Characterization of ethylene biosynthesis associated with ripening in banana fruit

Abstract

We investigated the characteristics of ethylene biosynthesis associated with ripening in banana (Musa sp. [AAA group, Cavendish subgroup] cv Grand Nain) fruit. MA-ACS1 encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase in banana fruit was the gene related to the ripening process and was inducible by exogenous ethylene. At the onset of the climacteric period in naturally ripened fruit, ethylene production increased greatly, with a sharp peak concomitant with an increase in the accumulation of MA-ACS1 mRNA, and then decreased rapidly. At the onset of ripening, the in vivo ACC oxidase activity was enhanced greatly, followed by an immediate and rapid decrease. Expression of the MA-ACO1 gene encoding banana ACC oxidase was detectable at the preclimacteric stage, increased when ripening commenced, and then remained high throughout the later ripening stage despite of a rapid reduction in the ACC oxidase activity. This discrepancy between enzyme activity and gene expression of ACC oxidase could be, at least in part, due to reduced contents of ascorbate and iron, cofactors for the enzyme, during ripening. Addition of these cofactors to the incubation medium greatly stimulated the in vivo ACC oxidase activity during late ripening stages. The results suggest that ethylene production in banana fruit is regulated by transcription of MA-ACS1 until climacteric rise and by reduction of ACC oxidase activity possibly through limited in situ availability of its cofactors once ripening has commenced, which in turn characterizes the sharp peak of ethylene production.

Figure 1

RNA blot showing the differential expression of three banana ACC synthase genes in the flesh tissue of preclimacteric, ripening, and wounded fruit. The lanes are: 1, preclimacteric fruit; 2, fruit ripened naturally; 3, fruit ripened by application of exogenous ethylene; and 4, fruit subjected to wounding. Each lane contains 5 μg of mRNA. MA-Actin was used as an internal control to normalize the amount of mRNA loaded.

Figure 2

Genomic Southern-blot analysis of banana ACC synthase genes. DNA purified from sprouting leaves was digested with EcoRI, HindIII, KpnI, or BamHI, fractionated on a 0.8% (w/v) agarose gel, and blotted to a nylon membrane. The membrane was hybridized to the ³²P-labeled MA-ACS1, MA-ACS2, and MA-ACS3 probes. Blots were washed with high-stringency buffer as described in “Materials and Methods,” and subsequently subjected to autoradiography.

Figure 3

A, Changes in ethylene biosynthesis in banana fruit ripened naturally. a, Ethylene production rate; b, ACC content; c, in vivo (●) and in vitro (○) ACC oxidase activities. B, Expression of ACC synthase and ACC oxidase genes. Individual fruit was separated from one hand and ripened naturally at 22°C. On each sampling day, ethylene production was determined in one fruit, and then flesh from the same fruit was used for the determination of ACC content, ACC oxidase activity assay, and RNA extraction for northern analysis. Each lane (B) contains 10 μg of mRNA for MA-ACS1 and MA-Actin and 1.5 μg for MA-ACO1. MA-Actin was used as an internal control to normalize the amount of mRNA loaded.

Figure 4

A, Changes in ethylene biosynthesis in banana fruit ripened by exogenous ethylene. a, Ethylene production rate; b, ACC content; c, in vivo ACC oxidase activity. B, Expression of ACC synthase and ACC oxidase genes. Individual fruit was separated from one hand, treated with 100 μL L⁻¹ ethylene for 18 h, and then ripened at 22°C. On each sampling day, ethylene production was determined in one fruit and then the flesh was used for determination of in vivo ACC oxidase activity, ACC content, and gene expression. Vertical bars represent means ± se (n = 3). Each lane (B) contains 10 μg of mRNA for MA-ACS1 and MA-Actin and 1.5 μg for MA-ACO1. MA-Actin was used as an internal control to normalize the amount of mRNA loaded.

Figure 5

Changes in free ascorbate (A), soluble iron content (B), in vivo ACC oxidase activity measured in the presence (○) and absence (●) of ascorbate and iron (C), and in vitro ACC oxidase activity (D) during ripening in banana fruit treated with ethylene. Individual fruit was separated from one hand, treated with 100 μL L⁻¹ ethylene for 18 h, and then ripened at 22°C. Vertical bars represents means ± se (n = 3).