Characterization of kiwifruit xyloglucan.

Structural characteristics of xyloglucan are constant in the pericarp cell walls of kiwifruit (Actinidia deliciosa) throughout fruit enlargement and maturation. Most of the xyloglucan (XG) persists in the cell walls of ripe kiwifruit. XG from the pericarp tissues of 36-h ethylene-treated kiwifruit was extracted as hemicellulose II (HC-II) with 4.28 M KOH containing 0.02% NaBH(4), and purified using iodine precipitation and subsequent anion-exchange chromatography. This purifying protocol increased XG purity from 50 mol% in HC-II fraction to 62 mol% in the purified XG powder. The molar ratio of glucose: xylose: galactose: fucose in the purified XG was 10: 6.9: 2.1: 0.3. Gel permeation chromatography indicated that purified XG had an average molecular-mass of 161 KDa, a value that exceeds the 95 KDa M(r) determined for total polymeric sugars. Sugar linkage analysis confirmed the lack of fucose in the kiwifruit XG, but a small amount of arabinoxylan and low M(r) glucomannan remained associated with this fraction.

Changes in the cell-wall polysaccharides of outer pericarp tissues of kiwifruit during development.

Changes in pectin, hemicelluloses and cellulose in the cell walls of outer pericarp tissues of kiwifruit (Actinidia deliciosa cv. Hayward) were determined during development. An extensive amylase digestion was employed to remove possible contaminating starch before and after fractionation of wall polysaccharides. An initial treatment of crude cell walls with alpha-amylase and iso-amylase or DMSO, was found to be insufficient removing the contaminating starch from wall polysaccharides. After EDTA and alkaline extraction, the pectic and hemicellulose fractions were again treated with the combination of alpha-amylase and iso-amylase. The amounts of predominant pectic sugars Gal, Rha and Ara, unaffected by the first and second amylase digestion, decreased markedly during the early fruit enlargement (8-12 weeks after anthesis, WAA), then increased during 16-20 WAA, and finally declined during fruit maturity (20-25 WAA). The molecular-mass of pectic polysaccharides decreased during fruit enlargement (8-16 WAA), and then changed little during fruit maturity. The higher molecular-mass components of hemicelluloses in HC-I and HC-II fractions detected at the early stage of fruit enlargement (8-12 WAA) were degraded at the late stage of fruit enlargement (16 WAA), but then remained stable at the much lower molecular-mass till fruit maturity. The amount of Xyl in the HC-II fraction decreased during the early fruit enlargement and fruit maturity, an observation that was consistent with xyloglucan (XG) content. The gel permeation profiles of XG showed a slight increase in higher molecular-mass components during 8-12 WAA, but thereafter there was no significant down-shift of molecular-mass until harvest time. The cellulose fraction increased steadily during fruit enlargement through maturity, but the XG contents in HC-I and HC-II fractions remained at a low level during these stages. Methylation analysis of HC-I and HC-II fractions confirmed the low level of XG in the hemicellulosic fractions. It was suggested that pectin in the outer pericarp of kiwifruit was degraded at the early stage of fruit enlargement, but XG remains constant during fruit enlargement and maturation.

Expression and function of cell wall-bound cationic peroxidase in asparagus somatic embryogenesis.

Cultured asparagus (Asparagus officinalis L. cv Y6) cells induced to regenerate into whole plants through somatic embryogenesis secreted a 38-kD protein into cell walls. The full-length cDNA sequence of this protein (Asparagus officinalis peroxidase 1 [AoPOX1]) determined by reverse transcriptase-polymerase chain reaction showed similarity with plant peroxidases. AoPOX1 transcripts were particularly abundant during early somatic embryogenesis. To evaluate the in vivo function of AoPOX1 protein, purified recombinant AoPOX1 protein was reacted with a series of phenolic substrates. The AoPOX1 protein was effective in the metabolism of feruloyl (o-methoxyphenol)-substituted substrates, including coniferyl alcohol. The reaction product of coniferyl alcohol was fractionated and subjected to gas chromatography-mass spectrometry analysis and (1)H-nuclear magnetic resonance analysis, indicating that the oxidation product of coniferyl alcohol in the presence of AoPOX1 was dehydrodiconiferyl alcohol. The concentration of dehydrodiconiferyl alcohol in the cultured medium of the somatic embryos was in the range of 10(-8) M. Functions of the AoPOX1 protein in the cell differentiation are discussed.

Constitutive overexpression of a ripening-related pepper endo-1,4-beta-glucanase in transgenic tomato fruit does not increase xyloglucan depolymerization or fruit softening.

The ripening-related pepper endo-1,4-beta-D-glucanase (EGase) CaCel1 was over-expressed in transgenic tomato plants under the control of the constitutive 35S promoter to investigate the effects on plant growth and fruit softening of high levels of a potential cell wall-degrading activity. In transgenic fruit, recombinant CaCel1 protein was associated with a high-salt putative cell wall fraction, and extractable CMCase activity was increased by up to 20-fold relative to controls. However, the effects of high levels of EGase activity on fruit cell wall metabolism were relatively small. The largest consequence observed was a decrease of up to 20% in the amount of matrix glycans in a 24% KOH-soluble fraction consisting of polysaccharides tightly bound to cellulose. This decrease was confined to polysaccharides other than xyloglucan, did not affect the size distribution of remaining molecules, and was not correlated with a corresponding increase in glycans in a 4% KOH-soluble fraction loosely bound to cellulose, suggesting that the missing polymers had been degraded to fragments small enough to be lost from the extracts. The amount of matrix glycans in the 4% KOH-soluble fraction was not substantially changed, but the size distribution showed a small relative increase in the amount of polymers in a peak eluting close to a linear dextran marker of 71 kDa. This could be due either to an increase in the amount of polymers of this size, or to a loss from the extract of other polymers present in peaks of higher molecular weight. Transgenic fruit were not softer than controls but appeared the same or slightly firmer at both green and red developmental stages, and no differences in plant vegetative growth were observed. CaCel1 did not cause depolymerization of tomato fruit xyloglucan in vivo, but differences in the amount or molecular weight profile of other matrix glycans were observed. The data suggest that degradation of a proportion of matrix glycans other than xyloglucan does not result in fruit softening, and that fruit softening is not limited by the amount of EGase activity present during ripening.