Teresa F. Wegrzyn

Apple beta-galactosidase. Activity against cell wall polysaccharides and characterization of a related cDNA clone.

A [beta]-galactosidase was purified from cortical tissue of ripe apples (Malus domestica Borkh. cv Granny Smith) using a procedure involving affinity chromatography on lactosyl-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that two polypeptides of 44 and 32 kD were present in the fraction that showed activity against the synthetic substrate p-nitrophenol-[beta]-D-galactopyranoside. The enzyme preparation was incubated with polysaccharide extracts from apple cell walls containing [beta]-(1–>4)-linked galactans, and products of digestion were analyzed by gas chromatography. Small amounts of monomeric galactose were released during incubation, showing that the enzyme was active against native substrates. Amino acid sequence information was obtained from the purified protein, and this showed high homology with the anticipated polypeptide coded by the ethylene-regulated SR12 gene in carnation (K.G. Raghothama, K.A. Lawton, P.B. Goldborough, W.R. Woodson [1991] Plant Mol Biol 17: 61–71) and a harvest-related pTIP31 cDNA from asparagus (G. King, personal communication). Using the asparagus cDNA clone as a probe, an apple homolog (pABG1) was isolated. This clone contains a 2637-bp insert, including an open reading frame that codes for a polypeptide of 731 amino acids. Cleavage of an N-terminal signal sequence would leave a predicted polypeptide of 78.5 kD. Genomic DNA analysis and the isolation of other homologous apple clones suggest that pABG1 represents one member of an apple [beta]-galactosidase gene family. Northern analysis during fruit development and ripening showed accumulation of pABG1-homologous RNA during fruit ripening. Enzyme activity as measured in crude extracts increased during fruit development to a level that was maintained during ripening.

Changes in Kiwifruit Cell Wall Ultrastructure and Cell Packing During Postharvest Ripening

Light, scanning electron, and transmission electron micrographs of ripening kiwifruit were compared at different stages of softening. The three tissues of the fruit, outer pericarp, inner pericarp, and core, differed in packing and cell size. Morphometric studies indicated that the volume of intercellular air spaces increased as the fruit softened. Cell walls swelled in the outer pericarp carlier than in the core and in both tissues well before the climacteric. Except for plasmodesmatal regions, staining of cell wall material markedly declined or was lost in ripe fruit. Middle lamella breakdown was evident in samples taken from both ripe fruit and fruit just prior to ripeness, and electron-dense bodies accumulated between the plasmalemma and cell wall. The results support studies on changes in chemical composition and indicate that kiwifruit cell walls undergo ultrastructural changes during ripening similar to other fruit. However, the extent of cell wall swelling, loss of stainability, and timing of the changes differs.

LeMAN4 endo-β-mannanase from ripe tomato fruit can act as a mannan transglycosylase or hydrolase

Mannan transglycosylases are cell wall enzymes able to transfer part of the mannan polysaccharide backbone to mannan-derived oligosaccharides (Schröder et al. in Planta 219:590–600, 2004). Mannan transglycosylase activity was purified to near homogeneity from ripe tomato fruit. N-terminal sequencing showed that the dominant band seen on SDS-PAGE was identical to LeMAN4a, a hydrolytic endo-β-mannanase found in ripe tomato fruit (Bewley et al. in J Exp Bot 51:529–538, 2000). Recombinant LeMAN4a protein expressed in Escherichia coli exhibited both mannan hydrolase and mannan transglycosylase activity. Western analysis of ripe tomato fruit tissue using an antibody raised against tomato seed endo-β-mannanase revealed four isoforms present after 2D-gel electrophoresis in the pH range 6–11. On separation by preparative liquid isoelectric focussing, these native isoforms exhibited different preferences for transglycosylation and hydrolysis. These results demonstrate that endo-β-mannanase has two activities: it can either hydrolyse mannan polysaccharides, or in the presence of mannan-derived oligosaccharides, carry out a transglycosylation reaction. We therefore propose that endo-β-mannanase should be renamed mannan transglycosylase/hydrolase, in accordance with the nomenclature established for xyloglucan endotransglucosylase/hydrolase. The role of endo-acting mannanases in modifying the structure of plant cell walls during cell expansion, seed germination and fruit ripening may need to be reinterpreted in light of their potential action as transglycosylating or hydrolysing enzymes.

Stability of antioxidants in an apple polyphenol-milk model system

The stability of antioxidants in an apple polyphenol-milk model system was examined. The model system consisted of skim milk fortified with pH-neutralised apple polyphenols (AP, 0-200mg per 100ml milk), with or without ascorbic acid (100mg per 100ml milk). Physical and chemical changes were evaluated after thermal treatment (120°C, 5min) and oxidative storage (20°C and 38°C, up to 12 weeks). Antioxidant capacity was determined using both oxygen radical absorbance capacity (ORAC) assay and ferric reducing antioxidant power (FRAP) assay. Significant antioxidant capacity was detected in the presence of milk. Antioxidant capacity was retained during thermal treatment but decreased slowly during storage. The concentration of ascorbic acid decreased rapidly, and was close to zero after 2-week storage at 38°C or 10-week storage at 20°C. The brownness of the polyphenol-milk system increased over storage duration of 0-12 weeks; this effect was retarded by the addition of ascorbic acid. This high polyphenol-milk has demonstrated good physical stability.

Alpha-amylase and starch degradation in kiwifruit

Summary Alpha-amylase activities and activity gel profiles were measured in kiwifruit Actinidia deliciosa var. deliciosa ([A. Chev.], C. F. Liang and Ferguson cv. Hayward) during fruit development and postharvest ripening. As fruit developed, alpha-amylase activity increased on a per fruit basis, mirroring the increase in starch content in the fruit. Alpha-amylase activity increased approximately 2-fold between 134 days after anthesis (DAA), (when fruit starch content is at its maximum before beginning to decline) and harvest. During postharvest ripening with and without ethylene treatment at 20 °C, or during postharvest storage at 0 °C, enzyme activity remained similar to harvest values until fruit were ripe and most of the starch converted to sugars. At this point activity decreased 4–5-fold. Several amylolytic bands as well as starch phosphorylase and debranching enzyme were visible in starch activity gels. Starch phosphorylase was lower in ripe fruit, as was the intensity of two amylolytic bands. None of the other amylolytic bands or the deb ranching enzyme changed in response to fruit development or ripening. The same results occurred if gels were incubated at pH 6.1 or pH 8.0. These results are discussed in relation to the initiation of net starch degradation at the commencement of fruit ripening.

Enthalpies of mixing of aqueous solutions of the amino acids glycine, l-alanine and l-serine

The enthalpies of mixing of aqueous solutions of the three amino acids glycine, L-alanine and L-serine have been determined at 25°C. These data have been analyzed, using the McMillan-Mayer theory, to obtain the various enthalpic pair interaction coefficients, hxy. The results are discussed in terms of the likely molecular interactions. The application of the Savage-Wood additivity principle to these solute systems is also considered.

Starch Degradation in Kiwifruit: In vivo and In vitro Ultrastructural Studies

Kiwifruit were examined at harvest and during postharvest ripening to quantify structural changes to starch granules as net starch degradation occurred. The changes were compared with those found in in vitro studies utilizing granules prepared from fruit at harvest. In in vitro studies starch granules were incubated with various cocktails of starch-degrading enzymes and examined by scanning and transmission electron microscope (TEM) at sequential points during enzyme incubation. Only α-amylase, or α-amylase in combination with other enzymes, was able to degrade the native granules. A combination of α-amylase and α-glucosidase resulted in less degradation than with α-amylase alone. Crude extracts from kiwifruit or pea leaf resulted in minimal degradation under the conditions tested. In all experiments α-amylase attack began by creating channels to the center of the granule, which was then followed by radial degradation from the center outward. An external shell was generally all that remained. This pattern was not detected in vivo. Here degradation appeared to be from the outside in. Often individual granules within a plastid appeared to resist degradation when adjacent ones were being hydrolyzed. Measurements of granule diameter, both in vivo and with extracted granules, also indicated that large granules may be preferentially degraded after fruit harvest. These results are discussed in relation to control of starch degradation.