Tomato (Solanum lycopersicum) is a model plant for studying fleshy fruit development. Several genetic and molecular approaches have been developed to increase our knowledge about the physiological basis of fruit growth, but very few data are yet available at the proteomic level. The main stages of fruit development were first determined through the dynamics of fruit diameter and pericarp cell number. Then, total proteins were extracted from pericarp tissue at six relevant developmental stages and separated by two-dimensional gel electrophoresis. Protein patterns were markedly different between stages.Proteins showing major variations were monitored. We identified 90 of 1,791 well-resolved spots either by matrix-assisted laser-desorption ionization time-of-flight peptide mass fingerprinting or liquid chromatography-mass spectrometry sequencing and expressed sequence tag database searching. Clustered correlation analysis results pointed out groups of proteins with similar expression profiles during fruit development. In young fruit, spots linked to amino acid metabolism or protein synthesis were mainly expressed during the cell division stage and down-regulated later. Some spots linked to cell division processes could be identified. During the cell expansion phase, spots linked to photosynthesis and proteins linked to cell wall formation transiently increased. In contrast, the major part of the spots related to C compounds and carbohydrate metabolism or oxidative processes were up-regulated during fruit development, showing an increase in spot intensity during development and maximal abundance in mature fruit. This was also the case for spots linked to stress responses and fruit senescence. We discuss protein variations, taking into account their potential role during fruit growth and comparing our results with already known variations at mRNA and metabolite-profiling levels.
SUMMARYThe GDP-D-mannose 3,5-epimerase (GME, EC 5.1.3.18), which converts GDP-D-mannose to GDP-L-galactose, is generally considered to be a central enzyme of the major ascorbate biosynthesis pathway in higher plants, but experimental evidence for its role in planta is lacking. Using transgenic tomato lines that were RNAi-silenced for GME, we confirmed that GME does indeed play a key role in the regulation of ascorbate biosynthesis in plants. In addition, the transgenic tomato lines exhibited growth defects affecting both cell division and cell expansion. A further remarkable feature of the transgenic plants was their fragility and loss of fruit firmness. Analysis of the cell-wall composition of leaves and developing fruit revealed that the cell-wall monosaccharide content was altered in the transgenic lines, especially those directly linked to GME activity, such as mannose and galactose. In agreement with this, immunocytochemical analyses showed an increase of mannan labelling in stem and fruit walls and of rhamnogalacturonan labelling in the stem alone. The results of MALDI-TOF fingerprinting of mannanase cleavage products of the cell wall suggested synthesis of specific mannan structures with modified degrees of substitution by acetate in the transgenic lines. When considered together, these findings indicate an intimate linkage between ascorbate and non-cellulosic cell-wall polysaccharide biosynthesis in plants, a fact that helps to explain the common factors in seemingly unrelated traits such as fruit firmness and ascorbate content.
To evaluate the genotypic variation of salt stress response in tomato, physiological analyses and a proteomic approach have been conducted in parallel on four contrasting tomato genotypes. After a 14 d period of salt stress in hydroponic conditions, the genotypes exhibited different responses in terms of plant growth, particularly root growth, foliar accumulation of Na(+), and foliar K/Na ratio. As a whole, Levovil appeared to be the most tolerant genotype while Cervil was the most sensitive one. Roma and Supermarmande exhibited intermediary behaviours. Among the 1300 protein spots reproducibly detected by two-dimensional electrophoresis, 90 exhibited significant abundance variations between samples and were submitted to mass spectrometry for identification. A common set of proteins (nine spots), up- or down-regulated by salt-stress whatever the genotype, was detected. But the impact of the tomato genotype on the proteome variations was much higher than the salt effect: 33 spots that were not variable with salt stress varied with the genotype. The remaining number of variable spots (48) exhibited combined effects of the genotype and the salt factors, putatively linked to the degrees of genotype tolerance. The carbon metabolism and energy-related proteins were mainly up-regulated by salt stress and exhibited most-tolerant versus most-sensitive abundance variations. Unexpectedly, some antioxidant and defence proteins were also down-regulated, while some proteins putatively involved in osmoprotectant synthesis and cell wall reinforcement were up-regulated by salt stress mainly in tolerant genotypes. The results showed the effect of 14 d stress on the tomato root proteome and underlined significant genotype differences, suggesting the importance of making use of genetic variability.
Phenol extraction of proteins is an alternative method to classical TCA-acetone extraction. It allows efficient protein recovery and removes nonprotein components in the case of plant tissues rich in polysaccharides, lipids, and phenolic compounds. We present here a tried and tested protocol adapted for two dimensional electrophoresis (2-DE) and further proteomic studies. After phenol extraction, proteins are precipitated with ammonium acetate in methanol. The pelleted proteins are then resuspended in isoelectric focusing buffer, and the protein concentration is measured with a modified Bradford assay prior to electrophoresis. The important points for successful use of this protocol are (1) keeping samples at very low temperature during the first step and (2) careful recovery of the phenolic phase after the centrifugations, which are major features of this protocol.
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 degrees 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 degrees 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.
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