An improved (13)C-density-labeling method was used to study cell wall synthesis in rapidly expanding, slowly expanding and recently mature internodes of Nitella translucens var axillaris (A.Br.) R.D.W. As cells matured, the rate of wall synthesis slowed and the deposition of cellulose microfibrils changed from a predominantly transverse direction in the primary wall of rapidly expanding internodes to a helicoidal array in the secondary wall of mature internodes. The secondary wall was characterized by relatively higher rates of cellulose synthesis and lower rates of pectin synthesis than the primary wall. The synthesis of xyloglucan also decreased markedly at the transition to secondary wall synthesis, while the synthesis of mannose-rich hemicellulose increased. Even though structural differences were striking between the primary and secondary walls of Nitella, compositional differences between the two types of wall were quantitative rather than qualitative.
A gas chromatographic-mass spectrometric technique utilizing D-[U-13C]glucose as a density label tracer was used to follow the synthesis of cell wall polysaccharides in pericarp discs that were excised from mature green tomato fruit (Lycopersicon esculentum) and allowed to ripen in culture. The biosynthetic capacity of discs from four different maturity stages was examined. Label was differentially incorporated into wall polysaccharides as the discs matured, indicating a change in the nature of wall polymers being synthesized. These differential rates of incorporation are consistent with descriptions of ripening-related cell wall compositional changes previously reported by other authors. Specific changes in wall biosynthesis noted include increased incorporation of xylosyl and mannosyl residues into hemicellulosic cell wall fractions as the discs mature and decreased incorporation of galactosyl residues into chelator-soluble pectins.Digestion of cell wall components has been the primary focus of research for several decades aimed at understanding ripening-related tissue softening of fruits (4,(11)(12)(13)21 saccharides well after the onset of ripening even though the total amount of cell wall material (per gram fruit fresh weight) is steadily decreasing. Synthesis continued into the red maturity stage, and they suggested that these synthetic events might be important in fruit softening. The results of this study generally indicate that cell wall polysaccharide synthesis continued well into the latter stages of tomato fruit ripening, but they provided no clear information concerning the nature of the polymers being synthesized or incorporation into individual monosaccharide residues. We have developed an analytical approach (9) (6). This combination of analytical approach and ripening system allows us to examine in detail incorporation at any stage of the ripening process. Detection of the label (and, thus, incorporation into the wall) is accomplished by means of a GC-MS analysis which compares the isotope ratio of selected ion pairs from individual monosaccharide residues. This method is similar to the technique of Sasaki et al. (18) who examined the fate of density-labeled inositol in the cell walls of germinating beans but is useful for the analysis of a greater range of monosaccharides because of the more general distribution of carbon from glucose into other sugars. The technique can also be applied to permethylated samples of cell wall polysaccharides and thus provide information regarding incorporation into specific cell wall polymer types. In this paper we demonstrate the utility of this density label technique in exploring cell wall synthesis potential and provide data that show that this potential is expressed well into advanced stages of ripening in the tomato fruit. MATERIALS AND METHODS Disc Preparation and TreatmentTomato pericarp discs were prepared from-field grown tomato fruit (Lycopersicon esculentum Mill. cv "Castlemart"), as described in detail by Campbell et al. (6). Briefly, discs (...
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