Akitsugu Inaba

Regulation of genes encoding ethylene biosynthetic enzymes in peach (Prunus persica L.) fruit by carbon dioxide and 1-methylcyclopropene

Abstract We have cloned one member (PP-ACS1) of the 1-aminocyclopropane-1-carboxylate (ACC) synthase and two members (PP-ACO1 and PP-ACO2) of the ACC oxidase gene families in peach (Prunus persica L.) fruit and studied their expression characteristics during fruit ripening and treatment with CO2 and 1-methylcyclopropene (MCP), inhibitors of ethylene action. Northern analysis showed that the abundance of PP-ACS1, PP-ACO1 and PP-ACO2 mRNAs increased with fruit ripening in parallel with increases in ethylene production and activities of ACC synthase and ACC oxidase. The abundance of PP-ACO2 mRNA was much lower than that of PP-ACO1. CO2 and MCP treatment inhibited ethylene production, ACC synthase activity, and accumulation of PP-ACS1 mRNA. Although CO2 had little effect on ACC oxidase activity, it inhibited the accumulation of PP-ACO1 and PP-ACO2 mRNAs to the same levels as MCP. Wound-induced ethylene production, ACC synthase activity, and the abundance of PP-ACS1 mRNA were blocked and stimulated by CO2 and MCP, respectively. CO2 and MCP had no effect on wound-induced ACC oxidase activity but inhibited the accumulation of its mRNA. Wound-induced activities of ACC synthase and ACC oxidase, and abundance of their mRNAs were inhibited and stimulated, respectively, by exogenous ethylene. The translational inhibitor, cycloheximide inhibited wound-induced ethylene biosynthesis but super-induced the accumulation of PP-ACS1 and PP-ACO1 mRNAs, suggesting that their induction is a primary response to the inducer. These results suggest that the expression of PP-ACS1 and PP-ACO1 genes play a key role in the regulation of ethylene biosynthesis in peach fruit during ripening and in response to wounding. The results also indicate that wound-induced PP-ACS1 and PP-ACO1 genes are under negative and positive control, respectively. Further, using MCP we provide evidence indicating that CO2 does not regulate ACC synthase activity and expression of the PP-ACS1 gene in peach fruit during ripening and in response to wounding by antagonizing ethylene action.

Ethylene Biosynthesis in Detached Young Persimmon Fruit Is Initiated in Calyx and Modulated by Water Loss from the Fruit

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 theDK-ACS andDK-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-ACS2transcripts or ethylene induction. Furthermore, the alleviation of water loss from the fruit significantly delayed the onset of ethylene production and the expression of DK-ACS2in 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 ofDK-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.

Ripening-associated ethylene biosynthesis in tomato fruit is autocatalytically and developmentally regulated

To investigate the regulatory mechanism(s) of ethylene biosynthesis in fruit, transgenic tomatoes with all known LeEIL genes suppressed were produced by RNA interference engineering. The transgenic tomato exhibited ethylene insensitivity phenotypes such as non-ripening and the lack of the triple response and petiole epinasty of seedlings even in the presence of exogenous ethylene. Transgenic fruit exhibited a low but consistent increase in ethylene production beyond 40 days after anthesis (DAA), with limited LeACS2 and LeACS4 expression. 1-Methylcyclopropene (1-MCP), a potent inhibitor of ethylene perception, failed to inhibit the limited increase in ethylene production and expression of the two 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) genes in the transgenic fruit. These results suggest that ripening-associated ethylene (system 2) in wild-type tomato fruit consists of two parts: a small part regulated by a developmental factor through the ethylene-independent expression of LeACS2 and LeACS4 and a large part regulated by an autocatalytic system due to the ethylene-dependent expression of the same genes. The results further suggest that basal ethylene (system 1) is less likely to be involved in the transition to system 2. Even if the effect of system 1 ethylene is eliminated, fruit can show a small increase in ethylene production due to unknown developmental factors. This increase would be enough for the stimulation of autocatalytic ethylene production, leading to fruit ripening.

Water stress-induced ethylene in the calyx triggers autocatalytic ethylene production and fruit softening in ‘Tonewase’ persimmon grown in a heated plastic-house

Abstract ‘Tonewase’ Japanese persimmon fruit (Diospyros kaki Thunb.) grown in a heated plastic-house softens rapidly within several days of harvest, which is a major problem in marketing of this cultivar. In this study, we elucidated the involvement of water stress-induced ethylene in fruit softening and investigated the induction mechanism of this ethylene biosynthesis occurring in specific tissues at the molecular level. Two instances of increase in ethylene production were observed in fruit held in ambient low humidity conditions (40–60% RH), an initial increase on the 1st and 2nd days and a second increase on the 6th and 8th days after harvest. Increase in ethylene production was accompanied by rapid softening in these fruit. Fruit held in high humidity conditions (>95%) neither produced detectable levels of ethylene nor softened rapidly. Moreover, treatment of the fruit held in low humidity with 1-methylcyclopropene (1-MCP), a strong inhibitor of ethylene action, inhibited fruit softening remarkably. These results suggest the involvement of water stress-induced ethylene in fruit softening. 1-MCP also suppressed the second increase in ethylene production but not the initial increase, indicating that the initial increase is induced directly in response to the primary water stress signal while the second is induced autocatalytically by the ethylene produced during the initial phase. During the initial increase in ethylene, the calyx produced more than 5 nl g−1 h−1 of ethylene accompanied by increased accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC) and expression of DK-ACS2. In pulp, 0.5 nl g−1 h−1 of ethylene was detected but no increase in ACC content or expression of any ethylene biosynthetic genes was observed. During the second increase in ethylene, ethylene production in the calyx was not detected whereas the pulp produced 0.2–0.4 nl g−1 h−1 of ethylene with a marked increase in ACC content and expression of the two ACC synthase (DK-ACS1, DK-ACS2) and one ACC oxidase (DK-ACO1) genes. These results suggest that in plastic-house ‘Tonewase’ persimmon fruit, ethylene production is initiated in the calyx in response to water stress through activated expression of DK-ACS2, and this ethylene in turn induces autocatalytic ethylene production in the pulp. As the flesh firmness decreased markedly just after the initial ethylene production, the results also indicate that the fruit softening is due to the action of ethylene produced in the calyx.

Extending shelf-life of astringent persimmon (Diospyros kaki Thunb.) fruit by 1-MCP

Abstract We investigated the potential for commercial use of 1-methylcyclopropene (1-MCP) to extend the shelf life of ‘Tonewase’ and ‘Saijo’ fruit, Japanese astringent persimmon cultivars, in combination with a de-astringency treatment using high CO 2 concentrations. The non-1-MCP treated fruit softened within 5 days after harvest, resulting in unacceptable quality. The 1-MCP treatments at more than 100 nl l −1 for 16–48 h inhibited fruit softening for 12 and 16 days after harvest at room temperature, for ‘Tonewase’ and ‘Saijo’, respectively. Treatment with 10 nl l −1 1-MCP had a limited inhibitory effect on softening. A time lag of up to 12 h from harvest to the beginning of 1-MCP treatment did not reduce the beneficial effects of 1-MCP. Fruit treated with 1-MCP remained insensitive to ethylene for 4 days after the end of the treatment. These results indicate that 1-MCP has the potential to extend the shelf life of Japanese persimmons.

Electrical Impedance Analysis of Tissue Properties Associated with Ethylene Induction by Electric Currents in Cucumber (Cucumis sativus L.) Fruit

A study based on electrical impedance analysis of tissue properties was conducted in order to understand the relationship between impedance components and ethylene biosynthesis induced by direct current in cucumber (Cucumis sativus L.) fruit. Impedances were measured at a range of alternating current frequencies from 500 Hz to 1 MHz. We calculated capacitances representing the plasma membranes, C1, and organelle membranes, C2, and resistances representing extracellular space, R1, cytoplasm, R2, and organelle interior, R3. Direct current of 1 to 3 mA induced ethylene synthesis with a sharp peak at 1 h. The rate of production was greater with a stronger current. This abrupt induction of ethylene synthesis was accompanied by an equally abrupt activation of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase within 1 h, but not that of ACC oxidase, which was activated only at the later stages of the treatment at a time when ethylene production and ACC synthase activity were declining. Using direct current of 0 to 3 mA, C2, R1, and R2 increased abruptly, and C1 increased gradually after 3 h. The rates of increases were greater with currents of larger magnitude, R3 was not affected during passage of the current. Diazocyclopentadiene, an inhibitor of ethylene action, eliminated the direct current induction of R1 but had no effect on the increases in C2 and R2. Diazocyclopentadiene counteracted the stimulative effects of exogenously applied ethylene with respect to respiration and activities of ACC oxidase and phenylalanine ammonia-lyase. These results indicate that an externally applied current may generate signal(s) by altering the functions of organelle membranes and/or cytoplasmic pH to induce ACC synthase.

Induction of ethylene biosynthesis and polyamine accumulation in cucumber fruit in response to carbon dioxide stress

Abstract Carbon dioxide stress-induced ethylene biosynthesis, respiration and polyamine accumulation in cucumber fruit ( Cucumis sativus L. cv. Sharp-1) held at 25 °C was investigated. Control fruit produced little ethylene and the respiration rate decreased with increase in incubation time while polyamine levels decreased. Elevated CO 2 induced ethylene production, respiration and polyamine accumulation. Putrescine and spermidine levels increased in response to CO 2 treatment, whereas spermine levels were not significantly affected. No cadaverine was detected in all treatments. The increase in ethylene production paralleled increases in 1-aminocyclopropane-1-carboxylic acid (ACC) and the activities of both ACC synthase and in vitro ACC oxidase. Infiltration of the fruit with aminooxyacetic acid, a potent inhibitor of the conversion of S-adenosylmethionine (AdoMet) to ACC completely blocked CO 2 stress-induced ethylene production. Similarly, cycloheximide, an inhibitor of nucleocytoplasmic protein synthesis effectively blocked CO 2 stress induction of polyamine accumulation, ethylene production, ACC formation and the development of ACC synthase. Withdrawal of CO 2 gas caused cessation of increases in ethylene production, respiration, ACC, putrescine and the activities of ACC synthase and ACC oxidase, but caused increase in spermidine and spermine levels. These data indicate that CO 2 induces de navo synthesis of ACC synthase thereby causing accumulation of ACC and increase in ethylene production and suggest that the conversion of AdoMet to ACC is the rate-limiting step in CO 2 stress-induced ethylene biosynthesis. The induction, however, requires continuous presence of the stimulus. The results also suggest that protein synthesis might be required for the CO 2 stress induction of polyamine biosynthesis. The results further suggest that in cucumber fruit under CO 2 stress, at least, the ethylene and polyamine biosynthetic pathways are not competitive.

Characterization of the regulation of ethylene biosynthesis in tomato fruit by carbon dioxide and diazocyclopentadiene

Abstract The regulation of ethylene biosynthesis by CO2 and diazocyclopentadiene (DACP), both inhibitors of ethylene action, was investigated in tomato (Lycopersicon esculentum Mill. cv. ‘Momotaro’) fruit held at 25 °C. When the tomato fruit at the pink stage of ripeness were treated with 20% CO2 (+ 20% O2 + 60% N2) or DACP, ethylene production by the fruit was rapidly decreased. The inhibition of ethylene production resulted primarily, if not solely, from the suppression of the activities of both 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. The inhibition of ACC synthase activity subsequently led to low levels of ACC. CO2 treatment further inhibited ACC conjugation into 1-(malonylamino) cyclopropane-1-carboxylic acid (MACC). By contrast, DACP-treated fruit maintained slightly higher levels of MACC relative to the control fruit. When the fruit were transferred from the CO2-enriched atmosphere to air, ethylene production, ACC and MACC contents and the activities of ACC synthase and ACC oxidase increased gradually to the control level after 24 h, while these values, except for MACC content, remained low in DACP-treated fruit throughout the experimental period. These results indicate that CO2 and DACP regulate ethylene production in tomato fruit by inhibiting ACC synthase and ACC oxidase activities and further support the hypothesis that the autocatalytic signal associated with ethylene action during fruit ripening stimulates the activities of both enzymes.

Regulation by carbon dioxide of wound-induced ethylene biosynthesis in tomato pericarp and winter squash mesocarp tissues

Abstract The effects of CO 2 treatment on wound-induced ethylene biosynthesis were investigated in excised pericarp and mesocarp tissues of tomato ( Lycopersicon esculentum Mill. cv. TVR-II) and winter squash ( Cucurbita maxima Duch. cv. Ebisu) respectively. Wounding caused increase in ethylene production rate, the levels of 1-aminocyclopropane-1-carboxylic acid (ACC) and 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) and the activities of ACC synthase and ACC oxidase. The increase in the rate of ethylene production and ACC synthase activity were suppressed by CO 2 treatment and was dependent on CO 2 concentration. CO 2 treatment also suppressed wound-induced increase in ACC level irrespective of the CO 2 concentration. The presence of 10% CO 2 had no effect on ACC conjugation to MACC induced by wounding while 60% CO 2 suppressed almost completely ACC conjugation. CO 2 promoted and inhibited induction of ACC oxidase activity in excised tomato and winter squash tissues respectively. These results suggest that CO 2 inhibits wound-induced ethylene biosynthesis by reducing the availability of ACC through repression of ACC synthase synthesis and/or inhibition of its activity. Also endogenous ethylene produced in response to wounding may play a role in the regulation of wound-induced ethylene production.

Physical measurement of firmness of banana fruit pulp: determination of optimum conditions for measurement

Abstract Stress-relaxation curves were obtained by plunging a conical probe into the pulp of green and yellow banana fruits [ Musa (AAA group, Cavendish subgroup) ‘Giant Cavendish’]. Three stress-relaxation parameters, minimum stress-relaxation time ( T 0 ), relaxation rate ( R ), and maximum stress-relaxation time ( T m ), were calculated from the stress-relaxation curve. Plunging depth and plunging speed varied the parameters. When parameters were fixed, with a plunging speed of 0.5 mm/s and the plunging depth of 0.6 mm, the yellow bananas showed significantly lower T 0 and T m than green bananas. The lower, T 0 and T m can predict the degradation of polymers responsible for the pulp texture. Measurements of stress-relaxation parameters in different parts of banana pulp revealed that the physical properties were not uniform within the same fruit.