In ripening fruits of tomato (Lycopersicon esdentum 1. var 83-C-38), the amounts of cellulose and xyloglucan (XC) remained constant during tissue softening, but the relative molecular weight (M,) of XG decreased markedly and the M, of cellulose declined slightly. These changes could have been due to adivities of nonspecific endo-1,4-@-glucanases and/or buffer-soluble XC endotransglycosylase, both of which increased when tissue firmness declined most rapidly. Tomato extracts also reduced the viscosity of XC solutions, especially in the presence of added XC oligosaccharides. This depolymerizing (XCase) capacity differed from 8-glucanase and XC transglycosylase activity (a) by being almost entirely buffer insoluble, and (b) by declining precipitously during fruit softening. Although it disappeared from ripe fruit, XCase may have functioned in promoting wall loosening at earlier stages of fruit development when its activity was highest. By contrast, during aging of fruit in the ripening-inhibited mutant rin there was no change in M, of XC or cellulose, and activities of B-glucanases and XC transglycosylase were lower than in wild-type tomato. Nevertheless, some softening of the fruit did take place over time and XC amounts declined, possibly because high XCase activity was maintained in the mutant, unlike in wild-type fruit.
Two distinct partial cDNAs, PRFl and PRF3, similar in sequence to previously described polygalacturonases, were amplified from ripe peach (Prunus persica 1. Batsch cv Flavorcrest) fruit cDNA by the polymerase chain reaction. PRF1-related RNA was present in fruit from early ripening at levels not detected by northern analysis. PRF3-related RNA was readily detectable in ripe fruit by northern analysis. PRF3 was used to isolate a cDNA with a complete open reading frame, PRFS, from a XZAP li cDNA library prepared from poly(A)+ RNA of ripe peach fruit. PRF5 coded for a predicted protein of 393 amino acids with a molecular mass of 41,500 D. The derived amino acid sequence of PRFS included a putative leader sequence of 23 amino acids, followed by a sequence that matched the N terminus of endopolygalacturonase protein purified from ripe peach fruit. By northern analysis, PRF3-related RNA was undetectable in firm, unripe Flavorcrest fruit. It appeared at low levels as a 1.7-kb transcript in fruit that had begun to ripen and soften and was very abundant in ripe fruit that had undergone the "melting" stage of softening. The marked increase in PRF3-related RNA levels took place over a period of less than 2 d at 20°C and coincided with the climacteric peak in ethylene evolution. Levels of 1 -aminocyclopropane-1 -carboxylate oxidase-related RNA increased during ripening at a much earlier stage than levels of PRF3-related RNA. Lower levels of 1.7-kb RNA transcript were detected by PRF3 in ripe fruit of the melting cultivar Fragar, which are firmer than Flavorcrest fruit. In ripe fruit of the nonmelting cultivar Carolyn, PRF3 detected a 1.45-kb RNA transcript that was present at low levels. Transcripts of a peach polygalacturonase-related genomic sequence were not detected in ripening fruit.
The use of the edible photosynthetic cyanobacterium Arthrospira platensis (spirulina) as a biomanufacturing platform has been limited by a lack of genetic tools. Here we report genetic engineering methods for stable, high-level expression of bioactive proteins in spirulina, including large-scale, indoor cultivation and downstream processing methods. Following targeted integration of exogenous genes into the spirulina chromosome (chr), encoded protein biopharmaceuticals can represent as much as 15% of total biomass, require no purification before oral delivery and are stable without refrigeration and protected during gastric transit when encapsulated within dry spirulina. Oral delivery of a spirulina-expressed antibody targeting campylobacter—a major cause of infant mortality in the developing world—prevents disease in mice, and a phase 1 clinical trial demonstrated safety for human administration. Spirulina provides an advantageous system for the manufacture of orally delivered therapeutic proteins by combining the safety of a food-based production host with the accessible genetic manipulation and high productivity of microbial platforms.
Post-phloem sugar transport in developing tomato (Lycopersicon esculentum Mill. cv. Flora-Dade) fruit follows an apoplastic route during the rapid phase of sugar accumulation. The pathway is characterized by sugar retrieval by the storage parenchyma cells from the fruit apoplast. Two tomato genotypes differing in fruit hexose content were compared in terms of the transport and transfer processes controlling fruit sugar levels. The genotypic difference in fruit sugar content was independent of photoassimilate export from source leaves. Discs of pericarp tissue were cultured in a medium based on analyses of the fruit apoplastic sap. The cultured discs maintained a composition, a relative growth rate and a respiration rate similar to those of the pericarp tissue of intact fruit. Estimates of hexose fluxes into metabolic and storage pools suggested that membrane transport regulated the genotypic difference in hexose accumulation. Short-term ['"^Clhexose uptake experiments demonstrated a genotypic difference in V^^^ for glucose, fructose and 3-O-methylglucose, and this difference was abolished in the presence of the inhibitor /7-chloromercuribenzenesulphonic acid (PCMBS). The results support the hypothesis that the activity of energized hexose carriers on the plasma membranes of storage parenchyma cells is a signiflcant determinate of the genotypic difference in hexose accumulation.
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