Structural characterization of cell wall pectin fractions in ripe strawberry fruits using AFM

Cell wall disassembly during fruit ripening is the main process leading to fruit softening. In strawberry fruit (Fragaria × ananassa, Duch.), functional analysis with transgenic plants have related the loss of firm texture to the metabolism of pectins. To gain insights into the role of pectins in strawberry softening we have analyzed physicochemical features of ionic and covalently bound pectins, isolated from ripe fruits, by Fourier transform infrared spectroscopy (FTIR), size exclusion chromatography (SEC) and atomic force microscopy (AFM). Cell wall material was isolated and chemically fractionated by sequential extraction with cyclohexane-trans-l,2-diamine tetraacetate (CDTA) and sodium carbonate (Na2CO3) in order to extract ionic and covalently bound pectins, respectively. Uronic acids (UA) were detected as the main component in CDTA samples, whilst a significantly higher content of neutral sugar was observed in the Na2CO3 samples, representing 33% of total sugars vs. 12% in the CDTA fraction. The spectral profile in the FTIR region 1200–900 cm−1 was similar in the case of both pectin fractions, although some minor changes in the band intensities suggest an enrichment of the carbonate fraction in rhamnogalacturonan-I pectin domains. SEC analysis showed that the average molecular weight of the CDTA pectin was higher than that of the carbonate extract. AFM histograms of polymer length were well approximated by a Log normal distribution function for both pectin fractions. The CDTA and Na2CO3 polymer length distributions were statistically different with mean values of ∼87 and 65 nm, respectively. AFM studies revealed the presence of sidechains and multiple branching, previously attributed to branching of the galacturonic acid backbone. Both fractions were about 9% branched, but the Na2CO3 samples showed a higher percentage of multi-branched polymers. These results demonstrate that AFM is an excellent tool revealing new information on pectin structure which, when combined with classical techniques such as FTIR or SEC, provides a deeper characterization of fruit pectins.