CONICET | Buscador de Institutos y Recursos Humanos

Cherry fruit firmness is a main quality attribute, and also is an important determinant of tolerance to handling and physical damage and decay. Firmness loss during fruit development is usually associated with cell wall polysaccharide turnover. Although texture has been suggested as one important goal for cherry improvement, few studies have attempted to characterize further the biochemical changes associated with cherry softening. Part of the genotypic variations in fruit firmness have been linked with differences in the patterns of cell wall disassembly. Soft cherry cultivars have more water soluble pectin upon ripening than firm cultivars, but in some cases the differences in texture among cultivars are already recorded at initial stages of development and persist throughout ripening, suggesting that early structural differences in wall architecture may be relevant. The general structural features of the polysaccharides that make up the plant cell wall are highly conserved among species. However, their degree of substitution, branching and integration in the cell wall may differ and have great impact in tissue properties found that firm immature cherries had higher neutral sugar/uronic acid ratios and suggested that this results from polyuronides with higher branching. However, it is not known whether or not these features may contribute to the differences in firmness among cultivars. In order to further characterize the cell wall changes occurring on cherry ontogeny and to identify putative factors associated with firmness variation in cherry fruit genotypes, we performed a detailed evaluation using UV-MALDI-TOF mass spectrometry of the changes in pectins fractions of five sweet cherry cultivars: ?Chelan?, ?Regina?, ?Sumele?, ?Sunburst? and ?Brooks? at two different developmental stages. Experiments were conducted as follows: Samples were analyzed on the Bruker Daltonics Ultraflex II TOF/TOF mass spectrometer (Leipzig, Germany). Mass spectra were acquired in linear positive ion mode. Solutions of samples were prepared in water. External mass calibration was made using b-cyclodextrin with nor-harmane (nHo) as matrix in positive ion mode. The matrix signal was used as an additional standard for calibration. Sample solutions were spotted on a MTP 384 target plate steel T F (Part No.: 209519; target frame # 74115) from Bruker Daltonics (Leipzig,Germany). For UV?MALDI MS matrix solutions were prepared by dissolving nHo (2 mg/ml) in methanol/water (2:1, v/v) solution and gentisic acid, GA (10 mg/ml) in methanol/water (1:1, v/v) solution. For UV-MALDI MS experiments dry droplet sample preparation or sandwich method was used, i.e., loading successively 0.5 ìl of matrix solution, analyte solution and matrix solution after drying each layer at normal atmosphere and room temperature. The ratio of matrix to analyte was 3:1 (v/v) and the matrix and analyte solution loading sequence was: i) matrix, ii) analyte, iii) matrix, iv) matrix. Desorption/ionization was obtained by using the frequency-tripled Nd:YAG laser (355-nm) with a 100 Hz shot frequency. The accelerating potential was 20 kV. Experiments were performed using, firstly, the full-range setting for laser firing position in order to select the optimal position for data collection, and secondly fixing the laser firing position in the sample sweet spots. The laser power was adjusted to obtain high signal-to-noise ratio (S/N) and each mass spectrum was generated by averaging 100 lasers pulses per spot. Spectra were obtained and analyzed with the programs FlexControl and FlexAnalysis, respectively. The UV-MALDI-TOF spectra of three pectin fractions (extracted sequentially by water, 50 mM CDTA aqueous solution and 0.1M sodium carbonate solution) isolated from five cultivars of sweet cherry (Regina, Sunburts, Chelan, Brooks and Sumele) in the first and last ontogenic stages were obtained, and compared with those obtained by gel-permeation chromatography. The pectins extracted with water were very consicuous by UV-MALDI MS, showing clear signals; those isolated by CDTA solution gave a poorer spectra, whereas those isolated after sodium carbonate extraction gave very poor mass spectra, with a very low signal-to-noise ratio. The UV-MALDI spectra allowed to determine that the fractions obtained from mature cherries gave rise to peaks clearly shift to lower molecular weights when compared to inmature cherries of the same cultivar. This shift was observable sometimes using GPC, but the results of UV-MALDI MS are sharper, and clearly more determinant. Although the molecular weights obtained by GPC and UV-MALDI MS are not coincident in absolute value in most cases, they follow the same trend, i.e., the cultivars which gave rise to higher average molecular weights by GPC originated more signals in the high-MW range of the UV-MALDI-TOF spectra (i.e, m/z range up to 160 kD).

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