ORIGINAL ARTICLES
Temporary Reduction and Control of Female Flower Expression in Cucumber (Cucumis sativus L.) by Application of 1-Methylcyclopropene
2022 Volume 91 Issue 1 Pages 42-48
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2022 Volume 91 Issue 1 Pages 42-48
A combination of phytohormones and ethylene, a key regulator of sex determination, induces female flowers in cucumber plants. Several inhibitors of ethylene biosynthesis or action affect plant sex determination. However, the use of ethylene inhibitors to control sex determination in commercial cucumber production has not been reported. 1-Methylcyclopropene (1-MCP), a commercial ethylene antagonist that inhibits ethylene signaling, is used to maintain the freshness of many harvested horticultural products. In this study, we evaluated the effect of 1-MCP on the sex ratio in cucumber plants to reduce production of cucumber fruit by inducing male flowers and reducing labor time for harvest. A concentration-dependent induction of male flowers was observed. Dose-response curves were obtained from the male flower induction rate (MFIR) and the range of nodes producing male flowers induced by 1-MCP treatment. We applied these models in independent experiments to predict the number of nodes and emergence day of male flowers induced by 1-MCP treatment and confirmed that the prediction fitted the results. Based on these results, production of cucumber fruit could be controlled by 1-MCP treatment and temporary excess labor for harvest could be avoided by using this model.
Most angiosperms produce hermaphroditic flowers with both male and female reproductive organs. However, many cucumber plants produce unisexual male and female flowers that have developed stamens/degenerated pistils or pistils/degenerated stamens, respectively. Cucumber plants show three patterns of sex expression: monoecious, gynoecious, and hermaphroditic (Malepszy and Niemirowicz-Szczytt, 1991). Monoecious plants, the most common pattern of sex expression, produce both male and female flowers on the same plant. Meanwhile, gynoecious plants produce only female flowers. Because cucumber fruits develop only from female flowers, the number of female flowers is an important trait for high yield in commercial cucumber production. However, an excess of female flowers and fruits on the plant tends to reduce plant growth and leads to self-topping, malformed fruits, and defects in fruit enlargement. Furthermore, in commercial cucumber production in Japan, growers have to harvest 20-cm long fruits twice a day during the warm season, at the most rapid growth stage, because Japanese consumers prefer immature cucumber fruits. When large numbers of female flowers are formed on cucumber plants, a transient reduction in female flower and fruit production is beneficial for the growers, as it contributes to controlled production and avoids excess labor requirements. The rate-limiting factor in cucumber production in Japan is labor, including harvesting. Therefore, in Japan, controlling the labor required to produce cucumber fruits is a major concern.
Phytohormones are considered the most important factors regulating sex determination in cucumber plants. Morphologically, hermaphroditic floral buds that have both staminate and pistillate primordia are formed during the first stage of flower development in cucumber plants. Ethylene positively and negatively regulates the development of the pistil and stamen, respectively, thereby inducing female flowers (Li et al., 2019). When silver nitrate (AgNO3), an ethylene action inhibitor, or aminoethoxyvinyl glycine (AVG), an ethylene biosynthesis inhibitor, was applied to gynoecious cucumber plants, sex expression shifted from female to male flowers (Atsmon and Tabbak, 1979; Yamasaki and Manabe, 2011). Although both AgNO3 and AVG are effective in changing sex expression from female to male flowers, they are not suitable for controlling sex expression in commercial cucumber production because their safety in humans has not been confirmed. Alternatively, 1-Methylcyclopropene (1-MCP) interacts with ethylene receptors, and it effectively inhibits the ethylene response (Sisler and Serek, 1997). Sold as an ethylene antagonist, 1-MCP is commercially available under the names EthylBloc® and SmartFreshTM and is used to maintain the freshness of harvested flowers, fruit and vegetables, such as orchids, apples and broccoli (Sisler and Serek, 2003).
In this study, we applied 1-MCP to cucumber plants and confirmed the changes in sex expression. Our results showed that cucumber fruit production can be conveniently modeled by using 1-MCP to control sex expression.
The monoecious, commercial cucumber cultivar ‘Fresco-dash’ (Kurumegenshyuikuseikai Co., Ltd., Fukuoka, Japan) was used in experiments performed three times using similar methods to examine the effect of concentration (1st and 2nd times), and for verification of the effect (3rd time). Seeds were sown in 12-cm (0.6 L) plastic pots containing commercial horticultural soil (Tane-Baido-1gou; Sumitomo Forestry Landscaping Co., Ltd., Tokyo, Japan) on September 27, 2019 for Experiment 1; then, on December 25, 2019 for Experiment 2, and then on March 6, 2020, for Experiment 3. Seedlings were grown in a seedling growth chamber (Nae Terrace; Mitsubishi Chemical Agri Dream Co., Ltd., Tokyo, Japan) illuminated with fluorescent lamps for a day-length of 16 h with a photosynthetic photon flux density (PPFD) of 350 μmol·m−2·s−1, a CO2 concentration of 1000 μmol·mol−1, and an air temperature of 22°C during daytime (16 h) and 19°C at night (8 h). Two weeks after sowing, plants were moved into a greenhouse (10 m long, 8 m wide, and 5 m high) at the National Agriculture and Food Research Organization Institute of Vegetable and Floriculture Science, in Tsukuba, Japan. The temperatures at which ventilation or heating began were set at 25°C and 15°C, respectively. Plants were watered ad libitum with Otsuka-SA nutrient solution (Otsuka AgriTechno, Tokyo, Japan), consisting of 12.2 mmol·L−1 NO3, 3.1 mmol·L−1 P, 7.1 mmol·L−1 K, 5.7 mmol·L−1 Ca, and 2.1 mmol·L−1 Mg, and adjusted to an electrical conductivity of 2 dS·m−1. In all three experiments, lateral branches that emerged from each node were removed and plants were grown until the flower at the 18th node was visible.
1-MCP effect on sex expressionEthylblocTM was purchased from Smithers-Oasis Japan Co., Ltd. (Tokyo, Japan) and was used to release 1-MCP gas. It was applied to the experimental plants when the fifth leaf was fully expanded (October 28, 2019, in Experiment 1; January 30, 2020, in Experiment 2; and April 9, 2020, in Experiment 3). Prior to the application of the treatment, plants were transferred into a sealable tunnel chamber covered with plastic film (8 m long, 1 m wide, and 2 m high). The concentration of 1-MCP was calculated from the volume of the chamber and the quantity of EthylblocTM dissolved in water. The EthylblocTM dissolved in water was transferred to the sealable tunnel chamber and the cover was quicky closed to fill with it 1-MCP gas at 16:30 each day. The EthylblocTM dissolved in water was not transferred to the sealable tunnel chamber as a control. At 08:30 the following day, the cover of the sealable tunnel chamber was opened and plants were returned to the greenhouse. To investigate the effect of 1-MCP concentration on sex determination, 1-MCP was adjusted to 0.1 ppm and 1 ppm in Experiment 1, and 1 ppm and 4 ppm in Experiment 2. To confirm the concentration-dependent effect of 1-MCP on sex determination, the concentration of 1-MCP was adjusted to 0.5 ppm and 2 ppm in Experiment 3. Although several male or female flowers were produced in a node, a node having a male flower would not produce fruit. Further, only one female flower enlarged when several female flowers were produced in the same node. Therefore, the sex of each flower up to node 18 was classified as either male or female on November 19, 2019, in Experiment 1, February 27, 2020, in Experiment 2, and May 8, 2020, in Experiment 3. When both male and female flowers were formed in the same node, the value was set at 50%.
Modelling of the relationship between 1-MCP concentration and male flower rateThe male flower induction rate (MFIR) is defined as the difference between the number of male flowers in 1-MCP treated plants and the number of male flowers in untreated plants. We investigated the relationship between MFIR for each plant and the 1-MCP concentration in Experiments 1 and 2. The dose-response curve was obtained and modeled from the MFIR of the 10th to 18th nodes in each 1-MCP treated plant (n = 4 to 6) as follows:
MFIR10-18 = Bm + (Tm − Bm)/(1 + 10(logECm50 − logC)), Eq. 1
where MFIR10-18 represents the MFIR of the 10th to 18th nodes, Bm represents the maximum inhibition response of MFIR, Tm represents the maximum response of MFIR, ECm50 represents the half-maximal effective concentration on MFIR, and C represents the 1-MCP concentration (ppm).
X = 5 + 9.68·ln(T1) − 20.15, Eq. 2
where X represents the number of the lowest nodes producing male flowers induced by 1-MCP treatment, and T1 represents the average daily temperature from 3 d before 1-MCP treatment to the day of 1-MCP treatment.
Y = Br + (Tr − Br)/(1 + 10(logECr50 − logC)), Eq. 3
where Y represents the range of nodes producing male flowers upon 1-MCP treatment, Br represents the maximum inhibition response of the range of nodes, Tr represents the maximum response of the range of nodes producing male flowers by 1-MCP treatment, and ECr50 represents the half-maximal effective concentration on the range of nodes producing male flowers by 1-MCP treatment.
To validate the reliability of these models using independent datasets, the average MFIR of the 10th to 18th nodes and the average lowest number and range of nodes producing male flowers by 1-MCP treatment were obtained from Experiment 3 and compared with these models.
Z = X + Y, Eq. 4
where Z represents the highest number of nodes that produce male flowers induced by 1-MCP treatment.
dNln/dt = 0.065·(Tn) − 0.511, Eq. 5
where dNln/dt represents the rate of leaf appearance n days after 1-MCP treatment, and Tn represents the daily average temperature at n days after 1-MCP treatment. The day of leaf emergence in the nodes producing male flowers was estimated from the daily temperature after 1-MCP treatment.
All results were analyzed using R version 3.6.3 <https://www.R-project.org/>.
As 1-MCP was applied only after the fifth leaf was fully expanded and the ninth leaf was already formed, we focused on sex expression of the flowers on the 10th to 18th nodes to evaluate the effect of 1-MCP on sex determination. Few male flowers were observed in 1-MCP untreated plants (Control) in Experiments 1 and 2 (Fig. 1A, D), as ‘Fresco-dash’ is a monoecious cultivar. The rates of male flowers on the 10th to 18th nodes in Experiments 1 and 2 are shown in Table 1. Although the rate of male flowers did not increase at 0.1 ppm 1-MCP, it did increase at 1 ppm 1-MCP in Experiment 1 (Fig. 1B, C; Table 1). Meanwhile, in Experiment 2, 80% of the control plants produced male flowers on the 11th node, and almost exclusively female flowers were produced on the 10th, and 12th to 18th nodes (Fig. 1D). The rate of male flowers on the 10th to 16th nodes increased with increasing concentrations of 1-MCP (Fig. 1E, F). Although the rate of male flowers induce by 4 ppm 1-MCP was higher than the value in control, there was no significant difference of the values between 1 and 4 ppm 1-MCP treatments.
Effect of 1-Methylcyclopropene (1-MCP) on sex expression in cucumber plants. The rates of male (closed box) and female flowers (open box) produced on each node of the plants are shown. In experiment 1, the greenhouses were filled with 0.1 (B) or 1 ppm (C) of 1-MCP. In experiment 2, the greenhouses were filled with 1 (E) or 4 ppm (F) of 1-MCP. n = 4 to 6. The error bars show SEs.
Average rate of male flowers from the 10th to the 18th node at different 1-MCP concentrations.
To quantitatively evaluate the concentration-dependent effect of 1-MCP on flower sex determination in cucumber plants, we plotted the dose-response curve of MFIR of each plant against common logarithms of 1-MCP concentration, modeled by this regression (Equation 1; Fig. 2). The results showed that MFIR increased with increasing 1-MCP concentration. To validate the reliability of this model, the effects of 0.5 and 2 ppm 1-MCP on sex determination were investigated in Experiment 3, and only female flowers were produced in control plants (Table 1). A few male flowers were observed between the 14th and 18th nodes in the treated plants as expected (Fig. 3), and the rates of male flowers between the 10th and 18th nodes were significantly increased by 1-MCP treatment (Table 1). The average values for MFIR induced by 0.5 and 2 ppm 1-MCP treatment in Experiment 3 were plotted and compared with the dose-response model (Equation 1), as shown in Figure 2. The values for MFIRs obtained in Experiment 3 were plotted close to the dose-response curve and were slightly lower than those of the dose-response model.
Effect of 1-MCP concentration on MFIR (male flower induction rate) in cucumber plants. The values of MFIR induced with 0.1 ppm, 1 ppm, and 4 ppm 1-MCP treatment are plotted (closed circle: Experiments 1 and 2) and the regression line assumed a dose-response curve with a standard slope (Equation 1); logECm50 is ˗0.13. The MFIR values induced by 0.5 ppm and 2 ppm 1-MCP are plotted (open circle: Experiment 3).
Effect of 1-Methylcyclopropene (1-MCP) on sex expression in cucumber plants in experiment 3. The rates of male (closed box) and female flowers (open box) produced on each node of the plants are shown. The greenhouses were filled with 0.5 (B) or 2 ppm (C) of 1-MCP. n = 5 to 6. The error bars show SEs.
The relationship between the daily average temperature before 1-MCP application and the number of first male flowers induced by 1-MCP was analyzed, and the regression line (Equation 2) for Experiments 1 and 2 was obtained (Fig. 4). Furthermore, we obtained a dose-response curve for the range of nodes producing male flowers in each plant against the common logarithms of 1-MCP concentration modeled by this regression (Equation. 3; Fig. 5). The independent dataset obtained from Experiment 3 was applied to these models and plotted on the regression line or dose-response curve (Figs. 4 and 5). The number of first male flowers and the range of nodes producing male flowers induced by 1-MCP were higher and lower than those of the models, respectively (Figs. 4 and 5). From these regressions (Equation 4), treatment with 0.5 ppm and 2 ppm 1-MCP was estimated to induce male flowers on nodes 13.7 to 15.2 and 13.7 to 17.3, respectively. The day of leaf emergence on nodes producing male flowers was estimated at 14 to 16 days and 14 to 19 days upon treatment with 0.5 ppm and 2 ppm 1-MCP, respectively, from the rate of leaf appearance after 1-MCP treatment (Equation 5).
Prediction of the lowest number of nodes producing male flowers with 1-MCP treatment. The average temperature for the three days before 1-MCP treatment and the lowest number of nodes producing male flowers induced by 1-MCP in Experiments 1 and 2 are plotted (closed circle) and the corresponding regression line was obtained (Equation 2). The average temperature of 3 days before 1-MCP treatment and the lowest number of nodes producing male flowers induced by 1-MCP in Experiment 3 are plotted (open circle).
Effect of 1-MCP concentration on the range of nodes producing male flowers in cucumber plants. The ranges of nodes producing male flowers induced by 0.1 ppm, 1 ppm, and 4 ppm 1-MCP treatment are plotted (closed circle: Experiments 1 and 2) and the regression line assumed a dose response curve with a standard slope (Equation 3); logECr50 is 0.15. The ranges of nodes producing male flowers induced by 0.5 ppm and 2 ppm 1-MCP are plotted (open circle: Experiment 3).
Although ethylene is the most important factor in determining sex expression in cucumber plants, other hormones such as auxins, brassinosteroids (BRs), and gibberellins are also involved in sex determination in cucumber. AgNO3 is an inhibitor of ethylene action that has also been shown to enhance indole-3-acetic acid (IAA) efflux in Arabidopsis roots (Strader et al., 2009). Therefore, IAA efflux caused by application of AgNO3 may also affect sex determination in cucumber flowers. Meanwhile, as 1-MCP is a specific inhibitor of ethylene action, the change in sex expression observed in this study was apparently caused by inhibition of ethylene signaling. As 1-MCP is currently used during fruit storage for apples and avocados (Sisler and Serek, 2003), its application for the control of sex determination can be easily applied to cucumber production. In contrast, as AgNO3 is a toxic substance, its application in commercial cucumber production is almost impossible. In turn, the safety of AVG in humans has not yet been confirmed. Thus, it is not possible to use either AgNO3 or AVG in commercial greenhouses. Therefore, at present, the use of 1-MCP to control flower sex determination is the only feasible alternative to influence productivity via the application of accumulated knowledge on growth regulator responses.
Previously, we reported an increase in cucumber fruit yield by manipulating environmental conditions (Higashide et al., 2021). However, such an increase in yield led to an increase in the labor required to harvest cucumber fruits. Because 47% of the total time for cucumber production consists of labor for harvesting, at 672 h/10 a per year, a transient reduction in yield could be an important strategy to control labor demand in cucumber production (NARO, 2020). Because male flowers do not develop into fruits, the change in sex expression from female to male caused by 1-MCP application could effectively reduce fruit production and hence, labor needs. Although more male flowers were produced in 1-MCP-treated cucumber plants, only female flowers were produced in the upper nodes (Fig. 1). Therefore, 1-MCP can transiently reduce fruit production. We focused on the concentration of 1-MCP and temperature, and constructed models to estimate the duration of reduced fruit production, as estimating the beginning and end time points of reduced fruit production are very important for controlling labor requirements in cucumber production.
The rate of male flowers increased in a 1-MCP concentration-dependent manner, and the regression model was obtained (Equation 1; Fig. 2). For the independent dataset obtained in Experiment 3, we successfully predicted the rate of male flowers using the model and 1-MCP concentration (Fig. 2). Thus, the rate of male flowers can be controlled by changing the concentration of 1-MCP based on the model. The daily average temperature before 1-MCP treatment seemed to correlate with the first nodes producing 1-MCP-induced male flowers (Equation 2; Fig. 4). Furthermore, the concentration of 1-MCP affected the number of nodes producing male flowers (Equation 3; Fig. 5). Because the rate of leaf appearance can be increased by an increase in daily average temperature, the day of leaf emergence on the nodes producing 1-MCP-induced male flowers can be predicted from Equation 5 and the daily average temperature. Based on our models, the day of leaf emergence on the nodes producing male flowers was estimated to be 14 to 19 days after treatment with 2 ppm 1-MCP in Experiment 3 (Equations 2, 3, 4, and 5). Moreover, since fruit require a period from fruit set to maturity, the duration of ripening should also be considered when estimating the duration of 1-MCP-induced yield reduction. Therefore, 1-MCP application would be effective in reducing fruit production from approximately 30 days after application and from 8 nodes above the treated nodes, at an average temperature of 18°C (Equation 2; Fig. 4). The application of 4 ppm 1-MCP temporarily reduced fruit production by 27.8% compared to the 12.2% reduction in control plants (Table 1). Based on these results and models, growers should maintain the daily average temperature at 18°C and treat their greenhouses with 4 ppm 1-MCP overnight to reduce yield and labor requirements for harvesting (e.g., one month later). Using such a strategy, fruit yield would decrease by approximately 16% for seven days from one month after treatment, thereby also reducing labor requirements. For example, to reduce yield and harvesting labor by 10%, the required 1-MCP concentration, as per the model, would be 1.2 ppm (Equation 1). In this case, the node range producing treatment-induced male flowers would be 2.7 (Equation 3). Treatment with 1.2 ppm 1-MCP would induce male flowers from nodes 11.1 to 13.8 at 15°C (Equations 2 and 4). Meanwhile, when the average temperature was set at 18°C, 1.2 ppm 1-MCP treatment would induce male flowers from nodes 12.8 to 15.6 (Equations 2 and 4). The emergence day of leaves on the nodes producing male flowers was estimated at 14 to 20 days or 12 to 17 days after treatment with 1.2 ppm 1-MCP at 15°C or 18°C, respectively (Equation 5). Therefore, 1.2 ppm 1-MCP application would be effective in reducing fruit production from about 40 or 30 days after 1-MCP application, for six or four days at 15°C or 18°C, respectively. Although the ripening period should be considered and the accuracy of regression lines estimating the nodes producing male flowers using 1-MCP should be revised, the duration of the decrease in cucumber fruit yield could be predicted. Thus, based on these models, 1-MCP concentration and temperature, we effectively manipulated the fruit yield and labor requirement in cucumber production.
Cucumber plants treated with 1-MCP showed a change in sex expression from female to male flowers under the experimental conditions (Fig. 1; Table 1). ‘Fresco-dash’ normally produces more female flowers compared to other Japanese cultivars. Since we confirmed the effect of 1-MCP on this cultivar, its effect on other Japanese cultivars with a low rate of female flowers may be equal to, or stronger than that of ‘Fresco-dash’.
Because 1-MCP is a gas, application of 1-MCP to plants should be conducted in a closed space, such as a small chamber. However, in a small chamber, the temperature increases due to solar radiation, leading to heat damage to the plants. Therefore, 1-MCP application was performed at night, for a duration of 16 h (between 16:30‒08:30). Based on our experiments, we were not able to come to a conclusion on the application times and duration of application of 1-MCP. Because 1-MCP quickly degrades, the concentration of 1-MCP will be quite low at the end of treatment. It was not clear whether 1-MCP had to be applied for 16 h or less, or whether it should be applied for 16 h continuously at one time or split into several times over the 16 h period of application. During the summer cropping season, nighttime is shorter than 16 h, e.g., 9 h; 20:00‒05:00. Therefore, the effect of 1-MCP application for fewer hours over a few days warrants further examination.
The change in sex determination induced by 1-MCP will reduce the number of fruits in each cucumber plant. Because total dry matter production is not affected by a reduction in the number of fruits, fruit enlargement could be affected by 1-MCP treatment. Therefore, further experiments are required to evaluate the effect of 1-MCP in terms of fruit enlargement for practical use of 1-MCP in cucumber production.
Here, we showed that 1-MCP induced male flower formation in a dose-dependent manner in cucumber plants. Because the induction of male flowers temporarily reduces the formation of fruit, 1-MCP should be useful to reduce fruit yield. Further, the duration of reduced yield could be estimated based on the 1-MCP concentration and daily average temperature. We constructed models to predict the duration of reduced fruit yield in cucumber by 1-MCP treatment; 1-MCP and temperature control may be useful tools for planning cucumber fruit production.
This work was partially supported by a grant from a commissioned project study on “The research project for the future agricultural production utilizing artificial intelligence”, Ministry of Agriculture, Forestry, and Fisheries. The authors gratefully acknowledge Dr. Hiroko Shimizu-Yumoto and Dr. Yusuke Kakei for their advice, and all members of the NARO plant factory in Tsukuba for their assistance.
