Differential Protein Accumulation in Banana Fruit during Ripening
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Abstract:
Banana (Musa acuminata, cv Dwarf Cavendish) proteins were extracted from pulp tissue at different stages of ripening and analyzed by two-dimensional electrophoresis. The results provide evidence of differential protein accumulation during ripening. Two sets of polypeptides have been detected that increase substantially in ripe fruit. These polypeptides were characterized as glycoproteins by western blotting and concanavalin A binding assays. Antibodies againts tomato polygalacturonase cross-react with one of these sets of proteins.Keywords:
Musaceae
Musa acuminata
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Cleavage (geology)
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The minimum treatment time necessary for ethylene to induce ripening (MTT) in banana fruit was examined in relation to the concentration of applied ethylene and temperature.Little difference was observed in the magnitude and rate of the climacteric between the fruits treated with ethylene only for the MTT period and those continuously exposed to ethylene. Evolution of ethylene from fruit treated with exogenous ethylene for a period less than the MTT may return to a preclimacteric level on removal to air. Applied ethylene probably induces an increase in both ethylene forming enzyme (EFE) and 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activity, with EFE activity increasing first. Although the actual MTT of fruit varied considerably in different clusters, the difference in MTT for different conditions was almost constant within one cluster. The data suggest that there may be a linear relation between the MTT of bananas and the logarithm of the applied ethylene concentration, and that the effect of treatment temperature may be expressed as a quadratic function.
Climacteric
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During ripening of bananas (Musa spp. [AAA group, Cavendish subgroup]), there is a massive conversion of starch to sucrose. Also during ripening there is a rise in respiration known as the respiratory climacteric. In this study changes in carbohydrate content, activities of starch and sucrose metabolizing enzymes, and respiration were measured to assess their potential interrelationships. Sucrose phosphate synthase activity increased dramatically during the first 4 days after initiation of ripening by ethylene treatment. Starch concentration decreased and sucrose concentration increased during this time period. Developmental changes in sucrose phosphate synthase activity were measured with limiting substrate (plus Pi) and saturating substrate concentrations. Activities were not parallel under the two assay conditions, providing tentative evidence that kinetically different forms of the enzyme may exist at different stages of ripening. Sucrose accumulation rate was most highly correlated with sucrose phosphate synthase activity assayed with limiting substrate concentrations (plus Pi). The cumulative amount of CO(2) respired during ripening was positively correlated with sugar accumulation (R(2) = 0.97). From this linear regression it was calculated that a constant 0.605 millimoles of CO(2) was evolved per mole of sucrose formed throughout ripening. Using this quantity, the percentage of the total respiratory ATP produced which was required for the conversion of starch to sucrose was calculated assuming different models for carbon export from the amyloplast. The results suggest that sucrose biosynthesis during ripening constitutes a significant sink for respiratory ATP.
Sucrose-phosphate synthase
Climacteric
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Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins (l), a number of modified analytical procedures ut.ilizing this reagent have been reported for the determination of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and in insulin (10).Although the reagent would seem to be recommended by its great sensitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes.In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard t.o effects of variations in pH, time of reaction, and concentration of reactants, permissible levels of reagents commonly used in handling proteins, and interfering subst.ances.Procedures are described for measuring protein in solution or after precipitation wit,h acids or other agents, and for the determination of as little as 0.2 y of protein. MethodReagents-Reagent A, 2 per cent N&OX in 0.10 N NaOH.Reagent B, 0.5 per cent CuS04.5Hz0 in 1 per cent sodium or potassium tartrabe.Reagent C, alkaline copper solution.Mix 50 ml. of Reagent A with 1 ml. of Reagent B. Discard after 1 day.Reagent D, carbonate-copper solution, is the same as Reagent C except for omission of NaOH.Reagent E, diluted Folin reagent.Titrate Folin-Ciocalteu phenol reagent ((II), Eimer and Amend, Fisher Scientific Company, New York) with NaOH t.o a phenolphthalein end-point.On the basis of this titration dilute the Folin reagent (about 2-fold) to make it 1 N in acid.Working standards may be prepared from human serum diluted IOO-to lOOO-fold (approximately 700 to 70 y per ml.).These in turn may be checked against a standard solution of crystalline bovine albumin (Armour and
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Lycopersicon
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Abstract During banana (Musa acuminata L.) fruit ripening ethylene production triggers a developmental cascade that is accompanied by a massive conversion of starch to sugars, an associated burst of respiratory activity, and an increase in protein synthesis. Differential screening of cDNA libraries representing banana pulp at ripening stages 1 and 3 has led to the isolation of 11 nonredundant groups of differentially expressed mRNAs. Identification of these transcripts by partial sequence analysis indicates that two of the mRNAs encode proteins involved in carbohydrate metabolism, whereas others encode proteins thought to be associated with pathogenesis, senescence, or stress responses in plants. Their relative abundance in the pulp and tissue-specific distribution in greenhouse-grown banana plants were determined by northern-blot analyses. The relative abundance of transcripts encoding starch synthase, granule-bound starch synthase, chitinase, lectin, and a type-2 metallothionein decreased in pulp during ripening. Transcripts encoding endochitinase, β-1,3-glucanase, a thaumatin-like protein, ascorbate peroxidase, metallothionein, and a putative senescence-related protein increased early in ripening. The elucidation of the molecular events associated with banana ripening will facilitate a better understanding and control of these processes, and will allow us to attain our long-term goal of producing candidate oral vaccines in transgenic banana plants.
Starch synthase
Musa acuminata
Musaceae
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Two exo‐acting polygalacturonase enzymes (exoPG, EC 3.2.1.67) increase in activity as peach ( Prunu persica L. Batsch cvs Coronet and Flavorcrest) fruits ripen. By examining populations of fruit, we show that the increase in activity occurs late in ripening when the fruit are very soft (below 2 kgf). The more abundant form of the enzyme, exoPG 2, was extensively purified and analyzed for its amino acid content and N‐terminal amino acid sequence. ExoPG 2 is a polypeptide of M, 66 000 and has a substantial excess of basic over acidic amino acids. Polyclonal antisera to exoPG 2 were raised in mice. The antisera inhibited the enzyme activity and recognized a M r 66 000 polypeptide in Western blots. Western blot analyses of extracts of fruit ranked for softness revealed a M r 66 000 polypeptide only in the softest fruit (less than 2.5 kgf). We conclude that the increased in exopolygalacturonase activity that occurs in very soft fruit is due to an increase in the amount of enzyme protein.
Polyclonal antibodies
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Coomassie Brilliant Blue
Bradford protein assay
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Expansin
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