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  • Author or Editor: Shohei Yamaki x
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The regulation of NAD+-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14) by sugar was investigated by using sliced tissues of japanese pear (Pyrus serotina Nakai cv. Kousui) fruit in order to determine its role in the mechanism of sugar accumulation in fruit tissue. The results of the activities and steady-state levels of the protein and mRNA indicate that NAD-SDH in japanese pear fruit is among the sugar-inducible genes. By preincubating the sliced tissues for 16 hours in a medium without sugar, NAD-SDH activity declined and reached a stable level that was maintained for up to 40 hours. The washing procedure also reduced the sugar concentration in the apoplast and cytosol of the sliced tissues to low concentrations and enabled them to be manipulated by exogenous applications of carbohydrate solutions. Incubation of tissues in 50 or 100 mm sorbitol for 8 hours led to enhanced expression of the NAD-SDH gene as determined by increased mRNA and protein levels and enhanced enzyme activity. The presence of 100 mm glucose, sucrose, or mannitol also gave significant stimulation on the levels of activity, protein, and mRNA of NAD-SDH compared with those of control tissues bathed in media in which the osmotic potential had been adjusted to that of the sugar solutions by adding polyethylene glycol. However, fructose was ineffective in stimulating NAD-SDH activities and the level of the protein was not enhanced but the level of mRNA was increased. Therefore, it is suggested that NAD-SDH gene transcription is enhanced by each sugar investigated, and fructose appears to be unique as it also influences NAD-SDH at a post-transcriptional level.

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A cDNA library was constructed from poly(A)+RNA extracted from pollinated fruit of `PMR-142' cucumber (Cucumis sativus L.). Subtraction hybridization was made between the cDNAs and poly(A)+RNA from unpollinated fruit to isolate cDNA clones that corresponded to the genes preferentially expressed in the pollinated fruit. We isolated three cDNAs, which were 756, 826, and 998 nucleotides long and designated Csf1, Csf2, and Csf3, respectively. When fruit growth was triggered by pollination, auxin treatment and natural parthenocarpy, Csf2 was always expressed. Time course of expression of the Csf2 gene was nearly parallel to that of the fruit growth. Nucleotide sequences of the Csf cDNAs were fully determined. Homology of the deduced amino acid sequence for Csf1 showed 75% identity with a pea extensin. Only 37%, 33%, and 26% homology was found between Csf2 and bell pepper CaSn-2, tobacco FB7-4, and opium poppy gMLP15, respectively. The Csf3 sequence showed 68% identity with the large subunit of 60S ribosomal protein L3 of Arabidopsis thaliana.

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Galactosidases are thought to play a key role in cell wall metabolism during fruit growth and ripening. In this study we cloned seven β-galactosidase (β-Gal) cDNAs from japanese pear fruit and designated them PpGAL2, PpGAL3, Pp-GAL4, PpGAL5, PpGAL6, PpGAL7, and PpGAL8, in addition to the previously described JP-GAL hereinafter termed PpGAL1. mRNA expression patterns of these clones were characterized throughout fruit growth and on-tree ripening, and in leaves and shoots in three japanese pear cultivars, `Housui', `Kousui', and `Niitaka'. The shared amino acid sequence identity among the eight japanese pear β-Gal (PpGAL) clones ranged from 50% to 60%. They all contained the putative active site containing consensus sequence pattern G-G-P-[LIVM](2)-x(2)-Q-X-E-N-E-[FY] belonging to glycoside hydrolase family 35. Expression of all the clones was both development- and tissue-specific. PpGAL1 and Pp-GAL4 were only expressed in the ripe fruit while PpGAL2 and PpGAL3 were expressed in both expanding and ripening fruit with their abundance being highest in the ripe fruit. The abundance of PpGAL5, PpGAL6, and PpGAL7 mRNAs was highest in expanding fruit but decreased drastically upon the onset of ripening. PpGAL8 was only detected in very young fruit (15 days after full bloom) and not in expanding and ripening fruit. These results indicate that in japanese pear fruit β-Gal is encoded by a multigene family whose members show distinct and overlapping expression during the various phases of fruit development. Some of the members are not only fruit-specific but also ripening-specific and, therefore, may play a crucial role in cell wall disassembly during japanese pear fruit softening.

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