Proteomic analysis of tobacco mosaic virus-infected tomato (Lycopersicon esculentum M.) fruits and detection of viral coat protein

Tobacco mosaic virus (TMV) is a widespread plant virus from the genus Tobamovirus that affects tobacco and tomato plants causing a pathology characterised by cell breakage and disorganisation in plant leaves and fruits. In this study we undertook a proteomic approach to investigate the molecular and biochemical mechanisms potentially involved in tomato fruit defence against the viral infection. The comparison of 2-D gels from control and TMV-infected but asymptomatic tomato fruits revealed changes in several proteins. The differential expression of peptidases, endoglucanase, chitinase and proteins participating in the ascorbate-glutathione cycle in infected fruits suggests that pathogenesis-related proteins and antioxidant enzymes may play a role in the protection against TMV infection. TMV coat protein appeared as a prominent spot in 2-D gels from TMV-infected asymptomatic fruits. A Triton X-114 phase-partitioning step of tomato protein extracts favoured the solubilisation of TMV coat protein and the enrichment of two aminopeptidases not present in control fruits. PMF and MS/MS data of the 2-D gel-isolated TMV coat protein is proposed as a powerful analysis method for the simultaneous tobamovirus detection, species determination and strain differentiation in virus-infected fruit commodities.     

Growth and proteomic analysis of tomato fruit under partial root-zone drying

The effects of partial root-zone drying (PRD) on tomato fruit growth and proteome in the pericarp of cultivar Ailsa Craig were investigated. The PRD treatment was 70% of water applied to fully irrigated (FI) plants. PRD reduced the fruit number and slightly increased the fruit diameter, whereas the total fruit fresh weight (FW) and dry weight (DW) per plant did not change. Although the growth rate was higher in FI than in PRD fruits, the longer period of cell expansion resulted in bigger PRD fruits. Proteins were extracted from pericarp tissue at two fruit growth stages (15 and 30 days post-anthesis [dpa]), and submitted to proteomic analysis including two-dimensional gel electrophoresis and mass spectrometry for identification. Proteins related to carbon and amino acid metabolism indicated that slower metabolic flux in PRD fruits may be the cause of a slower growth rate compared to FI fruits. The increase in expression of the proteins related to cell wall, energy, and stress defense could allow PRD fruits to increase the duration of fruit growth compared to FI fruits. Upregulation of some of the antioxidative enzymes during the cell expansion phase of PRD fruits appears to be related to their role in protecting fruits against the mild stress induced by PRD.     

Plant plasma membrane proteins : immunological characterization of a major 75 kilodalton protein group

A major 75 kD protein group from the tomato plasma membrane was semipurified on polyacrylamide gels and used to raise a rabbit antiserum. The resulting antiserum recognized a single 75 kilodalton band from phase partitioned tomato plasma membrane (from both suspension cells and mature, green fruit) after resolution on one-dimensional polyacrylamide gels. Two-dimensional polyacrylamide gel analysis of proteins from tomato plasma membrane showed that the 75 kilodalton antiserum recognized a group of proteins ranging from 63.1 to 88.2 kilodaltons (mean = 75.6 kilodaltons) and with isoelectric point values ranging from 5.7 to 6.3. No other spots were visible on the two-dimensional blots. This antiserum was shown to bind protoplast surface epitopes by indirect immunofluorescence. The presence of this protein group in both monocotyledonous and dicotyledonous plants was established by immunoblotting the tomato 75 kilodalton antiserum against proteins obtained from plasma membrane-enriched fractions from corn roots and soybean roots. The data suggest that this 75 kilodalton protein group is a major proteinaceous component of the plant plasma membrane.     

Purification of tomato sucrose synthase phosphorylated isoforms by Fe(III)-immobilized metal affinity chromatography

The major phosphorylation site of maize sucrose synthase (SuSy) is well conserved among plant species but absent in the deduced peptide sequence of the tomato SuSy cDNA (TOMSSF). In this study, we report the in vitro phosphorylation of 25-day-old tomato fruits SuSy on seryl residue(s) by an endogenous Ca2+-dependent protein kinase activity. Two distinct 32P-labeled peptides detected in the tryptic peptide map of in vitro 32P-radiolabeled tomato fruit SuSy were purified. Amino acid sequencing and phosphoamino acid analysis of the major 32P-labeled peptide revealed the presence of a SuSy isozyme in young tomato fruit having the N-terminus phosphorylation site present in other plant species. By using Fe(III)-immobilized metal affinity chromatography [Fe(III)-IMAC] as a final purification step of tomato fruit SuSy, two 32P-labeled tomato SuSy isoforms were separated from a nonradiolabeled SuSy fraction by using a pH gradient. The major 32P-SuSy isoform was phosphorylated exclusively at the seryl residue related to the phosphorylation site of maize SuSy. The multiphosphorylated state of the second radiolabeled SuSy fraction was indicated by a higher retention during Fe(III)-IMAC and by tryptic peptide mapping analysis. Kinetic analyses of SuSy isoforms purified by Fe(III)-IMAC have revealed that phosphorylation of the major phosphorylation site of tomato fruit SuSy was not sufficient by itself to modulate tomato SuSy activity, whereas the affinity for UDP increased about threefold for the multiphosphorylated SuSy isoform.     

Alignment and statistical difference analysis of complex peptide data sets generated by multidimensional LC-MS

A method for high-resolution proteomics analyses of complex protein mixtures is presented using multidimensional HPLC coupled to MS (MDLC-MS). The method was applied to identify proteins that are differentially expressed during fruit ripening of tomato. Protein extracts from red and green tomato fruits were digested by trypsin. The resulting highly complex peptide mixtures were separated by strong cation exchange chromatography (SCX), and subsequently analyzed by RP nano-LC coupled to quadrupole-TOF MS. For detailed quantitative comparison, triplicate RP-LC-MS runs were performed for each SCX fraction. The resulting data sets were analyzed using MetAlign software for noise and data reduction, multiple alignment and statistical variance analysis. For each RP-LC-MS chromatogram, up to 7000 mass components were detected. Peak intensity data were compared by multivariate and statistical analysis. This revealed a clear separation between the green and red tomato samples, and a clear separation of the different SCX fractions. MS/MS spectra were collected using the data-dependent acquisition mode from a selected set of differentially detected peptide masses, enabling the identification of proteins that were differentially expressed during ripening of tomato fruits. Our approach is a highly sensitive method to analyze proteins in complex mixtures without the need of isotope labeling.     

Ripening-related occurrence of phosphoenolpyruvate carboxykinase in tomato fruit

Phosphoenolpyruvate carboxykinase (PEPCK) is present in ripening tomato fruits. A cDNA encoding PEPCK was identified from a PCR-based screen of a cDNA library from ripe tomato fruit. The sequence of the tomato PEPCK cDNA and a cloned portion of the genomic DNA shows that the complete cDNA sequence contains an open reading frame encoding a peptide of 662 amino acid residues in length and predicts a polypeptide with a molecular mass of 73.5 kDa, which corresponds to that detected by western blotting. Only one PEPCK gene was identified in the tomato genome. PEPCK is shown to be present in the pericarp of ripening tomato fruits by activity measurements, western blotting and mRNA analysis. PEPCK abundance and activity both increased during fruit ripening, from an undetectable amount in immature green fruit to a high amount in ripening fruit. PEPCK mRNA, protein and activity were also detected in germinating seeds and, in lower amounts, in roots and stems of tomato. The possible role of PEPCK in the pericarp of tomato fruit during ripening is discussed.     

A cellular hypothesis for the induction of blossom-end rot in tomato fruit

BACKGROUND: The incidence of blossom-end rot (BER) is generally associated with a calcium (Ca) deficiency in the distal portion of tomato fruits. The visible symptom is a necrotic lesion, which is presumed to be a consequence of cell death and the subsequent leakage of solutes into the extracellular space. Environmental factors that affect either fruit cell expansion or Ca delivery to the distal portion of the fruit influence the occurrence of BER. However, since no absolute, critical fruit Ca concentration for the occurrence of BER has been identified, it is now important to define the role of Ca in fruit cell physiology and to seek the cause of BER at the cellular level. HYPOTHESIS: Here, it is suggested that BER is initiated by a cellular dysfunction in the distal portion of a young fruit during rapid cell expansion. It is proposed that insufficient Ca(2+) is available for critical apoplastic and cytoplasmic functions when the cellular Ca demand imposed by vacuolation exceeds the Ca delivery to an expanding cell. A local Ca deficiency, therefore, may result in aberrant intracellular Ca(2+) signals, a weakening of cell walls and a loss of cellular integrity. Ultimately it may lead to cell death and the visible symptoms of BER. Several experimental strategies are suggested to confirm the occurrence of aberrant Ca(2+) concentrations in cells contributing to BER. PERSPECTIVE: Many genetic and genomic resources are becoming available for tomato. Ultimately, these will allow genes affecting the occurrence of BER to be identified. Such knowledge will inform breeding strategies to eliminate BER. In the meanwhile, increasing the apoplastic Ca concentration in susceptible fruit tissue should provide a simple and reliable, practical solution for the prevention of BER in tomatoes. It is suggested that current horticultural practices, such as the manipulation of the mineral composition of the feed or the growth environment, are not completely effective in reducing BER because they affect apoplastic Ca concentration in fruit tissue indirectly. Therefore, spraying Ca directly onto young fruits is recommended for the prevention of BER.     

Tackling the plant proteome: practical approaches, hurdles and experimental tools

The study of complex biological questions through comparative proteomics is becoming increasingly attractive to plant biologists as the rapidly expanding plant genomic and expressed sequence tag databases provide improved opportunities for protein identification. This review focuses on practical issues associated with comparative proteomic analysis, including the challenges of effective protein extraction and separation from plant tissues, the pros and cons of two-dimensional gel-based analysis and the problems of identifying proteins from species that are not recognized models for functional genomic studies. Specific points are illustrated using data from an ongoing study of the tomato and pepper fruit proteomes.     

Molecular cloning of a ripening-specific lipoxygenase and its expression during wild-type and mutant tomato fruit development.

A 94-kD protein that accumulates predominately in tomato (Ly-copersicon esculentum) fruit during ripening was purified, and antibodies specific for the purified protein were used to isolate cDNA clones from a red-ripe fruit cDNA library. A sequence analysis of these cDNAs and cross-reactivity of the 94-kD-specific antibodies to the soybean lipoxygenase (LOX) L-1, L-2, and L-3 proteins and soybean LOX L-1-specific antibodies to the 94-kD protein identified it as a member of the LOX gene family. Maximum levels of the 94-kD LOX mRNA and protein are present in breaker to ripe and red-ripe stages, respectively. Expression of 94-kD LOX in different tissues from mature green and red-ripe tomato fruits was found to be greatest in the radial walls of ripe fruit, but immunocytolocalization using tissue printing suggests that the highest accumulation of its protein occurs in locular jelly. None of 94-kD LOX is expressed in nonripening mutant fruits of any age. Never-ripe mutant fruit accumulate the 94-kD LOX mRNA to levels similar to those obtained in wild-type fruit, but fail to accumulate the 94-kD LOX protein. Collectively, the results show that expression of 94-kD LOX is regulated by the ripening process, and ethylene may play a role in its protein accumulation.     

Protective proteins are differentially expressed in tomato genotypes differing for their tolerance to low-temperature storage

When stored at low temperature, tomato fruits exhibit chilling injury symptoms, such as rubbery texture and irregular ripening. To identify proteins related to chilling tolerance, we compared two tomato near isogenic lines differing for their texture phenotype at harvest in a fruit-storage trial including two temperatures (4 and 20°C) along several days of conservation. Fruit evolution was followed by assessing fruit color, ethylene emission and texture parameters. The most contrasted samples were submitted to proteomic analysis including two-dimensional electrophoresis and mass spectrometry of protein spots to identify the proteins, whose expression varied according to the genotype or the storage conditions. Unexpectedly, the most firm genotype at harvest was the most sensitive to cold storage. The other genotype exhibited a delay in fruit firmness loss leading to the texture differences observed after 20 days of 4°C storage. The proteome analysis of these contrasted fruits identified 85 proteins whose quantities varied with temperature or genotype. As expected, cold storage decreased the expression of proteins related to maturation process, such as acidic invertase, possibly controlled post-translational regulation of polygalacturonase and up-regulated proteins related to freezing tolerance. However, the study point out proteins involved in the differential resistance to chilling conditions of the two lines. This includes specific isoforms among the large family of small heat shocked proteins, and a set of proteins involved in the defense against of the reticulum endoplasmic stress.