# The Proteomic Changes inCynara Cardunculus L. var. altilis DC Following the Etiolation Phenomena Using De Novo Sequence Analysis

Authors: Carmine Guarino; Luciana De Simone; Simona Santoro; Simonetta Caira; Sergio Lilla; Maria Grazia  Calabrese; Lina Chianese; Francesco Addeo
Journal: Journal of Botany
 (2010)

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## Abstract

Etiolation of vegetables is a complex phenomenon implying qualitative and quantitative protein changes. The 2-DE protein profile of green and etiolated fleshy stalk samples has shown great differences in the 4–7 pH and 10 to 250 kDa mass range.  Currently, a shortage ofCynara DNA or protein sequences has required the use of de novo sequencing and BLAST similarity searches for protein identification. Highlighted herein is an application of proteomics to the identification of proteins, which in a great part matched those from the Viridiplantae order. Quantitative  evaluation, statistical analyses, and MALDI-TOF MS characterization of the resolved spots in green and etiolated samples of C. cardunculus enabled us to identify 46 out of 60 spots, containing 21 spots included conserved Viridiplantae domains.
The etiolation process is discussed in light of the identification of some proteins involved in specific biochemical and metabolic pathways.


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## Body

## 1. Introduction

In dark-grown angiosperm plants, chlorophyll biosynthesis is arrested at the Pchlide stage (etiolation) due to the deficiency of a light-requiring oxidoreductase enzyme which naturally converts Pchlide to chlorophylls. In parallel, the natural content of fibre, bitter, and aromatic compounds declines in vegetables [1–4]. By and large, the edible portion of plants corresponds to etiolated foliated rosettes or young stalks of biennials or perennials. Transient etiolation of leaves and other tissues takes place naturally during spring development under light-deficit. The same phenomena can be reproduced by transferring plants to a dark chamber at optimal temperature for growth. During etiolation, various biochemical and anatomical processes occur, implying mainly an increased synthesis and accumulation of plastidial proteins. Little information had thus far been available on the expressed plant proteins during etiolation and subsequent deetiolation. A more detailed characterization of vegetable proteins using proteomics is needed for a more comprehensive view of the physiological changes associated to the etiolation process. A deeper understanding of the cellular events is highly desirable also in view of manipulations aimed to improve sensorial, nutritional, and shelf life features.The cultivated cardoon (Cynara cardunculus L. var. altilis DC) is one of the vegetables most subjected to etiolation. Cynara cardunculus L. is a diploid (2n=34) out crossing perennial species native to the Mediterranean basin and comprising the globe artichoke (var. scolymus L.), the cultivated cardoon (var. altilis DC), and the wild cardoon (var. sylvestris (Lamk) Fiori). Cynara cardunculusvar. sylvestrisis considered the wild ancestor of the globe artichoke and cultivated cardoon [5–7]. The latter has been considered one of the most  important perennial herbaceous cultivated plants mainly for the blanched leaf petioles, regarded in the Mediterranean area as a delicious speciality. Interest towards this crop is growing in European countries, especially in Spain, France, and Italy. In Portugal, Cynara cardunculus L. cardoon is traditionally used as a source of two aspartic proteinases, Cardosins A and B, for milk clotting in the manufacturing of cheese [8].Mass spectrometry- (MS-) based proteomics has been used extensively to explore the proteomes of various cells and organisms. MS technology is now being applied to protein identification as it offers high sensitivity and, when coupled with liquid chromatography, reduces sample complexity and increases high-throughput capability [9].Recently, high-resolution tandem mass spectrometers have enhanced robustness of peptide identification [10] based on matches between the measured mass of the peptides resulting from the protein digestion with high specificity proteases and the corresponding fragments in the protein databases. This approach is unable, however, to deal with proteomes or genomes that have not yet been sequenced.In such cases,de novo sequence determination and sequence similarity searches provide alternative approaches to address the protein identification.  Several software tools have been developed to deduce an amino acid sequence from an MS/MS spectrum [11, 12]. Furthermore, database search algorithms like BLAST [13, 14], recently optimised to deal with the sequence query length typical of mass spectrometric data [15, 16], have been utilized in cross-species identification of unknown proteins. However, the quality of tandem MS data and both de novo sequence interpretation and similarity search algorithms is critical, possibly leading to false positive or false negative matches. Such approaches have been successfully applied to protein identification from various organisms with unsequenced genomes [17, 18], including that of vegetables [19–22]. However, no rule has been established indicating the length, the number of fragmented peptides, or the de novo sequencing accuracy for a positive identification of homologous proteins. Moreover, the sequence percentage required for the identification of homologous proteins is also undetermined. Simulation has suggested that more than 80% of mammalian proteins could be positively identified on the basis of similarity between orthologous proteins of different species [23]. Therefore, for a successful characterization of a proteome by mass spectrometry, availability of the relative genome may not be a prerequisite.On the basis of this background, the main objective of this study was to investigate the changes of the proteins ofCynara cardunculus L. var. in relation to the etiolation process. Therefore, we compared global protein expression profiles of green and etiolated fleshy stalks of cultivated cardoon.


## 2. Materials and Methods

### 2.1. Plant Materials

Green and etiolated fleshy stalks of cultivated cardoon (Cynara cardunculus L. var. altilis DC) were used for this study. The system was realized in the Agricultural Company situated near to San Nicola Manfredi in Benevento district (Campania region) in March with direct dwell seeds in distant rows with 80 cm between each plant in plots of 3×3 meters. They have placed three seeds per hole, which leads to a single plant. In the period of May-June, the plants were in the rosette phase.  The plants reached their complete development at the end of summer-beginning of autumn (August-September). At the end of autumn (October), some green fleshly stalks were collected from three adjacent plants, quickly washed in sterile distilled water, freeze-dried, and stored at -80°C until analysis. Continuous dark for 25/30 days provoked plant etiolation as the leaves became more tender, white, and sweet as well. At the end of December, the etiolated fleshly stalks were collected and quickly washed in sterile distilled water, freeze-dried, and stored at -80°C until analysis.


### 2.2. Protein Extraction

Total proteins were extracted by a phenol-based method described by Saravanan and Rose [24]. The fleshy stalks were finely powdered in liquid nitrogen, using a mortar, and suspended in 7.5 mL of extraction buffer (700 mM sucrose, 500 mM Tris-HCl, pH 8.0, 50 mM EDTA, 100 mM KCl, 2% v/v β-mercaptoethanol, and 1 mM phenylmethylsulphonylfluoride),  by vortexing for 15 minutes at 4°C. After addition of an equal volume of phenol saturated 500 mM Tris-HCl, pH 7.5, the mixture was vortexed extensively for 10 minutes and then centrifuged at 10000 g for 15 minutes at 4°C. The upper phenol phase was removed and extracted twice with the extraction buffer. Proteins were precipitated from the phenol phase by the addition of five volumes of saturated ammonium acetate in methanol, overnight at -20°C. Precipitated proteins were centrifuged at 10000 g for 30 minutes.


### 2.3. 2-DE

Protein pellets were washed with ice-cold methanol (once) and ice-cold acetone (three times) dried and solved in IEF buffer (9 M urea, 4% w/v CHAPS, 20 mM DTT and 1% w/v ampholyte pH 3–10) (Bio-Rad, Hercules, CA, USA). Protein concentration was quantified using the BioRad protein assay using BSA as a standard. IPG strips (18-cm pH 4–7 Bio-Rad ReadyStrip), rehydratated  overnight with 315μL of IEF buffer containing 400 μg of total proteins. Proteins were focussed using a Protean IEF Cell (Bio Rad) at 12°C, applying 250 V (1 h), 1000 V (5 h), and 8000 V for a total of 52 kVh. Prior to the second dimension, the gel strips were reduced with 2% w/v DTT in 6 M urea, 30% w/v glycerol, 2% w/v SDS, 50 mM Tris-HCl pH 8.8, containing 0.01% w/v bromophenol blue for 20 minutes, followed by 20 minutes in the same buffer containing 2.5% w/v iodoacetamide. Electrophoresis in the second dimension was carried out using a Protean apparatus (BioRad) and 12% polyacrylamide gels (18cm×24cm×1mM) in 25 mM Tris (pH 8.3), 1.92 M glycine, and 1% w/v SDS, with 70 V (l35 mA) being applied for 16 h. Each sample was run in triplicate. Protein spots were annotated only if detectable in all gels [25].


### 2.4. Image Acquisition and Analysis

2-DE gels were stained with colloidal Coomassie G250 and scanned using a GS-800 calibrated densitometer (Bio-Rad). Image analysis was performed using the PDQuest software (Bio-Rad). Spot detection and matching between gels were performed automatically, followed by manual verification. After normalization of the spot densities against the whole-gel densities, the percentage volume of each spot was averaged for six different (three replicates of two samples) gels and the Student’st-test analysis (P<.01) was performed to find out statistically significant protein fold changes associated to etiolation. A twofold change in normalized spot densities was considered indicative of a differentially expressed component.


### 2.5. In-Gel Tryptic Digestion

Spots in the Coomassie Brilliant Blue G-250-stained 2-DE gel were excised manually and before being subjected to in-gel trypsinolysis as previously reported by Mamone et al. [26],  reduced (10 mM DTT in 100 mM NH4HCO3 allowing to react for 45 min, at 55°C) and alkylated with 55 mM iodoacetamide in 100 mM NH4HCO3 for 30 min at 25°C, in the dark. The resulting tryptic digest was dissolved in 40 μL of acetonitrile and 5% formic acid solution (1 : 1 v/v) and then the sample concentrated with vacuum centrifuge on medium heat to one-tenth initial volume for mass spectrometric analysis.


### 2.6. Reverse Phase Capillary Chromatography

Reverse phase capillary chromatography coupled with quadrupole time-of-flight tandem mass spectrometry was carried out using a Waters CapLC ternary pump system connected through a stream-select valve module to the nano-Z-spray source of the mass spectrometer. The sample was injected into the system through the CapLC autosampler. Solvent A and C consisted of water with 0.1% TFA, and solvent B consisted of acetonitrile with 0.1% TFA.The protein digest was preconcentrated and desalted on a precolumn cartridge packed with Atlantis dC18 resin at flow rate of 20μL/min-1 over 3 minutes. After switching to pumps A and B, a linear gradient from 5 to 70% of B over 45 min at a flow-rate of 300 nL min-1 (2% B/min) was applied to the precolumn cartridge and then used to elute the sample from the Atlantis dC18 resin analytical column (15cm×75 μm i.d.).


### 2.7. Mass Spectrometry

All mass spectra were acquired using a quadrupole-TOF hybrid mass spectrometer Waters Q-TOF II equipped with a nano-Z-spray source operating in positive ion mode. The ionization conditions used included a capillary voltage of 2.6 kV, source temperature of 80°C, and no nebulising gas. External calibration was applied to all data using Glu1-Fibrinopeptide B (Sigma-Aldrich) MS/MS fragments over a mass range from 50 to 1600 m/z.Survey scan acquisition was done on-line with capillary chromatographic separation; an initial TOF-MS scan was acquired over the mass range of 400–1600 m/z each second, with switching criteria for MS to MS/MS that included ion intensity (10 counts/s) and charge state (+2 to +4). MS/MS of precursor ion selected was acquired over the mass range of 50–1600 m/z. The collision energy used varied automatically according to the mass and charge state of the eluting peptides.


### 2.8. Nanoflow LC-ESI-MSMS

LC-ESI-MSMS analysis was performed using a Q-TOF Ultima mass spectrometer (Waters/Micromass UK Ltd., Manchester, UK) utilizing automated data-dependent acquisition. A nanoflow HPLC system (Waters CapLC (Waters/Micromass UK Ltd., Manchester, UK) was used for chromatographic separation of the peptide mixtures prior to MS detection. The peptides were concentrated and desalted on a precolumn cartridge packed with Atlantis dC18 resin (Waters CapLC, Waters/Micromass UK Ltd., Manchester, UK) and eluted at 200 nL/min by an increasing concentration of acetonitrile (2%/min gradient) onto an analytical column (15cm×75μm i.d.) also packed with Atlantis dC18 resin. An MS-TOF survey spectrum was recorded for 1 s. The five most abundant ions present in the survey spectrum were automatically mass-selected and fragmented by collision-induced dissociation (4 s per MSMS spectrum). The MSMS data were converted to a pkl file format using the Mass-Lynx 3.5 ProteinLynx software, and the resulting pkl file was searched against the NCBInr protein sequence databases using the Waters ProteinLynx GlobalSERVER v2.2.5 (PLGS).


### 2.9. Protein Identification

Acquired raw data were deisotoped and converted to pkl files using Waters ProteinLynx GlobalSERVER v2.2.5 (PLGS). Database search was performed against the tryptic peptides of entire taxonomy orderViridiplantae of NCBI database using a peptide tolerance of 0.1 Da and fragment mass tolerance of 0.2 Da, allowing a maximum number of two missed cleavages. Carbamidomethylated cysteine was set as fixed modification, while oxidized methionine was set as variable modification. Automod algorithm was used to determine additional posttranslational modifications and/or single amino acid substitution. The MS/MS spectra of peptides not identified by the previous two algorithms were submitted to the PLGS “de novo” sequence algorithm and the sequences obtained were BLAST searched against of NCBI Viridiplantae database using the same software tool. Alignments parameters were adjusted to search for a short input sequence, that is, PAM30MS matrix and expected threshold set to 1000. Redundant database search and de novo sequence alignments match several homologous proteins of different species, making difficult to establish a scoring system at protein level. Data were filtered at peptide level allowing only peptide having at least three consecutive b- or y-fragment ions of an homologous protein sequence tag and a ladder score (percentage of y- and b-ions detected) greater than 40. Using a decoy database, generated randomising NCBI Viridiplantaedatabase, the calculated false positive discovery rate was limited to 6.7%.To detect the potential conserved domains included in the sequences deduced by mass spectrometry [27], the peptide sequences inferred were blast searched using the protein BLAST tools  limiting the results to the accession numbers of homologous proteins having the higher number of matches in each spot. Blast algorithm used was blastp with BLOSUM62 matrix and expected value set to 10.


## 2.1. Plant Materials

Green and etiolated fleshy stalks of cultivated cardoon (Cynara cardunculus L. var. altilis DC) were used for this study. The system was realized in the Agricultural Company situated near to San Nicola Manfredi in Benevento district (Campania region) in March with direct dwell seeds in distant rows with 80 cm between each plant in plots of 3×3 meters. They have placed three seeds per hole, which leads to a single plant. In the period of May-June, the plants were in the rosette phase.  The plants reached their complete development at the end of summer-beginning of autumn (August-September). At the end of autumn (October), some green fleshly stalks were collected from three adjacent plants, quickly washed in sterile distilled water, freeze-dried, and stored at -80°C until analysis. Continuous dark for 25/30 days provoked plant etiolation as the leaves became more tender, white, and sweet as well. At the end of December, the etiolated fleshly stalks were collected and quickly washed in sterile distilled water, freeze-dried, and stored at -80°C until analysis.


## 2.2. Protein Extraction

Total proteins were extracted by a phenol-based method described by Saravanan and Rose [24]. The fleshy stalks were finely powdered in liquid nitrogen, using a mortar, and suspended in 7.5 mL of extraction buffer (700 mM sucrose, 500 mM Tris-HCl, pH 8.0, 50 mM EDTA, 100 mM KCl, 2% v/v β-mercaptoethanol, and 1 mM phenylmethylsulphonylfluoride),  by vortexing for 15 minutes at 4°C. After addition of an equal volume of phenol saturated 500 mM Tris-HCl, pH 7.5, the mixture was vortexed extensively for 10 minutes and then centrifuged at 10000 g for 15 minutes at 4°C. The upper phenol phase was removed and extracted twice with the extraction buffer. Proteins were precipitated from the phenol phase by the addition of five volumes of saturated ammonium acetate in methanol, overnight at -20°C. Precipitated proteins were centrifuged at 10000 g for 30 minutes.


## 2.3. 2-DE

Protein pellets were washed with ice-cold methanol (once) and ice-cold acetone (three times) dried and solved in IEF buffer (9 M urea, 4% w/v CHAPS, 20 mM DTT and 1% w/v ampholyte pH 3–10) (Bio-Rad, Hercules, CA, USA). Protein concentration was quantified using the BioRad protein assay using BSA as a standard. IPG strips (18-cm pH 4–7 Bio-Rad ReadyStrip), rehydratated  overnight with 315μL of IEF buffer containing 400 μg of total proteins. Proteins were focussed using a Protean IEF Cell (Bio Rad) at 12°C, applying 250 V (1 h), 1000 V (5 h), and 8000 V for a total of 52 kVh. Prior to the second dimension, the gel strips were reduced with 2% w/v DTT in 6 M urea, 30% w/v glycerol, 2% w/v SDS, 50 mM Tris-HCl pH 8.8, containing 0.01% w/v bromophenol blue for 20 minutes, followed by 20 minutes in the same buffer containing 2.5% w/v iodoacetamide. Electrophoresis in the second dimension was carried out using a Protean apparatus (BioRad) and 12% polyacrylamide gels (18cm×24cm×1mM) in 25 mM Tris (pH 8.3), 1.92 M glycine, and 1% w/v SDS, with 70 V (l35 mA) being applied for 16 h. Each sample was run in triplicate. Protein spots were annotated only if detectable in all gels [25].


## 2.4. Image Acquisition and Analysis

2-DE gels were stained with colloidal Coomassie G250 and scanned using a GS-800 calibrated densitometer (Bio-Rad). Image analysis was performed using the PDQuest software (Bio-Rad). Spot detection and matching between gels were performed automatically, followed by manual verification. After normalization of the spot densities against the whole-gel densities, the percentage volume of each spot was averaged for six different (three replicates of two samples) gels and the Student’st-test analysis (P<.01) was performed to find out statistically significant protein fold changes associated to etiolation. A twofold change in normalized spot densities was considered indicative of a differentially expressed component.


## 2.5. In-Gel Tryptic Digestion

Spots in the Coomassie Brilliant Blue G-250-stained 2-DE gel were excised manually and before being subjected to in-gel trypsinolysis as previously reported by Mamone et al. [26],  reduced (10 mM DTT in 100 mM NH4HCO3 allowing to react for 45 min, at 55°C) and alkylated with 55 mM iodoacetamide in 100 mM NH4HCO3 for 30 min at 25°C, in the dark. The resulting tryptic digest was dissolved in 40 μL of acetonitrile and 5% formic acid solution (1 : 1 v/v) and then the sample concentrated with vacuum centrifuge on medium heat to one-tenth initial volume for mass spectrometric analysis.


## 2.6. Reverse Phase Capillary Chromatography

Reverse phase capillary chromatography coupled with quadrupole time-of-flight tandem mass spectrometry was carried out using a Waters CapLC ternary pump system connected through a stream-select valve module to the nano-Z-spray source of the mass spectrometer. The sample was injected into the system through the CapLC autosampler. Solvent A and C consisted of water with 0.1% TFA, and solvent B consisted of acetonitrile with 0.1% TFA.The protein digest was preconcentrated and desalted on a precolumn cartridge packed with Atlantis dC18 resin at flow rate of 20μL/min-1 over 3 minutes. After switching to pumps A and B, a linear gradient from 5 to 70% of B over 45 min at a flow-rate of 300 nL min-1 (2% B/min) was applied to the precolumn cartridge and then used to elute the sample from the Atlantis dC18 resin analytical column (15cm×75 μm i.d.).


## 2.7. Mass Spectrometry

All mass spectra were acquired using a quadrupole-TOF hybrid mass spectrometer Waters Q-TOF II equipped with a nano-Z-spray source operating in positive ion mode. The ionization conditions used included a capillary voltage of 2.6 kV, source temperature of 80°C, and no nebulising gas. External calibration was applied to all data using Glu1-Fibrinopeptide B (Sigma-Aldrich) MS/MS fragments over a mass range from 50 to 1600 m/z.Survey scan acquisition was done on-line with capillary chromatographic separation; an initial TOF-MS scan was acquired over the mass range of 400–1600 m/z each second, with switching criteria for MS to MS/MS that included ion intensity (10 counts/s) and charge state (+2 to +4). MS/MS of precursor ion selected was acquired over the mass range of 50–1600 m/z. The collision energy used varied automatically according to the mass and charge state of the eluting peptides.


## 2.8. Nanoflow LC-ESI-MSMS

LC-ESI-MSMS analysis was performed using a Q-TOF Ultima mass spectrometer (Waters/Micromass UK Ltd., Manchester, UK) utilizing automated data-dependent acquisition. A nanoflow HPLC system (Waters CapLC (Waters/Micromass UK Ltd., Manchester, UK) was used for chromatographic separation of the peptide mixtures prior to MS detection. The peptides were concentrated and desalted on a precolumn cartridge packed with Atlantis dC18 resin (Waters CapLC, Waters/Micromass UK Ltd., Manchester, UK) and eluted at 200 nL/min by an increasing concentration of acetonitrile (2%/min gradient) onto an analytical column (15cm×75μm i.d.) also packed with Atlantis dC18 resin. An MS-TOF survey spectrum was recorded for 1 s. The five most abundant ions present in the survey spectrum were automatically mass-selected and fragmented by collision-induced dissociation (4 s per MSMS spectrum). The MSMS data were converted to a pkl file format using the Mass-Lynx 3.5 ProteinLynx software, and the resulting pkl file was searched against the NCBInr protein sequence databases using the Waters ProteinLynx GlobalSERVER v2.2.5 (PLGS).


## 2.9. Protein Identification

Acquired raw data were deisotoped and converted to pkl files using Waters ProteinLynx GlobalSERVER v2.2.5 (PLGS). Database search was performed against the tryptic peptides of entire taxonomy orderViridiplantae of NCBI database using a peptide tolerance of 0.1 Da and fragment mass tolerance of 0.2 Da, allowing a maximum number of two missed cleavages. Carbamidomethylated cysteine was set as fixed modification, while oxidized methionine was set as variable modification. Automod algorithm was used to determine additional posttranslational modifications and/or single amino acid substitution. The MS/MS spectra of peptides not identified by the previous two algorithms were submitted to the PLGS “de novo” sequence algorithm and the sequences obtained were BLAST searched against of NCBI Viridiplantae database using the same software tool. Alignments parameters were adjusted to search for a short input sequence, that is, PAM30MS matrix and expected threshold set to 1000. Redundant database search and de novo sequence alignments match several homologous proteins of different species, making difficult to establish a scoring system at protein level. Data were filtered at peptide level allowing only peptide having at least three consecutive b- or y-fragment ions of an homologous protein sequence tag and a ladder score (percentage of y- and b-ions detected) greater than 40. Using a decoy database, generated randomising NCBI Viridiplantaedatabase, the calculated false positive discovery rate was limited to 6.7%.To detect the potential conserved domains included in the sequences deduced by mass spectrometry [27], the peptide sequences inferred were blast searched using the protein BLAST tools  limiting the results to the accession numbers of homologous proteins having the higher number of matches in each spot. Blast algorithm used was blastp with BLOSUM62 matrix and expected value set to 10.


## 3. Results and Discussion

### 3.1. Proteome Changes Observed during Etiolation

Proteins extracted from green and etiolated fleshy stalks of cultivated cardoon were separated by 2D-PAGE and spots were visualized by CBB staining. Analysis of spots on the 2-DE gel using the PDQuest 2D image analysis software (Bio-Rad) indicated approximately 200 proteins having anMr less than 100 kDa and a pI in the range 4–7 (Figure 1). Taking into account the spot areas and their intensity, 60 protein spots were selected along each 2-DE gel for green and etiolated fleshy stalks. 21 out of 60 proteins were differentially expressed in green and etiolated fleshy stalks (P<.01) while 23 protein spots were found to be common to two types of fleshy stalks. By capillary LC-nano-ESI Q-TOF MS/MS sequence analysis 46 proteins (82%) were successfully identified. Among the sequences inferred from Cynara cardunculus proteins identified within 60 electrophoretic spots, ~71% tryptic peptides exactly matched the molecular mass and sequence of orthologous peptides occurring in the Viridiplantae database, the remaining differed by one or more amino acid residues inferred by the de novo sequence. The identification of conserved domains along the protein stretches using sequence similarity searches provides additional confidence on the function of the identified proteins although additional experiments are necessary to obtain ultimate confirmation. Occurrence of false positive and false negative matches may affect identification even using robust high-resolution techniques. Error may be caused essentially by sequencing errors derived from incomplete fragmentation and/or interpretation of spectra and/or erroneous sequence alignment. Taking into account these limitations, sequence similarity searches based on capillary LC coupled with nano-ESI Q-TOF data seem to represent a valid analytical tool capable of extending proteomic analysis to unsequenced genome organisms. As expected, Ile and Leu residues were indistinguishable in low-energy collision-induced dissociation spectra. Similarly, neither Gln nor Lys differing in 0.036 Da was discriminated, except for the C-terminal Lys in tryptic peptides.Representative 2-DE gels of total protein extracts from fleshy stalks of cultivated cardoon (Cynara cardunculus L. var. altilis DC). (a) Etiolated fleshy stalks; (b) green fleshy stalks. The horizontal bar indicates pI and the vertical one Mr. Spot numbering refers to Table 1. Gels were stained with colloidal CBB G-250.
(a)(b)The relative quantitative variations of the identified protein are shown in Table1.  Among the identified upregulated proteins, a large portion of the spots is represented by proteins involved in carbon/energy metabolism, defense from environmental/pathogen stresses, and oxidative stress control.Table 1
Constantly and differently expressed proteins in green and etiolated fleshy stalks classified according to functional categories. (a) Differentially expressed proteins  and  (b) constantly expressed proteins.  Spot number, experimentalpI and Mr values, protein description, accession number, hortologous organism, peptide sequences, and conserved domain are listed. Increasing/decreasing index (fold change) was calculated as the ratio of spot intensities (relative volumes) for green and etiolated fleshy stalks. Proteins were considered as differentially expressed when a relative fold change >2.0 or <0.5 was measured. (c) Abbreviated names of organisms: AT: Arabidopsis thaliana; CI × CE: Cichorium intybus×Cichorium endivia; CJ: Cercidiphyllum japonicum; CR:  Catharanthus roseus; CS: Cucumis sativus; DC: Daucus carota; FB: Flaveria bidentis; GM: Glycine max; HA: Helianthus annuus; LS: Lactuca sativa; MT: Medicago truncatula; NP: Narcissus pseudonarcissus; NPl: Nicotiana plumbaginifolia; NS: Nicotiana sylvestris; NT: Nicotiana tabacum; OS: Oryza sativa; OSj: Oryza sativa  japonica; OT: Ostreococcus tauri; PB: Pimpinella brachycarpa; PG: Panax ginseng; PS: Pisum sativum; PT: Populus trichocarpa; SA: Sinapis alba; SP: Schizosaccharomyces pombe; ST: Solanum tuberosum; TA: Triticum aestivum; TS: Tsuga heterophylla; VV: Vitis vinifera; ZH: Zinnia elegans.
Carbon∖Nitrogen metabolism or energySpotpI /Mr (kDa)Relative Fold change Green/EtiolatedDescriptionAccession numberHortologous Organisms (c)Exact Match sequence“de novo” sequenceConserved domains(a) Differentially expressed proteins106.1/305.30Triosephosphate isomerase cytosolicP48493LS(K)VASPAQAQEVHAGLR(K)(K)VIACmVGETLEQR(E) (L)AYEPVWAIGTGK(V) (R)EAGTTMoxEVVAAQTK(A) (R)IIYGGSVSGSNCmK(E)N(I/L)SADVAGDTR VASPA(Q/K)A(Q/K)EVYAE(I/L)Rcd00311165.7/560.40Malate dehydrogenase cytoplasmicQ7XDC8OSj(E)FISTVQQR(G)(R)ALGQISEK(L)(Q/K)G(I/L)(Q/K)(I/L)DEFSRARMDA255.7/103.00Vacuolar membrane ATPase subunit GAAD54418.1AT—R(I/L)E(Q/K)ETDE(Q/K)(I/L)AG(I/L)(I/L)TREAGAA(I/L)EA(Q/K)(Q/K)(I/L)VNAAR (Q/K)(I/L)TETSGDSGANV(Q/K)R(I/L)SSDVVEMox(I/L)(I/L)KA(Q/K)MEADY(Q/K)R306.8/113.60Nucleoside diphosphate kinase BQ39839GM(-)MDEQTFIMIKPDGVQR(G)M(I/L)WEG(Q/K)NVV(I/L)TGR(Q/K)(I/L)(I/L)GATNPAASEPGT(I/L)Rcd04413Nucleoside diphosphate kinase BP47920FB(E)IIGATNPAESAPGTIR(G) (E)IIGATNPAESAPGTIRGDFAIDIGR(N) (E)PGTIR(G) (K)NVVTTGR(E) (L)VGEILGRFEK(K) (R)GDFAIDIGR(N)  (R)GDFAIDIGRNVIHGSDAVESAKK(E) (R)GLVGEIIGRFEK(K) (R)GLVGEILGR(F) (R)NVIHGSDAVESAK(K) (R)NVIHGSDAVESAKK(E) (V)IHGSDAVESAKK(E)RNA binding translational regulation protein of the SUA5 family  ISSCAL50272.1OT(R)EGLLGR(I)214.3/110.08Acyl CoA binding proteinBAB85987.1PG(K)QATVGPVNTSR(P)(K)TLPESSTTNENK(L) (K)LILYGL(Y)G(I/L)(K/Q)EDFEEHAEKcd00435606.6/123.10Nucleoside diphosphate kinase BP47920FB(N)PAESAPGTIR(G)(V)IHGSDAVESAK(K) (A)TNPAESAPGTIR(G) (R)NVIHGSDAVESAK(K) (K)IIGATNPAESAPGTIR(G)cd04413365.8/272.40Oxygen evolving enhancer protein 2, chloroplastQ40407NP(K)EVEFPGQVLR(Y)(K)VDYLLGK(Q) (K)WNPSKEVEFPGQVLR(Y)EATDFGPPEEMox(I/L)SKAYGEAANVFG(Q/K)PKGDDTAEHSHSAT(I/L)TATVSGGKpfam01789455.2/382.40Oxygen-evolving enhancer protein 1, chloroplastQ40459NT(R)GSSFLDPK(G)(K)RLTFDEIQSK(T)465.3/382.40Oxygen evolving enhancer protein 1, chloroplastP26320ST(R)GGSTGYDNAVALPAGGR(G)(R)LTFDEIQSK(T) (R)VPFLFTIK(Q) (S)SFLDPK(E) (T)YLEVK(G) (V)PFLFTIK(Q)GDEEE(I/L)(I/L)(Q/K)EN(I/L)Kpfam01716475.4/382.30Oxygen evolving enhancer protein 1, chloroplastP26320ST(R)VPFLFTIK(Q)(K)RLTFDEIQSK(T) (K)FCmLEPTSFTVK(A)Oxidative stress386.0/323.10Ascorbate peroxidaseAAF22246.1PB(K)EGLLQLPTDK(A)(K)TGGPFGTMR(L) (K)TGGPFGTMoxR(L)WGEVRVD(Q/K)EEPP(Q/K)NGREEPP(I/L)EGR557.0/800.40Catalase 4AAF61734.1HA(F)AYSDTQR(H)(K)YPINPAR(V) (L)GPNYLQLPVNAPK(C) (N)FPVFF(V) (N)FPVFFTR(D) (N)YLQLPVNAPK(C) (R)DEEVDYFPSR(Y) (R)EGNFDIVGNNFPVFFTR(D) (R)FSTVIHER(G) (R)LGPNYLQLPVNAPK(C) (S)SVNTLTLVNK(E) (Y)TLVNK(A)APGV(Q/K)TPT(I/L)VRS(Q/K)AD(Q/K)S(I/L)G(Q/K)Kcd00328Stress and defense175.1/510.09beta-1,3-glucanaseCAA09765.1CI×CEYVAVGNEVDPNK(I/L)YDPD(Q/K)NT(I/L)(Q/K)A(I/L)(Kbeta-1,3-glucanaseCAB55309.1CI×CEDN(I/L)(Q/K)NYPDVK185.0/510.03beta-1,3-glucanaseBAA31142.1CS(R)TYNNNLIQHVK(Q)—485.5/303.00Putative chaperonin 21AAT80888.1VV(V)SPGNTVLYSK(Y)YAGNDF(Q/K)GPDGS(I/L)F(I/L)DTRcd00320(Y)TSLKPLGDR(V)VGA(Q/K)VVYSK(K)YTSLKPLGDR(V)Putative chaperonin 21BAD35227.1OSj—E(Q/K)PS(I/L)GTV(I/L)AVGPGP(I/L)DDEG(Q/K)RTriose phosphate isomeraseAAB23371.1LS(R)IIYGGSVSGSNCmK(E)(R)EAGTTMoxEVVAAQTK(A)Miscellaneous54.9/292.50Proteasome subunit alpha typeQ9M4T8GM(K)EGVVLAVEK(R)(R)TLVEHAR(V) (K)VTPNNVDIAK(V) (R)TEYDRGVNTFSPEGR(L) (K)QVMoxEEKVTPNNVDIAK(V) (R)ITSPLLEPSSVEK(I) (R)FGEGDEESMoxSRPF(G)T(Q/K)DGVV(I/L)AVE(Q/K)RDGVV(I/L)AVE(Q/K)RVAPTYH(I/L)YTPAEVEAV(I/L)SRLDET(Q/K)F(Q/K)(Q/K)EYYN(Q/K)A(I/L) CmD(I/L)PSRcd03753126.6/192.50Transcription factorCAA70323.1NPI(R)LQSTLKR(L)(K)TTTTDDKRLQSTLKR(I) (Q)ANTWVVSGSPQTK(N)(Q/K)(I/L)GPDN(I/L)DN(I/L)R245.4/124.30Glycine rich RNA binding proteinQ03878DC(I)NDRETGR(S)(I)INDRETGR(S) (R)EGGGGGYGGGGGYGGR(R) (R)CmFVGGLAWATNDE(S)AAADVEYR(I/L)DGRN(I/L)TVNEA(Q/K)SRUnidentified proteins35.3/230.40Unidentified proteins(I/L)APYFCRTPPN(I/L)DPPRPYVRYMPAVFPEVF(Q/K)ADKPEVTKPFP(I/L)(I/L)VTPS(Q/K)YF(Q/K)T(Q/K)MDYPPPGGTKT(I/L)DAGGSWY(I/L)VT(I/L)VEN40.20Unidentified proteinsPVP(Q/K)KSEFT(Q/K)K64.6/302.50Unidentified proteinsAANNETGRGEFSPYGESYPCRPVP(Q/K)K84.4/4513.00Unidentified proteinsMSCVSACGANFCKS(I/L)NYGAAMERCGPCNSDFR(b) Constantly expressed proteins155.8/722.00Mitochondrial processing peptidaseCAA56520.1ST(R)IVLAASGVEHEELLK(V)DV(I/L)S(I/L)PTYDVVNEVEA(I/L)GGNVTASASR356.5/290.50Triosephosphate isomerase  cytosolicP48493LS(K)VASPAQAQEVHAGLRK(W)(K)VASPAQAQEVHAGLR(K) (L)AYEPVWAIGTGK(V)  (K)ISSWDNVVLAYEPVWAIGTGK(V)  (R)ALLNETNEFVGDKVAYALSQGLK(V)  (K)AIADKISSWDNVVLAYEPVWAIGTGK(V)cd00311395.9/290.60Triosephosphate isomerase  cytosolicP48493LS(K)VASPAQAQEVH(A)(A)YEPVWAIGTGK(V) (R)IIYGGSVSGSNCmK(E) (K)VIACmVGETLEQR(E) (R)ALLNETNEFVGDK(V) (R)EAGTTMoxEVVAAQTK(A) (V)AAQTK(A)MoxYEVHAG(I/L)Rcd00311Triosephosphate isomerase  cytosolicP21820OSj(F)VGGNWK(C)PCmGTSEDV(Q/K)K405.4/190.80ATP synthase D chainQ9FT52AT(K)SLKESERLEK(E)(K)FDALLVELK(E) (L)LVELK(E) (I)ADVAFK(A) (K)LSTMoxTADEYFEK(H)FS(Q/K)EPEP(I/L)DWEYYRKVTDEA(Q/K)R(Q/K)VADVAFK415.3/201.90ATP synthase D chainQ9FT52AT(R)TIDWDGMoxAK(V) (L)LVELKEAEQK(S) (I)ADVAFK(A) (K)LSTMoxTADEYFEK(H) (K)MoxLVSDEAR(K) (K)MoxLVSDEARK(E)SVE(I/L)PKFS(Q/K)EPEP(I/L)DWEYYRKFS(Q/K)EPEP(I/L)DWEYYR496.6/511.00Malate dehydrogenaseQ7XDC8OS(K)NVSIYK(A)(R)ALGQISEK(L) (A)SSACmDHIR(-)F(I/L)STV(Q/K)(Q/K)RMalate dehydrogenaseAAP70009.1TA(R)LGVQVSDVK(N)Malate dehydrogenaseBAA97412.1AT(Q)ASALEK(H)Malate dehydrogenaseAAF69802.1VV(K)TFYAGK(A)506.6/510.90Malate dehydrogenaseAAF69802.1VV(L)FGVTTLDVVR(A)(T)FYAGK(A) (R)LFGVTTLDVVR(A) (R)TQDGGTEVVEAK(A) (T)EVVEAK(A) (K)TFYAGK(A)  (S)SLSLYDIAGTPGVAADVSHINTR(S)  (K)VAVLGAAGGIGQPLALLMoxK(L)R(I/L)FGVDS(I/L)DVVRRT(Q/K)DGGTEVVEAKA(I/L)EGADVV(I/L)(I/L)PAGVPRSMCmTA(I/L)AK(I/L)FGVTT(I/L)DVVRAGA(I/L)FADACm(I/L)KN(I/L)SNEE(I/L)VA(I/L)TKNGVEEV(I/L)G(I/L)GS(I/L)SDYEKcd01337536.9/520.80Malate dehydrogenaseAAC19244.1GM(R)LFGVTTLDVVR(A)(R)DDLFNINAGIVK(T) (K)ALEGADVVIIPAGVPR(K)cd01337Malate dehydrogenaseBAA97412.1AT(K)ASALEK(G)(K)SQASALEK(H) (K)NVSIYK(A)F(I/L)STV(Q/K)(Q/K)RMalate dehydrogenaseQ7XDC8OS(R)ALGQISEK(L)Malate dehydrogenaseAAP70009.1TA(R)LGVQVSDVK(N)547.0/530.50Malate dehydrogenaseAAF69802.1VV(R)TQDGGTEVVEAK(A)(R)DDLFNINAGIVK(T) (K)TFYAGK(A) (K)RTQDGGTEVVEAK(A) (K)SDYEK(M) (R)FYAGK(V) (C)TAIAK(Y)VSS(I/L)S(I/L)YD(I/L)AGTPGVAADVSHMNAPANGVEEV(I/L)G(I/L)GS(I/L)SDYEKcd01337Malate dehydrogenaseAAD56659.1GM(N)VPVAGVNVPVVGGH(A)(K)ALEGADVVIIPAGVPR(K) (N)VPVAGV(N)SEVVGY(Q/K)GDEEMGK565.5/740.50ATPase beta subunitAAD03393.1NS(R)IINVIGEPIDER(G) (R)EAPAFVEQATEQQILVTGIK(V) (R)DAEGQDVLLFIDNIFR(F) (I)GLFGGAGVGK(T) (G)SPITVPVGR(A) (N)VIGEPIDER(G) (Y)GQMoxNEPPGAR(A) (R)TIAMoxDGTEGLVR(G) (R)VLNTGSPITVPVGR(A) (K)TVLIMoxELINNVAK(A) (R)FTQANSEVSALLGR(I) (K)CmALVYGQMoxNEPPGAR(A)  (R)IPSAVGYQPTLATDLGGLQER(I) (L)GEDHYNTAR(G)(Q/K)NVA(Q/K)H(I/L)GYMoxMRcd01133Ribulose bisphosphate carboxylase RuBisCO large subunitQ05987CJ(R)MSGGDHLHSGTVVGK(L)(L)GLSAK(N)Oxidative stress346.2/301.90Glutathione S transferase PM24P46422AT(G)EHKKEPFLSR(N)A(I/L)T(Q/K)Y(I/L)AHRYEG(Q/K)GTN(I/L)(I/L)PADSKcd03053Stress and defence336.9/200.70Pathogenesis-related protein 5-1AAM21199.1HA(R)NNCmPYTVWAGAVPGGGR(Q)AVFT(I/L)RNNCPYTVWAGAVPGGGRTGCmNFDGSGRpfam00314425.0/160.70Pathogenesis-related proteinBAC10911.1ZH(K)VFSDFDNIAPK(V)(L)FTFGDAVPFTSGK(C) (K)QTIVYNCK(G) (K)LFTFGDAVPFTSGK(C)434.8/161.70Pathogenesis-related proteinBAC10911.1ZH(K)VFSDFDNIAPK(V)(K)QTIVYNCK(G)—595.1/1001.50Stromal 70 kDa heat shock related proteinQ02028PS(R)IPAVQELVK(K)(T)TLPTSK(S) (K)NQADSVVYQTEK(Q) (K)GPEGDVIDADFTDSK(-)(R)QAVVNPENTFFSVK(R) (R)IINEPTAA(S) (R)IINEPTAASLAYGFER(K) (V)PAYFNDSQR(T)NTT(I/L)PTSKMoxVSEAE(Q/K)FAK (Q/K)VSYTVVRDDNNNV(Q/K)(I/L)ECPA(I/L)GK(I/L)PAV(Q/K)EVVKCOG0443516.6/421.00Phenylcoumaran benzylic ether reductaseCAA06706.1PT(K)ILIIGGTGYIGK(F)(I)LGDGNAK(A) (F)IVEASAK(S) (I)ILGDGNAK(A) (V)VEASAK(S)(I/L)VA(I/L)KDVSDPSKD(Q/K)VV(I/L)(I/L)GDGNAKSFNE(I/L)VS(I/L)WEK(I/L)PT(I/L)A(Q/K)PAADGPPR526.7/420.50Phenylcoumaran benzylic ether reductase homolog TH2AAF64177.1TH(K)VVILGDGNAK(A)(L)GDGNAK(A) (R)DKVVILGDGNAK(A) (I)LGDGNAK(A) (V)ILGDGNAK(A)(I/L)VA(I/L)KFGNDVDRYTTVDEY(I/L)TRMiscellaneous265.7/131.10Glycine rich RNA binding proteinQ03878DC(I)NDRETGR(S)(I)INDRETGR(S) (K)IINDRETGR(S)DV(I/L)DS(Q/K)(I/L)(I/L)TDRDA(I/L)EGMSGEE(I/L)DGRTAFA(Q/K)YGDV(I/L)DSKGFGFVTF(Q/K)DE(Q/K)SMRN(I/L)TVNEA(Q/K)SRCmFVGG(I/L)AWATGDAEpfam00076Glycine rich RNA binding protein GRP2AP49311SA(M)ASPDVEYR(C)RNA binding glycine rich protein 1  RGP 1aBAA03743.1NS(G)SGGGGGGGGFRGGRREGGG(G)T(I/L)GEAFS(Q/K)FGE(I/L)(I/L)DSK275.9/170.70Protein At5g10860  mitochondrial precursorQ9LEV3AT(R)AMoxQLMoxTDNR(I)H(I/L)R(Q/K)(I/L)(I/L)V(Q/K)GRVGD(I/L)MoxTEEN(Q/K)(I/L)(I/L)TVTPDTKTVTPDTKRegulator of ribonuclease like protein 1Q9M8R9AT(R)VLVVDGGGSLR(C)VFEDNV(I/L)(I/L)R(R)ALQPVFQIYGR(R)Eukaryotic translation initiation factor 5A 1  eIF 5A 1Q9XI91AT(K)VVEVSTSK(T)             (K)TYPQQAGTIR(K)375.8/301.40Proteasome subunit alpha type 2A subunit BO23708AT(K)ASNGVVLATEK(K)(T)AVGSGQTSLGIK(A) (K)EPIPVTQLVR(E)  (D)DKGPQLYQVDPSGSYFSWK(A) (R)YTEDMELDDAIHTAILTLK(E)EGFEG(Q/K)(I/L)SGKPAAPS(I/L)(I/L)VDETEG(Q/K)K(I/L)YEEP(I/L)PVT(Q/K)(I/L)VRACmSNCmSESMTTFSPSGKcd03750Triosephosphate isomerase cytosolicP48493LS(R)IIYGGSVSGSNCmK(E) (R)EAGTTMoxEVVAAQTK(A)575.4/681.3026S protease regulatory subunit 6A homologO04019AT(K)LAGPQLVQMoxFIGDGAK(L) (Q)MoxFIGDGAK(L) (K)GVLLYGPPGTGK(T)COG1222445.0/411.40atranbp1b (Ran-binding protein)CAA66046.1AT(K)LYRFDK(D)(R)FGSVENCmK(A)Unidentified proteins585.0/640.70Unidentified proteinsAPDKDDPGR


### 3.2. Proteins Associated with Carbon and Energy Metabolism

Different key enzymes of glycolysis related to starch catabolism are upregulated during etiolation. Their abundance likely reflected the pivotal role of this pathway that fuels substrates for respiration and organic acid and pigment synthesis from imported sugars during etiolation, as shown by cytosolic malate dehydrogenase (spot 16) that are implicated in the glycolyis and gluconeogenesis. Interestingly, levels of glycolytic enzymes on the whole decreased in etiolated fleshy stalks of cultivated cardoon (spot 10). In fact triosephosphate isomerase catalyses the reversible interconversion of the triose phosphate isomers dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate. It plays an important role in glycolysis and it is essential for efficient energy production. When the cytosolic triosephosphate accumulation inhibits the respiration, it triggers malate efflux from mitochondria and its oxidation. In fact, while starch is the predominant storage form in green fleshy stalks, etiolation is characterized by a decline in starch content, sucrose import, and accumulation of hexoses. Therefore, in photosynthetic tissues, mitochondria are obligatory partners of chloroplasts with respect toCO2 light-dependent assimilation, due to their implication in the photorespiration pathway.Spot 21 was identified as Acyl-CoA-binding protein (ACBP), homologous to that fromPanax ginseng, and is a 10 kDa protein that binds with high affinity to C12–C22 acyl-CoA esters. In vitro and in vivo experiments suggest that it is involved in multiple cellular tasks including modulation of fatty acid biosynthesis, enzyme regulation, regulation of the intracellular acyl-CoA pool size, donation of acyl-CoA esters for β-oxidation, vesicular trafficking, complex lipid synthesis, and gene regulation. In higher plants, de novo fatty acid biosynthesis occurs in the chloroplasts. Plastid-synthesized fatty acyl-CoAs, palmitoyl-CoA (16 : 0-CoA), and oleoyl-CoA (18 : 1-CoA) are likely exported by intracellular lipid transporters to the endoplasmic reticulum (ER) for the “eukaryotic pathway” in lipid biosynthesis. The process as to how the fatty acyl-CoAs are exported from the chloroplasts to the ER remains unclear to date. The ACBP that was first reported in Arabidopsis was proposed to facilitate transport and maintenance of long-chain acyl-CoA esters in the cytosol. This assumption was based on a lack of knowledge at that time that five other ACBPs exist in Arabidopsis[28]. Recently studies suggest that those proteins are light-regulated [29]; in fact we observed an increased value of this protein in the etiolated flesh stalks.Moreover, some enzymes involved in electron transport and energy production are  downregulated. When exposed to dark, photosynthetically competent chloroplast developed into photosynthetically incompetent etioplast. This involved the destruction of component of  the photosynthetic apparatus and prononouced alterations in plastid ultrastrutture that include the conversion of stromal and stacked thylakoid structures characteristic of chloroplast into distinctive prolamellar body. In this study, vacuolar ATP synthase (spot 25), the photosystem II oxygen-evolving complex protein 1 (spots 45, 46, 47), and photosystem II oxygen-evolving complex protein 2 (spot 36) were downregulated (Table1). Infact, the oxygen evolving protein plays an important role in stabilizing, binding, and maintaining functional conformation of the manganese cluster which directly catalyzes the H2O-splitting reaction [30]. After exposure to the dark the protein decreases significantly, indicating that the oxygen evolving complex of PSII was flourished by exposure to light.


### 3.3. Proteins Associated with Oxidative Stresses

Etiolation has been described as an oxidative process which might lead to the production or accumulation of a large amount of harmful active oxygen species (AOS) or, more specifically, of hydrogen peroxide as a by-product of increased electron transport activity in the mitochondria. The presence of damaging hydrogen peroxide might explain the strong presence of ascorbate peroxidase, with the function of contrasting toxic peroxide [31].L-ascorbate peroxidase activity (spot 38) showed a remarkable decreasing value in etiolated as compared to green fleshy stalks. Ascorbate is a major metabolite in plants. It is an antioxidant and, in association with other components of the antioxidant system, protects plants against oxidative damage resulting from aerobic metabolism, photosynthesis, and a range of pollutants. In fact, the degradation of the photosynthetic apparatus reduces the presence of the L-ascorbate peroxidase.Catalase (cd00328) (spot 55) was identified as a homologue ofHeliantus annuus catalase 4 and was also upregulated in etiolated fleshy stalks. The increase in antioxidant enzymes was indicative of a pro-oxidant signal under a nonphotosynthetic condition. Besides, glutathione S-transferase (spot 34) was similar in green and etiolated fleshy stalks.


### 3.4. Proteins Associated with Stress and Defense

Proteins homologous to stromal 70 kDa heat-shock protein (spot 59) were detected in both green and etiolated fleshy stalks. The chaperonin 21 (spot 48) was downregulated; it has been demonstrated that this protein has a more general role in plastid development and may influence the folding of numerous chloroplast protein such RuBisCO (EC̲ 4.1.1.39̲) [32].The beta 1,3 glucanase (spots 17,18) proteins are upregulated and are involved in the response to biotic stresses that are differentially expressed during etiolation.Besides, phenylcoumaran benzylic ether reductase (PCBER) (spots 51, 52) was identified as protein constantly expressed, which plays a significant role in the biosynthesis of important plant defense phenylpropanoid-derived compounds, based on known catalytic function component presiding over defense mechanisms. These proteins possess NADPH binding sites and appear to be cytosolic enzymes, since they lack secretory pathway signal sequences. In addition, they may be regulated in a comparable manner, not only at the transcriptional level but also at the enzymatic level by protein phosphorylation, as evidenced by several potential phosphorylation sites that are conserved among all of the members [33].Enzymatic assay with recombinant Bet v 6 and Pyr c 5 showed  PCBER catalytic activity for both recombinant allergens [34]. Therefore, PCBER may represent a new class of cross-reactive allergens of birch pollen and plant food.


### 3.5. Miscellaneous

Information on several identified proteins that did not group in the above classes was very poor; in particular, their function was postulated on the basis of the homology sequence criteria. In only a few cases, important functions in some physiological processes have emerged for definite proteins. In this respect, it is worth noting that three proteins are involved in the control of programmed cell death (PCD), namely, the proteasome alpha subunit type 2A (spot 37), 26S protease regulatory subunit 6A (spot 57), and proteasome alpha subunit type 5 (spot 5) containing a ubiquitin-like domain interacting with catalytically active proteasomes [35]. Increasing evidence indicates that PCD plays a pivotal role in diverse plant processes, such as embryogenesis, senescence, or pathogen defense [36, 37], and we find out that the proteasome alpha subunit type 5 (spot 5) is downregulated and decreased its value in the etiolating process but data are lacking on its involvement in this process.Ran-binding protein 1b, homologous to that fromArabidopsis(AtRanBP1b), was also identified (spot 44). Ran-binding proteins are activators of Ras-related nuclear small GTP-binding proteins (Ran) and in Arabidopsis have been shown to be involved in the regulation of auxin-induced mitotic progression [38]. Another identified protein for which information is very scarce or missing was the glycine-rich RNA-binding protein (spot 24) which decreases its value in the etiolation process. Besides, two proteins are exclusively expressed during etiolation (spots 29 and 32; Table 2) in the fleshy stalks.Table 2
Protein expressed exclusively in etiolated cultivated cardoon. Spot number, experimentalpI and Mr values, protein description, accession number, hortologous organism, peptide sequences, and conserved domain are listed. (a) Abbreviated names of organisms: AT: Arabidopsis thaliana; GM: Glycine max; SS: Solenostemon scutellarioides.
MiscellaneousSpotpI/Mr (kDa)DescriptionAccession numberHortologous Organism (a)Exact Match Sequence“de novo” SequenceConserved domains296.1/19CBS domain-containing proteinABK64186.1SS(K)LITVTPDTK(V)AVVSEHREE(I/L)DR(R)AMQLMTDNR(I)(K)VGDIMTEENKLITVTPDTK(V)Eukaryotic translation initiation factor 5AQ9XI91AT(K)VVEVSTSK(T)(K)TYPQQAGTIR(K)326.9/20Protein P21P25096GM(R)VVFCP(-)(R)TGCNFDGSGR(G)Spot 29 is a cystathionine beta-synthase (CBS), domain-containing protein also present in the proteome of archaeobacteria, prokaryotes, and eukaryotes, having divergent functions ranging from metabolic enzymes and transcriptional regulators to ion channels and transporters. Although the precise function has not yet been elucidated, CBS domains could have physiological importance because point mutations are responsible for several hereditary diseases in humans [39].Protein P21 (P25096) (spot 32) is classified as a ras-oncogene-encoded p21 protein in humans and is known to cause large numbers of tumours. The protein differs from its healthy counterpart in eukaryotic cells in that it contains single amino acid substitutions at critical positions of the polypeptide chain, such asGly12, Gly13, Ala59, and Gln61 [40]. In a recent study [41], one quantitative trait locus (QTL), fw2.2, has been demonstrated as being responsible for the domestication of the tomato. When transformed into large-fruited cultivars, a cosmid derived from the fw2.2 region of a small-fruited wild species reduced fruit size by the predicted amount and had the gene action expected for fw2.2. The cause of the QTL effect is a single gene, ORFX, expressed early in the floral development. It controls carpel cell number and its sequence has structural features similar to that of human oncogene c-H-ras p21


## 3.1. Proteome Changes Observed during Etiolation

Proteins extracted from green and etiolated fleshy stalks of cultivated cardoon were separated by 2D-PAGE and spots were visualized by CBB staining. Analysis of spots on the 2-DE gel using the PDQuest 2D image analysis software (Bio-Rad) indicated approximately 200 proteins having anMr less than 100 kDa and a pI in the range 4–7 (Figure 1). Taking into account the spot areas and their intensity, 60 protein spots were selected along each 2-DE gel for green and etiolated fleshy stalks. 21 out of 60 proteins were differentially expressed in green and etiolated fleshy stalks (P<.01) while 23 protein spots were found to be common to two types of fleshy stalks. By capillary LC-nano-ESI Q-TOF MS/MS sequence analysis 46 proteins (82%) were successfully identified. Among the sequences inferred from Cynara cardunculus proteins identified within 60 electrophoretic spots, ~71% tryptic peptides exactly matched the molecular mass and sequence of orthologous peptides occurring in the Viridiplantae database, the remaining differed by one or more amino acid residues inferred by the de novo sequence. The identification of conserved domains along the protein stretches using sequence similarity searches provides additional confidence on the function of the identified proteins although additional experiments are necessary to obtain ultimate confirmation. Occurrence of false positive and false negative matches may affect identification even using robust high-resolution techniques. Error may be caused essentially by sequencing errors derived from incomplete fragmentation and/or interpretation of spectra and/or erroneous sequence alignment. Taking into account these limitations, sequence similarity searches based on capillary LC coupled with nano-ESI Q-TOF data seem to represent a valid analytical tool capable of extending proteomic analysis to unsequenced genome organisms. As expected, Ile and Leu residues were indistinguishable in low-energy collision-induced dissociation spectra. Similarly, neither Gln nor Lys differing in 0.036 Da was discriminated, except for the C-terminal Lys in tryptic peptides.Representative 2-DE gels of total protein extracts from fleshy stalks of cultivated cardoon (Cynara cardunculus L. var. altilis DC). (a) Etiolated fleshy stalks; (b) green fleshy stalks. The horizontal bar indicates pI and the vertical one Mr. Spot numbering refers to Table 1. Gels were stained with colloidal CBB G-250.
(a)(b)The relative quantitative variations of the identified protein are shown in Table1.  Among the identified upregulated proteins, a large portion of the spots is represented by proteins involved in carbon/energy metabolism, defense from environmental/pathogen stresses, and oxidative stress control.Table 1
Constantly and differently expressed proteins in green and etiolated fleshy stalks classified according to functional categories. (a) Differentially expressed proteins  and  (b) constantly expressed proteins.  Spot number, experimentalpI and Mr values, protein description, accession number, hortologous organism, peptide sequences, and conserved domain are listed. Increasing/decreasing index (fold change) was calculated as the ratio of spot intensities (relative volumes) for green and etiolated fleshy stalks. Proteins were considered as differentially expressed when a relative fold change >2.0 or <0.5 was measured. (c) Abbreviated names of organisms: AT: Arabidopsis thaliana; CI × CE: Cichorium intybus×Cichorium endivia; CJ: Cercidiphyllum japonicum; CR:  Catharanthus roseus; CS: Cucumis sativus; DC: Daucus carota; FB: Flaveria bidentis; GM: Glycine max; HA: Helianthus annuus; LS: Lactuca sativa; MT: Medicago truncatula; NP: Narcissus pseudonarcissus; NPl: Nicotiana plumbaginifolia; NS: Nicotiana sylvestris; NT: Nicotiana tabacum; OS: Oryza sativa; OSj: Oryza sativa  japonica; OT: Ostreococcus tauri; PB: Pimpinella brachycarpa; PG: Panax ginseng; PS: Pisum sativum; PT: Populus trichocarpa; SA: Sinapis alba; SP: Schizosaccharomyces pombe; ST: Solanum tuberosum; TA: Triticum aestivum; TS: Tsuga heterophylla; VV: Vitis vinifera; ZH: Zinnia elegans.
Carbon∖Nitrogen metabolism or energySpotpI /Mr (kDa)Relative Fold change Green/EtiolatedDescriptionAccession numberHortologous Organisms (c)Exact Match sequence“de novo” sequenceConserved domains(a) Differentially expressed proteins106.1/305.30Triosephosphate isomerase cytosolicP48493LS(K)VASPAQAQEVHAGLR(K)(K)VIACmVGETLEQR(E) (L)AYEPVWAIGTGK(V) (R)EAGTTMoxEVVAAQTK(A) (R)IIYGGSVSGSNCmK(E)N(I/L)SADVAGDTR VASPA(Q/K)A(Q/K)EVYAE(I/L)Rcd00311165.7/560.40Malate dehydrogenase cytoplasmicQ7XDC8OSj(E)FISTVQQR(G)(R)ALGQISEK(L)(Q/K)G(I/L)(Q/K)(I/L)DEFSRARMDA255.7/103.00Vacuolar membrane ATPase subunit GAAD54418.1AT—R(I/L)E(Q/K)ETDE(Q/K)(I/L)AG(I/L)(I/L)TREAGAA(I/L)EA(Q/K)(Q/K)(I/L)VNAAR (Q/K)(I/L)TETSGDSGANV(Q/K)R(I/L)SSDVVEMox(I/L)(I/L)KA(Q/K)MEADY(Q/K)R306.8/113.60Nucleoside diphosphate kinase BQ39839GM(-)MDEQTFIMIKPDGVQR(G)M(I/L)WEG(Q/K)NVV(I/L)TGR(Q/K)(I/L)(I/L)GATNPAASEPGT(I/L)Rcd04413Nucleoside diphosphate kinase BP47920FB(E)IIGATNPAESAPGTIR(G) (E)IIGATNPAESAPGTIRGDFAIDIGR(N) (E)PGTIR(G) (K)NVVTTGR(E) (L)VGEILGRFEK(K) (R)GDFAIDIGR(N)  (R)GDFAIDIGRNVIHGSDAVESAKK(E) (R)GLVGEIIGRFEK(K) (R)GLVGEILGR(F) (R)NVIHGSDAVESAK(K) (R)NVIHGSDAVESAKK(E) (V)IHGSDAVESAKK(E)RNA binding translational regulation protein of the SUA5 family  ISSCAL50272.1OT(R)EGLLGR(I)214.3/110.08Acyl CoA binding proteinBAB85987.1PG(K)QATVGPVNTSR(P)(K)TLPESSTTNENK(L) (K)LILYGL(Y)G(I/L)(K/Q)EDFEEHAEKcd00435606.6/123.10Nucleoside diphosphate kinase BP47920FB(N)PAESAPGTIR(G)(V)IHGSDAVESAK(K) (A)TNPAESAPGTIR(G) (R)NVIHGSDAVESAK(K) (K)IIGATNPAESAPGTIR(G)cd04413365.8/272.40Oxygen evolving enhancer protein 2, chloroplastQ40407NP(K)EVEFPGQVLR(Y)(K)VDYLLGK(Q) (K)WNPSKEVEFPGQVLR(Y)EATDFGPPEEMox(I/L)SKAYGEAANVFG(Q/K)PKGDDTAEHSHSAT(I/L)TATVSGGKpfam01789455.2/382.40Oxygen-evolving enhancer protein 1, chloroplastQ40459NT(R)GSSFLDPK(G)(K)RLTFDEIQSK(T)465.3/382.40Oxygen evolving enhancer protein 1, chloroplastP26320ST(R)GGSTGYDNAVALPAGGR(G)(R)LTFDEIQSK(T) (R)VPFLFTIK(Q) (S)SFLDPK(E) (T)YLEVK(G) (V)PFLFTIK(Q)GDEEE(I/L)(I/L)(Q/K)EN(I/L)Kpfam01716475.4/382.30Oxygen evolving enhancer protein 1, chloroplastP26320ST(R)VPFLFTIK(Q)(K)RLTFDEIQSK(T) (K)FCmLEPTSFTVK(A)Oxidative stress386.0/323.10Ascorbate peroxidaseAAF22246.1PB(K)EGLLQLPTDK(A)(K)TGGPFGTMR(L) (K)TGGPFGTMoxR(L)WGEVRVD(Q/K)EEPP(Q/K)NGREEPP(I/L)EGR557.0/800.40Catalase 4AAF61734.1HA(F)AYSDTQR(H)(K)YPINPAR(V) (L)GPNYLQLPVNAPK(C) (N)FPVFF(V) (N)FPVFFTR(D) (N)YLQLPVNAPK(C) (R)DEEVDYFPSR(Y) (R)EGNFDIVGNNFPVFFTR(D) (R)FSTVIHER(G) (R)LGPNYLQLPVNAPK(C) (S)SVNTLTLVNK(E) (Y)TLVNK(A)APGV(Q/K)TPT(I/L)VRS(Q/K)AD(Q/K)S(I/L)G(Q/K)Kcd00328Stress and defense175.1/510.09beta-1,3-glucanaseCAA09765.1CI×CEYVAVGNEVDPNK(I/L)YDPD(Q/K)NT(I/L)(Q/K)A(I/L)(Kbeta-1,3-glucanaseCAB55309.1CI×CEDN(I/L)(Q/K)NYPDVK185.0/510.03beta-1,3-glucanaseBAA31142.1CS(R)TYNNNLIQHVK(Q)—485.5/303.00Putative chaperonin 21AAT80888.1VV(V)SPGNTVLYSK(Y)YAGNDF(Q/K)GPDGS(I/L)F(I/L)DTRcd00320(Y)TSLKPLGDR(V)VGA(Q/K)VVYSK(K)YTSLKPLGDR(V)Putative chaperonin 21BAD35227.1OSj—E(Q/K)PS(I/L)GTV(I/L)AVGPGP(I/L)DDEG(Q/K)RTriose phosphate isomeraseAAB23371.1LS(R)IIYGGSVSGSNCmK(E)(R)EAGTTMoxEVVAAQTK(A)Miscellaneous54.9/292.50Proteasome subunit alpha typeQ9M4T8GM(K)EGVVLAVEK(R)(R)TLVEHAR(V) (K)VTPNNVDIAK(V) (R)TEYDRGVNTFSPEGR(L) (K)QVMoxEEKVTPNNVDIAK(V) (R)ITSPLLEPSSVEK(I) (R)FGEGDEESMoxSRPF(G)T(Q/K)DGVV(I/L)AVE(Q/K)RDGVV(I/L)AVE(Q/K)RVAPTYH(I/L)YTPAEVEAV(I/L)SRLDET(Q/K)F(Q/K)(Q/K)EYYN(Q/K)A(I/L) CmD(I/L)PSRcd03753126.6/192.50Transcription factorCAA70323.1NPI(R)LQSTLKR(L)(K)TTTTDDKRLQSTLKR(I) (Q)ANTWVVSGSPQTK(N)(Q/K)(I/L)GPDN(I/L)DN(I/L)R245.4/124.30Glycine rich RNA binding proteinQ03878DC(I)NDRETGR(S)(I)INDRETGR(S) (R)EGGGGGYGGGGGYGGR(R) (R)CmFVGGLAWATNDE(S)AAADVEYR(I/L)DGRN(I/L)TVNEA(Q/K)SRUnidentified proteins35.3/230.40Unidentified proteins(I/L)APYFCRTPPN(I/L)DPPRPYVRYMPAVFPEVF(Q/K)ADKPEVTKPFP(I/L)(I/L)VTPS(Q/K)YF(Q/K)T(Q/K)MDYPPPGGTKT(I/L)DAGGSWY(I/L)VT(I/L)VEN40.20Unidentified proteinsPVP(Q/K)KSEFT(Q/K)K64.6/302.50Unidentified proteinsAANNETGRGEFSPYGESYPCRPVP(Q/K)K84.4/4513.00Unidentified proteinsMSCVSACGANFCKS(I/L)NYGAAMERCGPCNSDFR(b) Constantly expressed proteins155.8/722.00Mitochondrial processing peptidaseCAA56520.1ST(R)IVLAASGVEHEELLK(V)DV(I/L)S(I/L)PTYDVVNEVEA(I/L)GGNVTASASR356.5/290.50Triosephosphate isomerase  cytosolicP48493LS(K)VASPAQAQEVHAGLRK(W)(K)VASPAQAQEVHAGLR(K) (L)AYEPVWAIGTGK(V)  (K)ISSWDNVVLAYEPVWAIGTGK(V)  (R)ALLNETNEFVGDKVAYALSQGLK(V)  (K)AIADKISSWDNVVLAYEPVWAIGTGK(V)cd00311395.9/290.60Triosephosphate isomerase  cytosolicP48493LS(K)VASPAQAQEVH(A)(A)YEPVWAIGTGK(V) (R)IIYGGSVSGSNCmK(E) (K)VIACmVGETLEQR(E) (R)ALLNETNEFVGDK(V) (R)EAGTTMoxEVVAAQTK(A) (V)AAQTK(A)MoxYEVHAG(I/L)Rcd00311Triosephosphate isomerase  cytosolicP21820OSj(F)VGGNWK(C)PCmGTSEDV(Q/K)K405.4/190.80ATP synthase D chainQ9FT52AT(K)SLKESERLEK(E)(K)FDALLVELK(E) (L)LVELK(E) (I)ADVAFK(A) (K)LSTMoxTADEYFEK(H)FS(Q/K)EPEP(I/L)DWEYYRKVTDEA(Q/K)R(Q/K)VADVAFK415.3/201.90ATP synthase D chainQ9FT52AT(R)TIDWDGMoxAK(V) (L)LVELKEAEQK(S) (I)ADVAFK(A) (K)LSTMoxTADEYFEK(H) (K)MoxLVSDEAR(K) (K)MoxLVSDEARK(E)SVE(I/L)PKFS(Q/K)EPEP(I/L)DWEYYRKFS(Q/K)EPEP(I/L)DWEYYR496.6/511.00Malate dehydrogenaseQ7XDC8OS(K)NVSIYK(A)(R)ALGQISEK(L) (A)SSACmDHIR(-)F(I/L)STV(Q/K)(Q/K)RMalate dehydrogenaseAAP70009.1TA(R)LGVQVSDVK(N)Malate dehydrogenaseBAA97412.1AT(Q)ASALEK(H)Malate dehydrogenaseAAF69802.1VV(K)TFYAGK(A)506.6/510.90Malate dehydrogenaseAAF69802.1VV(L)FGVTTLDVVR(A)(T)FYAGK(A) (R)LFGVTTLDVVR(A) (R)TQDGGTEVVEAK(A) (T)EVVEAK(A) (K)TFYAGK(A)  (S)SLSLYDIAGTPGVAADVSHINTR(S)  (K)VAVLGAAGGIGQPLALLMoxK(L)R(I/L)FGVDS(I/L)DVVRRT(Q/K)DGGTEVVEAKA(I/L)EGADVV(I/L)(I/L)PAGVPRSMCmTA(I/L)AK(I/L)FGVTT(I/L)DVVRAGA(I/L)FADACm(I/L)KN(I/L)SNEE(I/L)VA(I/L)TKNGVEEV(I/L)G(I/L)GS(I/L)SDYEKcd01337536.9/520.80Malate dehydrogenaseAAC19244.1GM(R)LFGVTTLDVVR(A)(R)DDLFNINAGIVK(T) (K)ALEGADVVIIPAGVPR(K)cd01337Malate dehydrogenaseBAA97412.1AT(K)ASALEK(G)(K)SQASALEK(H) (K)NVSIYK(A)F(I/L)STV(Q/K)(Q/K)RMalate dehydrogenaseQ7XDC8OS(R)ALGQISEK(L)Malate dehydrogenaseAAP70009.1TA(R)LGVQVSDVK(N)547.0/530.50Malate dehydrogenaseAAF69802.1VV(R)TQDGGTEVVEAK(A)(R)DDLFNINAGIVK(T) (K)TFYAGK(A) (K)RTQDGGTEVVEAK(A) (K)SDYEK(M) (R)FYAGK(V) (C)TAIAK(Y)VSS(I/L)S(I/L)YD(I/L)AGTPGVAADVSHMNAPANGVEEV(I/L)G(I/L)GS(I/L)SDYEKcd01337Malate dehydrogenaseAAD56659.1GM(N)VPVAGVNVPVVGGH(A)(K)ALEGADVVIIPAGVPR(K) (N)VPVAGV(N)SEVVGY(Q/K)GDEEMGK565.5/740.50ATPase beta subunitAAD03393.1NS(R)IINVIGEPIDER(G) (R)EAPAFVEQATEQQILVTGIK(V) (R)DAEGQDVLLFIDNIFR(F) (I)GLFGGAGVGK(T) (G)SPITVPVGR(A) (N)VIGEPIDER(G) (Y)GQMoxNEPPGAR(A) (R)TIAMoxDGTEGLVR(G) (R)VLNTGSPITVPVGR(A) (K)TVLIMoxELINNVAK(A) (R)FTQANSEVSALLGR(I) (K)CmALVYGQMoxNEPPGAR(A)  (R)IPSAVGYQPTLATDLGGLQER(I) (L)GEDHYNTAR(G)(Q/K)NVA(Q/K)H(I/L)GYMoxMRcd01133Ribulose bisphosphate carboxylase RuBisCO large subunitQ05987CJ(R)MSGGDHLHSGTVVGK(L)(L)GLSAK(N)Oxidative stress346.2/301.90Glutathione S transferase PM24P46422AT(G)EHKKEPFLSR(N)A(I/L)T(Q/K)Y(I/L)AHRYEG(Q/K)GTN(I/L)(I/L)PADSKcd03053Stress and defence336.9/200.70Pathogenesis-related protein 5-1AAM21199.1HA(R)NNCmPYTVWAGAVPGGGR(Q)AVFT(I/L)RNNCPYTVWAGAVPGGGRTGCmNFDGSGRpfam00314425.0/160.70Pathogenesis-related proteinBAC10911.1ZH(K)VFSDFDNIAPK(V)(L)FTFGDAVPFTSGK(C) (K)QTIVYNCK(G) (K)LFTFGDAVPFTSGK(C)434.8/161.70Pathogenesis-related proteinBAC10911.1ZH(K)VFSDFDNIAPK(V)(K)QTIVYNCK(G)—595.1/1001.50Stromal 70 kDa heat shock related proteinQ02028PS(R)IPAVQELVK(K)(T)TLPTSK(S) (K)NQADSVVYQTEK(Q) (K)GPEGDVIDADFTDSK(-)(R)QAVVNPENTFFSVK(R) (R)IINEPTAA(S) (R)IINEPTAASLAYGFER(K) (V)PAYFNDSQR(T)NTT(I/L)PTSKMoxVSEAE(Q/K)FAK (Q/K)VSYTVVRDDNNNV(Q/K)(I/L)ECPA(I/L)GK(I/L)PAV(Q/K)EVVKCOG0443516.6/421.00Phenylcoumaran benzylic ether reductaseCAA06706.1PT(K)ILIIGGTGYIGK(F)(I)LGDGNAK(A) (F)IVEASAK(S) (I)ILGDGNAK(A) (V)VEASAK(S)(I/L)VA(I/L)KDVSDPSKD(Q/K)VV(I/L)(I/L)GDGNAKSFNE(I/L)VS(I/L)WEK(I/L)PT(I/L)A(Q/K)PAADGPPR526.7/420.50Phenylcoumaran benzylic ether reductase homolog TH2AAF64177.1TH(K)VVILGDGNAK(A)(L)GDGNAK(A) (R)DKVVILGDGNAK(A) (I)LGDGNAK(A) (V)ILGDGNAK(A)(I/L)VA(I/L)KFGNDVDRYTTVDEY(I/L)TRMiscellaneous265.7/131.10Glycine rich RNA binding proteinQ03878DC(I)NDRETGR(S)(I)INDRETGR(S) (K)IINDRETGR(S)DV(I/L)DS(Q/K)(I/L)(I/L)TDRDA(I/L)EGMSGEE(I/L)DGRTAFA(Q/K)YGDV(I/L)DSKGFGFVTF(Q/K)DE(Q/K)SMRN(I/L)TVNEA(Q/K)SRCmFVGG(I/L)AWATGDAEpfam00076Glycine rich RNA binding protein GRP2AP49311SA(M)ASPDVEYR(C)RNA binding glycine rich protein 1  RGP 1aBAA03743.1NS(G)SGGGGGGGGFRGGRREGGG(G)T(I/L)GEAFS(Q/K)FGE(I/L)(I/L)DSK275.9/170.70Protein At5g10860  mitochondrial precursorQ9LEV3AT(R)AMoxQLMoxTDNR(I)H(I/L)R(Q/K)(I/L)(I/L)V(Q/K)GRVGD(I/L)MoxTEEN(Q/K)(I/L)(I/L)TVTPDTKTVTPDTKRegulator of ribonuclease like protein 1Q9M8R9AT(R)VLVVDGGGSLR(C)VFEDNV(I/L)(I/L)R(R)ALQPVFQIYGR(R)Eukaryotic translation initiation factor 5A 1  eIF 5A 1Q9XI91AT(K)VVEVSTSK(T)             (K)TYPQQAGTIR(K)375.8/301.40Proteasome subunit alpha type 2A subunit BO23708AT(K)ASNGVVLATEK(K)(T)AVGSGQTSLGIK(A) (K)EPIPVTQLVR(E)  (D)DKGPQLYQVDPSGSYFSWK(A) (R)YTEDMELDDAIHTAILTLK(E)EGFEG(Q/K)(I/L)SGKPAAPS(I/L)(I/L)VDETEG(Q/K)K(I/L)YEEP(I/L)PVT(Q/K)(I/L)VRACmSNCmSESMTTFSPSGKcd03750Triosephosphate isomerase cytosolicP48493LS(R)IIYGGSVSGSNCmK(E) (R)EAGTTMoxEVVAAQTK(A)575.4/681.3026S protease regulatory subunit 6A homologO04019AT(K)LAGPQLVQMoxFIGDGAK(L) (Q)MoxFIGDGAK(L) (K)GVLLYGPPGTGK(T)COG1222445.0/411.40atranbp1b (Ran-binding protein)CAA66046.1AT(K)LYRFDK(D)(R)FGSVENCmK(A)Unidentified proteins585.0/640.70Unidentified proteinsAPDKDDPGR


## 3.2. Proteins Associated with Carbon and Energy Metabolism

Different key enzymes of glycolysis related to starch catabolism are upregulated during etiolation. Their abundance likely reflected the pivotal role of this pathway that fuels substrates for respiration and organic acid and pigment synthesis from imported sugars during etiolation, as shown by cytosolic malate dehydrogenase (spot 16) that are implicated in the glycolyis and gluconeogenesis. Interestingly, levels of glycolytic enzymes on the whole decreased in etiolated fleshy stalks of cultivated cardoon (spot 10). In fact triosephosphate isomerase catalyses the reversible interconversion of the triose phosphate isomers dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate. It plays an important role in glycolysis and it is essential for efficient energy production. When the cytosolic triosephosphate accumulation inhibits the respiration, it triggers malate efflux from mitochondria and its oxidation. In fact, while starch is the predominant storage form in green fleshy stalks, etiolation is characterized by a decline in starch content, sucrose import, and accumulation of hexoses. Therefore, in photosynthetic tissues, mitochondria are obligatory partners of chloroplasts with respect toCO2 light-dependent assimilation, due to their implication in the photorespiration pathway.Spot 21 was identified as Acyl-CoA-binding protein (ACBP), homologous to that fromPanax ginseng, and is a 10 kDa protein that binds with high affinity to C12–C22 acyl-CoA esters. In vitro and in vivo experiments suggest that it is involved in multiple cellular tasks including modulation of fatty acid biosynthesis, enzyme regulation, regulation of the intracellular acyl-CoA pool size, donation of acyl-CoA esters for β-oxidation, vesicular trafficking, complex lipid synthesis, and gene regulation. In higher plants, de novo fatty acid biosynthesis occurs in the chloroplasts. Plastid-synthesized fatty acyl-CoAs, palmitoyl-CoA (16 : 0-CoA), and oleoyl-CoA (18 : 1-CoA) are likely exported by intracellular lipid transporters to the endoplasmic reticulum (ER) for the “eukaryotic pathway” in lipid biosynthesis. The process as to how the fatty acyl-CoAs are exported from the chloroplasts to the ER remains unclear to date. The ACBP that was first reported in Arabidopsis was proposed to facilitate transport and maintenance of long-chain acyl-CoA esters in the cytosol. This assumption was based on a lack of knowledge at that time that five other ACBPs exist in Arabidopsis[28]. Recently studies suggest that those proteins are light-regulated [29]; in fact we observed an increased value of this protein in the etiolated flesh stalks.Moreover, some enzymes involved in electron transport and energy production are  downregulated. When exposed to dark, photosynthetically competent chloroplast developed into photosynthetically incompetent etioplast. This involved the destruction of component of  the photosynthetic apparatus and prononouced alterations in plastid ultrastrutture that include the conversion of stromal and stacked thylakoid structures characteristic of chloroplast into distinctive prolamellar body. In this study, vacuolar ATP synthase (spot 25), the photosystem II oxygen-evolving complex protein 1 (spots 45, 46, 47), and photosystem II oxygen-evolving complex protein 2 (spot 36) were downregulated (Table1). Infact, the oxygen evolving protein plays an important role in stabilizing, binding, and maintaining functional conformation of the manganese cluster which directly catalyzes the H2O-splitting reaction [30]. After exposure to the dark the protein decreases significantly, indicating that the oxygen evolving complex of PSII was flourished by exposure to light.


## 3.3. Proteins Associated with Oxidative Stresses

Etiolation has been described as an oxidative process which might lead to the production or accumulation of a large amount of harmful active oxygen species (AOS) or, more specifically, of hydrogen peroxide as a by-product of increased electron transport activity in the mitochondria. The presence of damaging hydrogen peroxide might explain the strong presence of ascorbate peroxidase, with the function of contrasting toxic peroxide [31].L-ascorbate peroxidase activity (spot 38) showed a remarkable decreasing value in etiolated as compared to green fleshy stalks. Ascorbate is a major metabolite in plants. It is an antioxidant and, in association with other components of the antioxidant system, protects plants against oxidative damage resulting from aerobic metabolism, photosynthesis, and a range of pollutants. In fact, the degradation of the photosynthetic apparatus reduces the presence of the L-ascorbate peroxidase.Catalase (cd00328) (spot 55) was identified as a homologue ofHeliantus annuus catalase 4 and was also upregulated in etiolated fleshy stalks. The increase in antioxidant enzymes was indicative of a pro-oxidant signal under a nonphotosynthetic condition. Besides, glutathione S-transferase (spot 34) was similar in green and etiolated fleshy stalks.


## 3.4. Proteins Associated with Stress and Defense

Proteins homologous to stromal 70 kDa heat-shock protein (spot 59) were detected in both green and etiolated fleshy stalks. The chaperonin 21 (spot 48) was downregulated; it has been demonstrated that this protein has a more general role in plastid development and may influence the folding of numerous chloroplast protein such RuBisCO (EC̲ 4.1.1.39̲) [32].The beta 1,3 glucanase (spots 17,18) proteins are upregulated and are involved in the response to biotic stresses that are differentially expressed during etiolation.Besides, phenylcoumaran benzylic ether reductase (PCBER) (spots 51, 52) was identified as protein constantly expressed, which plays a significant role in the biosynthesis of important plant defense phenylpropanoid-derived compounds, based on known catalytic function component presiding over defense mechanisms. These proteins possess NADPH binding sites and appear to be cytosolic enzymes, since they lack secretory pathway signal sequences. In addition, they may be regulated in a comparable manner, not only at the transcriptional level but also at the enzymatic level by protein phosphorylation, as evidenced by several potential phosphorylation sites that are conserved among all of the members [33].Enzymatic assay with recombinant Bet v 6 and Pyr c 5 showed  PCBER catalytic activity for both recombinant allergens [34]. Therefore, PCBER may represent a new class of cross-reactive allergens of birch pollen and plant food.


## 3.5. Miscellaneous

Information on several identified proteins that did not group in the above classes was very poor; in particular, their function was postulated on the basis of the homology sequence criteria. In only a few cases, important functions in some physiological processes have emerged for definite proteins. In this respect, it is worth noting that three proteins are involved in the control of programmed cell death (PCD), namely, the proteasome alpha subunit type 2A (spot 37), 26S protease regulatory subunit 6A (spot 57), and proteasome alpha subunit type 5 (spot 5) containing a ubiquitin-like domain interacting with catalytically active proteasomes [35]. Increasing evidence indicates that PCD plays a pivotal role in diverse plant processes, such as embryogenesis, senescence, or pathogen defense [36, 37], and we find out that the proteasome alpha subunit type 5 (spot 5) is downregulated and decreased its value in the etiolating process but data are lacking on its involvement in this process.Ran-binding protein 1b, homologous to that fromArabidopsis(AtRanBP1b), was also identified (spot 44). Ran-binding proteins are activators of Ras-related nuclear small GTP-binding proteins (Ran) and in Arabidopsis have been shown to be involved in the regulation of auxin-induced mitotic progression [38]. Another identified protein for which information is very scarce or missing was the glycine-rich RNA-binding protein (spot 24) which decreases its value in the etiolation process. Besides, two proteins are exclusively expressed during etiolation (spots 29 and 32; Table 2) in the fleshy stalks.Table 2
Protein expressed exclusively in etiolated cultivated cardoon. Spot number, experimentalpI and Mr values, protein description, accession number, hortologous organism, peptide sequences, and conserved domain are listed. (a) Abbreviated names of organisms: AT: Arabidopsis thaliana; GM: Glycine max; SS: Solenostemon scutellarioides.
MiscellaneousSpotpI/Mr (kDa)DescriptionAccession numberHortologous Organism (a)Exact Match Sequence“de novo” SequenceConserved domains296.1/19CBS domain-containing proteinABK64186.1SS(K)LITVTPDTK(V)AVVSEHREE(I/L)DR(R)AMQLMTDNR(I)(K)VGDIMTEENKLITVTPDTK(V)Eukaryotic translation initiation factor 5AQ9XI91AT(K)VVEVSTSK(T)(K)TYPQQAGTIR(K)326.9/20Protein P21P25096GM(R)VVFCP(-)(R)TGCNFDGSGR(G)Spot 29 is a cystathionine beta-synthase (CBS), domain-containing protein also present in the proteome of archaeobacteria, prokaryotes, and eukaryotes, having divergent functions ranging from metabolic enzymes and transcriptional regulators to ion channels and transporters. Although the precise function has not yet been elucidated, CBS domains could have physiological importance because point mutations are responsible for several hereditary diseases in humans [39].Protein P21 (P25096) (spot 32) is classified as a ras-oncogene-encoded p21 protein in humans and is known to cause large numbers of tumours. The protein differs from its healthy counterpart in eukaryotic cells in that it contains single amino acid substitutions at critical positions of the polypeptide chain, such asGly12, Gly13, Ala59, and Gln61 [40]. In a recent study [41], one quantitative trait locus (QTL), fw2.2, has been demonstrated as being responsible for the domestication of the tomato. When transformed into large-fruited cultivars, a cosmid derived from the fw2.2 region of a small-fruited wild species reduced fruit size by the predicted amount and had the gene action expected for fw2.2. The cause of the QTL effect is a single gene, ORFX, expressed early in the floral development. It controls carpel cell number and its sequence has structural features similar to that of human oncogene c-H-ras p21


## 4. Concluding Remarks

The 634,985 proteins of theViridiplantae database, including the complete genome of Arabidopsis thaliana and other important plants, have allowed identification of protein expression profiles of Cynara cardunculus under usual light or dark conditions.In this study, constitutive and differentially expressed proteins in green and etiolated fleshy stalks of cultivated cardoon (Cynara cardunculus L. var. altilis DC) have been discussed in relation to the plant etiolation process. In fact, the proteomic study indicated that, during etiolation, the differentially expressed proteins were involved mainly in starch metabolism  and stress response. The major group in response to dark exposure was found to be proteins related to PSII complex. Thus groups comprised four proteins (spots 36; 45; 46; 47) encoded by two nuclear genes (PSBO; PSBP). The  decreasing values of these proteins explain the role that the PSII complex plays in the early photosynthetic phase. In fact its downregulation during the etiolating process is correlated to a progressive loose of function such as the oxidation of water to molecular oxygen. Therefore, the rapid decrease in the relative amount of these proteins following dark exposure suggests that the PSII complex is the light-driven water: plastoquinone oxidoreductase.In conclusion, this study allowed to identify the proteins that are implicated in different physiological and defence process besides their involvement in the response to stress, as the etiolation process. This one is to be considered as a reaction that helps plants to survive in the dark condition, contributing to the response at the physiological effects due to this exposure.



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*Source: 496893-2010-07-18.xml*