Ethylene biosynthesis in detached young persimmon fruit is initiated in calyx and modulated by water loss from the fruit

Abstract

Persimmon (Diospyros kaki Thunb.) fruit are usually classified as climacteric fruit; however, unlike typical climacteric fruits, persimmon fruit exhibit a unique characteristic in that the younger the stage of fruit detached, the greater the level of ethylene produced. To investigate ethylene induction mechanisms in detached young persimmon fruit, we cloned three cDNAs encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (DK-ACS1, 2, and -3) and two encoding ACC oxidase (DK-ACO1 and -2) genes involved in ethylene biosynthesis, and we analyzed their expression in various fruit tissues. Ethylene production was induced within a few days of detachment in all fruit tissues tested, accompanied by temporally and spatially coordinated expression of all the DK-ACS and DK-ACO genes. In all tissues except the calyx, treatment with 1-methylcyclopropene, an inhibitor of ethylene action, suppressed ethylene production and ethylene biosynthesis-related gene expression. In the calyx, one ACC synthase gene (DK-ACS2) exhibited increased mRNA accumulation accompanied by a large quantity of ethylene production, and treatment of the fruit with 1-methylcyclopropene did not prevent either the accumulation of DK-ACS2 transcripts or ethylene induction. Furthermore, the alleviation of water loss from the fruit significantly delayed the onset of ethylene production and the expression of DK-ACS2 in the calyx. These results indicate that ethylene biosynthesis in detached young persimmon fruit is initially induced in calyx and is modulated by water loss through transcriptional activation of DK-ACS2. The ethylene produced in the calyx subsequently diffuses to other fruit tissues and acts as a secondary signal that stimulates autocatalytic ethylene biosynthesis in these tissues, leading to a burst of ethylene production.

Figure 1

Expression of DK-ACS and DK-ACO gene families during ripening in persimmon fruit. Fruit harvested at mature stage (156 d AFB) were held at 20°C in ambient laboratory humidity for 30 d or treated with 50 μL L⁻¹ of exogenous ethylene for 2 d. Lane 1, Fruit at harvest; lane 2, fruit held for 30 d; lane 3, ethylene-treated fruit. Each lane contained 5 μg of total RNA, and the transcript levels of 17S rRNA are shown as an internal loading control.

Figure 2

Schematic diagrams showing a perspective view and a longitudinal section of persimmon fruit.

Figure 3

Effects of 1-MCP treatment on the rate of whole-fruit ethylene production (A) and flesh firmness (B) in detached young persimmon fruit during storage at 20°C. Fruit were detached at young stage (65 d AFB) and held at 20°C in ambient laboratory humidity (40%–60% RH). Immediately after detachment, fruit were treated with 200 nL L⁻¹ 1-MCP for 3 h. Each point represents the mean value for three fruits and vertical bars represent ±se.

Figure 4

Effect of 1-MCP treatment on the rate of ethylene production in various tissues of young persimmon fruit during storage at 20°C. Each point represents the mean of three replications and vertical bars represent ±se.

Figure 5

Northern-blot analysis of the expression of DK-ACS and DK-ACO genes in the pulp of young persimmon fruit treated with or without 1-MCP during storage at 20°C. Each lane contained 5 μg of total RNA, and the transcript levels of 17S rRNA are shown as an internal loading control.

Figure 6

Expression of DK-ACS and DK-ACO gene families in various tissues of young persimmon fruit at 3 d after detachment with or without 1-MCP treatment. Each lane contained 5 μg of total RNA, and the transcript levels of 17S rRNA are shown as an internal loading control. AZ, Abscission zone.

Figure 7

Changes in the accumulation of mRNAs corresponding to DK-ACS and DK-ACO genes in the calyx of young persimmon fruit treated with or without 1-MCP during storage at 20°C. Each lane contained 5 μg of total, and the transcript levels of 17S rRNA are shown as an internal loading control.

Figure 8

Effects of packaging fruit in perforated polyethylene bags with different numbers of holes on weight loss (A) and the rate of ethylene production (B) in persimmon fruit detached at a young stage (70 d AFB). Ten fruit per treatment were packaged individually in perforated polyethylene bags (150 × 130 mm) with two, eight, or 18 holes (4-mm diameter) equivalent to 0.03%, 0.15%, or 0.3% of the TFSA, respectively. Non-packaged fruit were used as control. Each column represents the mean of 10 fruits and vertical bars represent ±se.

Figure 9

Changes in the rate of ethylene production (A) and the accumulation of mRNAs corresponding to members of the DK-ACS and DK-ACO gene families (B) in the calyx of young persimmon fruit held in low- or high-humidity conditions. Low humidity was equivalent to ambient laboratory conditions (40%–60% RH), and high-humidity conditions (95% RH) were maintained in a container through which humidified air was passed at 250 mL min⁻¹. Each point in A represents the mean of three replications and vertical bars represent ±se. Each lane in B contained 5 μg of total RNA, and the transcript levels of 17S rRNA are shown as an internal loading control.

Figure 10

Changes in the rate of ethylene production (A) and the accumulation of mRNAs corresponding to DK-ACS and DK-ACO genes (B) in the pulp of young persimmon fruit held in low- (40%–60% RH) or high- ( 95% RH) humidity conditions. Each point in A represents the mean of three replications and vertical bars represent ±se. Each lane in B contained 5 μg of total RNA, and the transcript levels of 17S rRNA are shown as an internal loading control.