anne-Marie Faber1, Nicolas Dauchot2, Dominique Mingeot3,Bernard Watillon3, Pierre Van Cutsem2, Pierre Morsomme1 and Marc Boutry1

1 Universite catholique de Louvain, Institut des Sciences de la Vie, Louvain-la-Neuve, Belgium2 Unite de Recherche en Biologie Cellulaire et Moleculaire Vegetale, Facultes Universitaires Notre-Dame de la Paix

Namur, Namur, Belgium3 Centre wallon de Recherches agronomiques, Biotechnology Department, Gembloux, Belgium

Received: September 18, 2008

Revised: January 21, 2009

Accepted: January 24, 2009

Chicory (Cichorium intybus) roots contain high amounts of inulin, a fructose polymer used as

a storage carbohydrate by the plant and as a human dietary and prebiotic compound. We

performed 2-D electrophoretic analysis of proteins from root material before the first freezing

period. The proteins were digested with trypsin and the peptides analyzed by MS (MALDI-

TOF/TOF). From the 881 protein spots analyzed, 714 proteins corresponded to a database

accession, 619 of which were classified into functional categories. Besides expected proteins

(e.g. related to metabolism, energy, protein synthesis, or cell structure), other well-represented

categories were proteins related to folding and stability (49 spots), proteolysis (49 spots), and

the stress response (67 spots). The importance of abiotic stress response was confirmed by the

observation that 7 of the 21 most intense protein spots are known to be involved in cold

acclimation. These results suggest a major effect of the low temperature period that preceded

root harvesting.

Keywords:

Abiotic stress / Anaerobic stress / Oxidative stress / Vegetative storage protein

Chicory (Cichorium intybus) is a biannual crop plant. During

the first year, the roots accumulate inulin-type fructans that

are soluble fructose polymers (mainly b (2-1)). They are

not digested and thus represent interesting dietary

compounds that cause significant changes in the composi-

tion of the gut microflora and can thus be considered as a

prebiotic [1].

At the end of the growing season, inulin is the most

abundant storage carbohydrate molecule in chicory root (up

to 20% of the root fresh weight). In chicory, fructans have

been proposed to serve as an energy reserve (chicory

contains little starch) and osmolyte source and also to play a

role during abiotic stress [1].

Although some major proteins involved in inulin meta-

bolism in chicory roots have been characterized [1] little is

known about other proteins typical of chicory roots. In

particular, as a biannual crop plant, chicory goes through a

period of cold and freezing temperatures, which induces

expression of proteins that protect the plant against cold

stress. Indeed, proteomic analyses of different species have

shown an increased abundance of several classes of proteins

[2]. Data concerning the proteins involved in the cold stress

response of chicory are limited. A few enzymes, e.g. fructan

1-exohydrolase [3] and pectin acetylesterase [4], have been

shown to be synthesized in chicory in higher amounts

during cold stress. However, no comprehensive analysis of

the proteins expressed in chicory roots has been performed.

In this report, we analyzed the proteome complement of a

chicory root (Sample P, Supporting Information Fig. 1)

harvested after a period with several days with a minimum

temperature below 51C and before a period with freezing

Abbreviations: 1-FFT, fructan:fructan 1-fructosyl transferase;

G3PDH, glyceraldehyde 3-phosphate dehydrogenase; VSP,

vegetative storage protein

Correspondence: Dr. Marc Boutry, Universite catholique de

Louvain, Institut des Sciences de la Vie, Croix du Sud 5–15, 1348

Louvain-la-Neuve, Belgium

E-mail: [email protected]

Fax: 132-10-473872

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Proteomics 2009, 9, 2903–2907 2903DOI 10.1002/pmic.200800744

temperatures. Because chicory is not a clonal variety, we

performed protein identification on a single root, thus

avoiding mixing roots with possibly different genotypes.

Root proteins were analyzed by 2-DE (Fig. 1). From this

gel, 881 protein spots were excised and digested with trypsin

and the peptides analyzed by MALDI-TOF/TOF. Identifica-

tion of the proteins (Supporting Information Fig. 2 and

Supporting Information Table 1) was performed in two ways.

We recently reported an EST database of 12 226 EST clones

from chicory, most of them from root tissues [5]; hence,

the MS data were compared with these EST data and the

proteins identified according to the EST annotation. A

parallel search was performed using the NCBI database

restricted to green plants. The majority of the spots were

identified by both methods. In most of these cases, the same

protein was identified although the accession numbers, and

sometimes the protein names, were different. This is

most likely due to the differences between these approaches:

comparison with the NCBI databases was based on

tryptic fragments scattered all along the protein while

comparison with the EST database was restricted to the

C-terminal region of the protein for which the sequence is

available. In some cases, identification did not match between

both methods. However, in most of these cases, inspection of

the MS data indicated that both proteins were identified by

each approach, but in a different order of score (only the

highest score is displayed in Supporting Information Table

1). This is a strong indication of the presence of two distinct

proteins in the same spot. Finally, 26% of the spots were not

identified, either because MS data were available for only a

very few peptides or because protein identification gave too

low a score.

Functional classification of the proteins was performed

according to [6] (Supporting Information Table 1). The

distribution of the identified proteins according to this clas-

sification is shown in Supporting Information Table 2. Not

surprisingly, the most highly represented categories were

‘‘metabolism’’ and ‘‘energy’’. Of these, the majority were

involved in sugar/polysaccharide metabolism or glycolysis,

respectively. The vast majority (98/105) of the proteins in the

category ‘‘Protein destination and storage’’ were subcategor-

ized into ‘‘Folding and stability’’ (49 proteins) or ‘‘Proteolysis’’

(49 proteins). Another well-represented subcategory was

‘‘Stress responses’’ (67 proteins).

The identity of 21 intense spots (labeled in Fig. 1) is

reported in Table 1. The most intense spots (452, 473, and

485 in Fig. 1) were very close together and were identified as

fructan:fructan 1-fructosyl transferase (1-FFT). This enzyme

is involved in chain elongation of inulin-type fructans, but

can also use sucrose as an acceptor for fructosyl units from

larger fructans, resulting in shorter polymers and an

increased fructan concentration. This might contribute to

cold resistance (discussed in [1]). Whether these spots

represent different products of different genes or different

post-translational modifications of the same protein cannot

yet be unambiguously determined, since the chicory

genome has not yet been sequenced. However, currently,

extensive investigation of 1-FFT polymorphism or chicory

EST library data mining does not substantiate the existence

of a 1-FFT multigenic family (N. Dauchot and P. Van

Cutsem, personal communication).

Another protein represented as several spots (circled in

Fig. 2A), including three intense ones (962, 966, and 968), is

the vegetative storage protein (VSP), which is thought to

Figure 1. 2-DE image of chicory root proteins.

Sample P (Supporting Information Fig. 1) was

analyzed as indicated in the Supporting

Information Materials and methods. The

most abundant spots are circled and their

identification number indicated. Their func-

tional category is defined in Table 1. The

dotted rectangles and the letters indicate the

regions shown in detail in the corresponding

panels in Fig. 2.

2904 H. Degand et al. Proteomics 2009, 9, 2903–2907


play a role in the storage of nitrogen. Several VSP spots

were previously identified on 2-D gel analysis and

considered as resulting from post-translational modifica-

tions of a unique protein [7, 8]. However, examination of the

chicory EST databases indicated that seven different

sequences presenting significant identity with VSP are

expressed in chicory root tissues (Supporting Information

Fig. S3). These seven sequences could all be detected in a

subset of the EST library obtained from a single root. Gene

polymorphism can therefore explain, in part, VSP protein

polymorphism.

Not surprisingly, intense spots were seen corresponding

to the cytoskeleton proteins actin (spot 567) and tubulin

(942) and to glycolytic enzymes: enolase (388), triosepho-

sphate isomerase (881), and glyceraldehyde 3-phosphate

dehydrogenase (G3PDH) (683 and 690). G3PDH is also

present in more than 20 spots of lower intensity (Fig. 2B).

Three major spots, superoxide dismutase (953), catalase

(425), and ascorbate peroxidase (874), are antioxidant

enzymes, particularly involved in the abiotic stress response.

The root sample used for proteomic analysis was subjected

to temperatures below 51C and it is known that cold accli-

mation induces the expression of anti-oxidative stress

proteins [9, 10]. Another abundant spot (983), nucleotide

diphosphate kinase B, can be added to this list of abiotic

stress response proteins. Although this enzyme is primarily

involved in the synthesis of nucleoside triphosphates other

than ATP, it also regulates the expression of genes involved

in the oxidative stress response [11].

An abundant cysteine proteinase inhibitor was also observed

(spot 989). The physiological role of this protein is not clearly

defined, but might involve the regulation of endogenous

proteases, programmed cell death, and the response to biotic as

well as abiotic stress. In particular, cold stress has been shown

to upregulate the expression of this protein [12, 13].

Other major spots include proteins known to be involved in

water stress, which is related to cold stress. A dehydrin protein

was identified as a major chicory root spot (829). Among water

stress proteins, dehydrins are believed to form complexes with

other macromolecules and protect them from freeze-induced

desiccation [14]. Another major spot (936) is similar to Medicagotruncatula cold acclimation-specific protein 15 (CAS15). This

protein contains the characteristic dehydrin K- and S-segments

and is highly cold-responsive [15]. Spot 938 is homologous to a

water stress-induced protein, which shows high identity with

CAS15. Although not major spots, other proteins related to

water stress were identified, such as the 25 kDa dehydrin-like

protein. Another dehydrin, ERD14, was identified in nine spots

(Supporting Information Table 1). In conclusion, of the 21

major spots analyzed, 7 were putatively involved in oxidative,

water or cold stress, all possibly related to cold acclimation. This

abundance is in agreement with the large number (67 spots) of

proteins classified as ‘‘Stress response’’ (Supporting Informa-

tion Table 1).

Focusing only on major spots can be misleading, as some

proteins might be scattered over a large number of spots,

either because they are encoded by a multigene family or

because they undergo various post-translational modifica-

tions. We have already mentioned the VSP and G3PDH

families. Other protein families represented by a large

number of spots are shown in Fig. 2. These include the

metabolic enzymes enolase (C), phosphoglycerate kinase

(D), methionine synthase (E), S-adenosylmethionine

synthase (F), and alcohol dehydrogenase (H). Alcohol

dehydrogenase together with pyruvate dehydrogenase and

lactate dehydrogenase both seen as several spots (Support-

ing Information Table 1), is probably involved in the

fermentation pathway, which occurs in root tissues subjec-

ted to low oxygen stress. Many HSPs were also identified,

with the HSP70 family being the most abundant (Fig. 2G),

and representing another example of proteins involved in

the stress response. HSP70 has been shown to be a mole-

cular chaperone during cold stress [16]. A final example of a

large protein family is the 14-3-3 protein cluster (Fig. 2I),

which are regulatory proteins involved in many different

signaling pathways, including the cold stress response [17].

In conclusion, investigation of the proteome complement of

chicory root led to the identification of major spots corre-

sponding to 1-FFT, the enzyme involved in inulin synthesis.

More surprising was the large number of proteins known to be

involved in the abiotic stress response, several of which were

Table 1. Identification of the most abundant spots

Spota) Protein identification Functional category

388 Enolase Glycolysis425 Catalase 3 Stress response452, 473, 485 Fructan:fructan 1-

fructosyltransferasePolysaccharide

metabolism519 Alcohol

dehydrogenaseshomologous

Fermentation

567 Actin Cytoskeleton683, 690 Glyceraldehyde 3-

phosphatedehydrogenase

Glycolysis

829 Dehydrin 2 Stress response874 Ascorbate peroxidase Stress response881 Triosephosphate

isomeraseGlycolysis

936 Cold-acclimationspecific protein 15

Stress response

938 Water stress-inducedprotein, putative

Stress response

942 Beta-7 tubulin Cytoskeleton953 Superoxide

dismutase [Cu–Zn]Stress response

962, 966, 968 Vegetative storageprotein

Storage

983 Nucleosidediphosphatekinase B

Nucleosidemetabolism andstress response

S989 Cysteine proteinaseinhibitor

Proteolysis

Proteomics 2009, 9, 2903–2907 2905


found in the most abundant protein spots or in clusters of more

than ten protein spots. This load might be related to the cold

temperatures before root harvesting. As a biannual species,

chicory has to adapt to the winter freezing period. Cold accli-

mation therefore occurs when temperature is still above the

freezing point.

This work was supported by a grant from the Ministere de laRegion wallonne-DGTRE, the Inter-university Attraction Poles,the Program-Belgian Science Policy, and the Belgian Fund forScientific Research.

The authors have declared no conflict of interest.

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Proteomic analysis of chicory root identifies proteins typically involved in cold acclimation