Effect of Exogenous Plant Hormones on Parthenocarpy and Fruit Quality in Tropical Squash (Cucurbita moschata L.)

Abstract

Artificially occurring parthenocarpy can be induced by exogenous application of plant hormones and is useful in the fruit production of many fruit crops and fruit trees. In cucumber (Cucumis sativus L.), a model species in the Cucurbitaceae family, the plant hormones auxin, gibberellin, cytokinin, and brassinosteroids are known to induce parthenocarpy. In tropical squash (Cucurbita moschata L.), synthetic auxins are known to induce parthenocarpy, but the effects of gibberellin, cytokinin, and brassinosteroids are still unknown. In addition, there are few published reports on the quality of parthenocarpic fruits induced by plant hormones in tropical squash, and the effects of these hormones remain largely unknown. In this study, we examined the effects of gibberellin, cytokinin, and brassinosteroids on the parthenocarpy of tropical squash and characterized the parthenocarpic fruits induced by the plant hormones. First, we evaluated fruit set and development in unpollinated fruits of ‘Kogiku’, a tropical squash cultivar, treated with gibberellic acid (GA₃), a synthetic cytokinin-like substance, N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU), and brassinolide (BL). CPPU promoted parthenocarpy, but GA₃ and BL did not, showing that cytokinin works as an important factor for parthenocarpy in tropical squash. Next, we investigated the quality of parthenocarpic fruits from ‘Kogiku’ induced by a synthetic auxin, 1-naphthylacetic acid (NAA), and CPPU. Total soluble solids and myo-inositol of NAA-treated parthenocarpic fruit were higher than those of pollinated fruits, whereas fructose of NAA-treated parthenocarpic fruit and fructose and glucose of CPPU-treated parthenocarpic fruits were significantly lower than those of pollinated fruits. These results showed that parthenocarpy of tropical squash induced by auxin and cytokinin had differing effects on fruit quality.

Introduction

The development of fruits without fertilization is called parthenocarpy, which can be classified into naturally occurring parthenocarpy and artificially occurring parthenocarpy. Naturally occurring parthenocarpy has been observed in many plants, including Citrus spp., Vitis spp., cucurbit, and solanaceous crops. The parthenocarpic cultivars of these crops are used for seedless fruit production, reducing the financial and labor costs for fruit set, or for stabilization of fruit production under unfavorable conditions. Among cucurbit crops, parthenocarpy is widespread in cucumber (Cucumis sativus L.), and it has been reported that melon (Cucumis melo L.) (Yoshioka et al., 2018), summer squash (Cucurbita pepo L.) (den Nijs and Balder, 1983; Robinson, 1993; Robinson and Reiners, 1999; de Menezes et al., 2005; Martínez et al., 2014), and tropical squash (Cucurbita. moschata L.) (Takisawa et al., 2021) have natural parthenocarpic cultivars. In cucumber, genetic analyses to determine the loci involved in parthenocarpy showed that genetic inheritance for parthenocarpy is quantitative (Sun et al., 2006; Lietzow et al., 2016; Wu et al., 2016).

Artificially occurring parthenocarpy is mainly induced by exogenous application of plant hormones and is used for the production of seedless fruits or for promoting fruit set. Seedless grape fruits are produced by gibberellic acid (GA₃) treatment, and the flowers of solanaceous crops are treated with auxin to promote fruit set. In species of the Cucurbitaceae family, auxin, cytokinin, gibberellin (GA), or brassinosteroids (BR) can induce parthenocarpy. In cucumber, indole-3-acetic acid (IAA), an inhibitor of polar auxin transport, 2,3,5-triiodobenzoic acid, a synthetic cytokinin-like substance, N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU), benzyl adenine (BA), GA₃, and 24-epibrassinolide, induce parthenocarpy (Kim et al., 1992; Fu et al., 2008). In melon and watermelon, CPPU, BA, and synthetic auxins induce parthenocarpy (Camacho et al., 2003; Hayata et al., 1995a, b, 2000, 2002; Li et al., 2002). However, although synthetic auxins are known to induce parthenocarpy in tropical squash (Takashima and Hatta, 1955), the effects of GA, cytokinin, and BR on parthenocarpy are still unknown.

The effect of plant hormones on fruit quality has been investigated in some cucurbit crops. In cucumber, NAA treatment did not affect the nutritional characteristics at harvest and after storage, whereas CPPU treatment decreased phenolic acid and ascorbic acid levels after storage (Qian et al., 2018). Huitrón et al. (2007) reported that CPPU-treated fruits of a triploid watermelon have lower or equivalent Brix values compared to fruits treated with the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Hayata et al. (1995a) reported that the soluble solid contents of fruits treated with 20 ppm CPPU were lower than those of pollinated fruits, and there was no difference in soluble solid contents between fruits treated with 200 ppm CPPU and pollinated fruits. In addition, parthenocarpic fruits treated with CPPU had lower soluble solid contents compared to pollinated fruits in muskmelon (Hayata et al., 2000). As described above, although the effect of parthenocarpy induced by plant hormones on fruit quality has already been reported in watermelon, melon, and cucumber, the effects of plant hormones on fruit quality remain unknown in tropical squash.

In this study, to obtain basic knowledge on the effects of plant hormones on parthenocarpy and fruit quality in tropical squash, we first investigated the effects of GA, cytokinin, and BR on the induction of parthenocarpy and then examined the fruit quality of parthenocarpic fruits induced by the plant hormones.

Materials and Methods

Plant materials and growth conditions

We used a C. moschata cultivar, ‘Kogiku’, in the 2018 spring experiment and ‘Kogiku’ and a C. maxima cultivar, ‘Tokyo’, in the 2018 fall experiment. Seeds were sown in 10.5 cm plastic pots filled with a mixture of bark compost, decomposed granite soil, and smoked rice hulls (2:2:1, v/v/v) on April 25, 2018, and August 29, 2018, for the spring and fall experiments, respectively. In the spring experiment, 30 seedlings of ‘Kogiku’ were transplanted to rows 300 cm in length, spaced 75 cm apart, in a field located at the Kizu Experimental Farm of Kyoto University at Kizugawa, Japan (34°73' N, 135°84' E), on May 22, 2018. The main shoot was topped, and two lateral shoots were trained horizontally without topping. In the fall experiment, 33 seedlings of ‘Kogiku’ and 24 seedlings of ‘Tokyo’ were transplanted in a greenhouse at the Kizu Experimental Farm. The plants were planted in rows 300 cm in length, spaced 30 cm apart, on September 20 and 21, 2018, and the main shoot was trained horizontally without topping. We performed irrigation, fertilization, and pest control according to standard procedures, and lateral buds on the lower nodes that bore fruit were removed.

Pollination and plant growth regulator treatments

The plant growth regulators used were as follows: a synthetic auxin (1-naphthaleneacetic acid [NAA]), a cytokinin-like substance (1-(2-chloro-4-pyridyl)-3-phenylurea [CPPU]), gibberellic acid (GA₃), and brassinolide (BL). In the spring experiment, female flowers of ‘Kogiku’ were bagged one day before anthesis to prevent pollination, and the following treatments were conducted at anthesis: negative control (water-applied), positive control (pollinated with pollen from male flowers of ‘Kogiku’ early in the morning), NAA (100 ppm, ca. 1 mL), CPPU (20 ppm, ca. 500 μL), GA₃ (10, 100, and 1000 ppm, ca. 1 mL), and BL (1, 10, and 100 ppm, ca. 500 μL). After treatment, the female flowers were bagged again. In the fall experiment, the following treatments were conducted at anthesis on the female flowers of ‘Kogiku’: negative control (water-applied), positive control (pollinated with pollen from male flowers of ‘Kogiku’ early in the morning), NAA (100 ppm, ca. 1 mL), and CPPU (20, 200, or 2000 ppm, ca. 500 μL) and ‘Tokyo’: negative control (water-applied) and CPPU (20, 200, or 2000 ppm, ca. 500 μL).

Investigation of fruit traits

To characterize parthenocarpic fruits of ‘Kogiku’ or ‘Tokyo’ induced by plant growth regulators, we used 5–6 fruits of ‘Kogiku’ in each treatment of the spring experiment and 5–7 fruits of ‘Kogiku’ and six fruits of ‘Tokyo’ in each treatment of the fall experiment. We examined the fruit set rates of treated flowers and harvested fruits 30 days after anthesis in the spring experiment and 40 days after anthesis in the fall experiment. After harvest, we measured fruit weight and transverse diameter at the equatorial plane of all harvested fruits, and flesh (mesocarp) thickness of pollinated, NAA-treated, and 2000 ppm CPPU-treated fruits in ‘Kogiku’.

Measurement of water content and total soluble solids (TSS)

To evaluate parthenocarpic fruit quality at harvest, in the fall experiment, we used 7 ‘Kogiku’ fruits that were pollinated, NAA-treated, or 2000 ppm CPPU-treated. The fruit was cut into small pieces, and approximately 5 g of sample dried at 80°C for four days. We measured the samples weights before and after drying and calculated water content. TSS was measured using a digital refractometer (PAL-1; Atago Co., Ltd., Tokyo, Japan) after grating the sample.

Analysis of soluble carbohydrates

For analysis of soluble carbohydrates, we used 7 ‘Kogiku’ fruits that were pollinated, NAA-treated, and 2000 ppm CPPU-treated. After removing seeds and placenta, we cut out flesh from the equatorial part of the fruit and sliced the flesh with a slicer. The 5.11–5.61 g sample was frozen using liquid nitrogen and disrupted with a MULTI-BEADS SHOCKER^® (Yasui Kikai Corporation, Osaka, Japan). The analysis of soluble carbohydrates was conducted according to Takisawa et al. (2021). Briefly, from the disrupted samples, soluble carbohydrates were extracted with 80% (v/v) ethanol, and the extracted solution filtered through a 0.2 μm Minisart Syringe Filter (Sartorius AG, Goettingen, Germany). Glucose, fructose, sucrose, and myo-inositol contents were determined by high-performance liquid chromatography (HPLC) using a Shodex Asahipak NH2P-50 4E column (Showa Denko K. K., Tokyo, Japan). The mobile phase was acetonitrile:water (75:25, v/v) at a flow rate of 1 mL·min⁻¹ at 40°C. Quantification of soluble carbohydrates was performed by comparison with external standards.

β-carotene analysis

For β-carotene analysis, we applied the disrupted samples, which were treated in the same way as in the soluble carbohydrates analysis, and the analysis was conducted according to Takisawa et al. (2021). Briefly, the 5.12–5.58 g of frozen sample, β-carotene was extracted with 100% acetone, and the extracted solution was filtered through a 0.2 μm Minisart Syringe Filter (Sartorius AG). β-carotene content was determined by HPLC. Separation of carotenoids was performed in a Kinetex C18 column (150 × 4.5 mm) (Phenomenex, Le Pecq, France) by gradient elution of acetonitrile and ethanol. The elution started with a mixture of 90% acetonitrile and 10% ethanol, which was changed to 10% acetonitrile and 90% ethanol at 10 min and returned to the initial condition at 14 min. The flow rate was 1 mL·min⁻¹. Quantification of β-carotene was performed by comparison with external standards.

Ascorbic acid analysis

Ascorbic acid content was determined using a reflectometer (RQflex^® 20; Merck KGaA, Darmstadt, Germany) and an ascorbic acid test (Reflectoquant ascorbic acid test; Kanto Chemical Co., Inc., Tokyo, Japan) according to the manufacturer’s instructions.

Statistical analysis

Statistical analysis of the data was performed using Welch’s t-test, one-way ANOVA and Tukey’s test to determine differences among treatments at 5% levels of significance.

Results and Discussion

Effect of exogenous plant growth regulators on parthenocarpy

To clarify the effect of plant hormones on parthenocarpy in tropical squash, water (negative control), NAA, GA₃, CPPU, and BL were used to treat female flowers of ‘Kogiku’ in the spring experiment. All female flowers treated with NAA formed parthenocarpic fruits, and there was no significant difference in fruit weight between pollinated fruits and NAA-treated fruits (Table 1). In agreement with our data, Takashima and Hatta (1955) reported that NAA induces parthenocarpy in C. maxima, C. pepo, and C. moschata. On the other hand, fruit set and development were not observed in the negative control, GA₃, CPPU, and BL treatments. With regard to the CPPU treatment, we expected 20 ppm CPPU to induce parthenocarpy because the instructions for the CPPU-containing pesticide, Flumet (Kyowa Hakko Bio Co., Ltd., Tokyo, Japan), states that spray treatments from 10 to 20 ppm promote fruit set in squashes; however, no female flowers treated with 20 ppm CPPU formed parthenocarpic fruits in this study. Therefore, we examined the effect of CPPU on parthenocarpy at multiple concentrations as with GA₃ and BL in the fall experiment. In the fall experiment, the fruit set rate for the parthenocarpic fruits of ‘Kogiku’ was unexpectedly 3/5 for the negative control (Table 2), showing that ‘Kogiku’ naturally exhibits parthenocarpy in the fall condition. Takisawa et al. (2021) suggested that low temperatures in fall increase the parthenocarpic ability of a C. moschata parthenocarpic cultivar, ‘Miyazaki-wase No. 1’. Therefore, ‘Kogiku’ may have a weak ability to induce parthenocarpy and this parthenocarpic ability was promoted under low-temperature conditions in fall. In fruit weight, although the one-way ANOVA showed significant difference (Table 2), no significant difference was detected among treatments by Tukey’s test. In CPPU treatment, the fruit set rate and the average fruit weight increased as CPPU concentration increased up to 200 ppm and 2000 ppm, respectively, and the fruit set rate was 7/7 at 2000 ppm (Table 2). These results suggest that CPPU promotes parthenocarpy and that cytokinin is an important factor for fruit set and development in tropical squash. We conducted the same experiment in a C. maxima cultivar, ‘Tokyo’. In ‘Tokyo’, the fruit set rate of parthenocarpic fruits was 5/6 in the 200 and 2000 ppm treatments, and no parthenocarpic fruits were formed with treatments of negative control and 20 ppm CPPU. The fruit set rate and fresh weight were not significantly different between the 200 and 2000 ppm treatments (Table 3). Li et al. (2005) reported that treatment with 500 ppm CPPU induced fruit set and development in zucchini (C. pepo L. ‘Beruna’), and we showed that CPPU promoted and induced parthenocarpy in C. moschata and C. maxima. Together, these results show that cytokinin is a common factor involved in parthenocarpy in these three species. Furthermore, in cucumber, auxin, cytokinin, GA, and BR can induce parthenocarpy (Kim et al., 1992; Fu et al., 2008). Here, we showed that auxin and cytokinin induced or promoted parthenocarpy, but GA and BR did not induce parthenocarpy, in tropical squash, suggesting that the plant hormones involved in parthenocarpy differ among Cucurbitaceae family species.

Table 1

Number of fruit set and fruit weight with water applied, pollinated, NAA-treated, CPPU-treated, GA₃-treated, and BL-treated fruits of ‘Kogiku’ in the spring 2018 experiment.

Table 2

Number of fruit set and fruit weight with water-applied, pollinated, NAA-treated, and CPPU-treated fruits of ‘Kogiku’ in the fall 2018 experiment.

Table 3

Number of fruit set and fruit weight with water-applied and CPPU-treated fruits of ‘Tokyo’ in the fall 2018 experiment.

Next, we compared the transverse diameter, flesh thickness, and seed morphology of pollinated fruits and parthenocarpic fruits induced by NAA and 2000 ppm CPPU in ‘Kogiku’ to characterize the fruit and seed morphology of parthenocarpic fruits induced by these hormones. There was no significant difference in both transverse diameter and flesh thickness among the treatments in the spring and fall experiments (Table 4). In addition, the seed sizes of parthenocarpic fruits induced by NAA and CPPU were almost the same as those of pollinated fruits, although they were empty (Fig. 1). These results suggest that the parthenocarpy induced by plant hormones did not affect fruit and seed morphology.

Table 4

Transverse diameter and flesh (mesocarp) thickness of pollinated, NAA-treated, and 2000 ppm CPPU-treated fruits of ‘Kogiku’ in the spring and fall 2018 experiments.

Fig. 1

Seeds pollinated and artificial parthenocarpic fruits of ‘Kogiku’ in the 2018 fall experiment. (A) Pollinated fruits. (B) NAA-treated fruits. (C) 2000 ppm CPPU-treated fruits. Scale bars: 1 cm.

Nutritional composition of parthenocarpic fruits induced by NAA and CPPU

We examined the water, TSS, soluble carbohydrates, β-carotene, and ascorbic acid contents in pollinated, NAA-, and CPPU-treated parthenocarpic fruits in the fall experiment. The one-way ANOVA showed no significant differences in water content, sucrose, β-carotene, and ascorbic acid contents among treatments (Table 5). The Tukey’s test results revealed that TSS and myo-inositol of NAA-treated parthenocarpic fruits were higher than those of pollinated fruits, and fructose of NAA-treated parthenocarpic fruit was lower than that of pollinated fruits (Table 5). Fructose and glucose of CPPU-treated parthenocarpic fruits were significantly lower than those in pollinated fruits, and there was no significant difference in myo-inositol content between CPPU-treated fruits and pollinated fruits. These results suggest that parthenocarpy induced by plant hormones affects fruit quality, and that the effect depends on the type of plant hormone. Previous studies on cucumber have shown that CPPU treatment decreases the levels of phenolic acid and ascorbic acid after storage (Qian et al., 2018). Hayata et al. (1995a) reported that soluble solid contents of fruits treated with 20 ppm CPPU were lower than those of pollinated fruits. Furthermore, parthenocarpic fruits treated with CPPU had lower soluble solid contents compared with pollinated fruits in muskmelon (Hayata et al., 2000). These reports show that CPPU treatment negatively affects fruit quality in cucumber, watermelon, and muskmelon. In this study, fructose and glucose contents were lower in CPPU-treated fruits than pollinated fruits, which suggests that CPPU treatment also decreases fruit quality in tropical squash.

Table 5

Water content, total soluble solids (TSS), soluble carbohydrate, β-carotene, and ascorbic acid content of pollinated, NAA-treated, and 2000 ppm CPPU-treated fruits of ‘Kogiku’ in the fall 2018 experiment.

In conclusion, we showed that CPPU promoted parthenocarpy, whereas GA and BR did not induce parthenocarpy in topical squash, suggesting that cytokinin is an important factor for fruit set and development in tropical squash, as with auxin. In addition, parthenocarpy induced by NAA and CPPU gave different effects on fruit quality, suggesting that the effect depends on the type of plant hormone.

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