The Plant Growth Regulator 14-OH BR Can Minimize the Application Content of CPPU in Kiwifruit (Actinidia chinensis) ‘Donghong’ and Increase Postharvest Time without Sacrificing the Yield

Abstract

The application of the plant growth regulator 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU) is extensively used for red-fleshed kiwifruits or ‘Donghong’, but it has toxicological properties. Extra plant growth regulators (PGRs) were screened for partial substitution of CPPU (10 mg L⁻¹) to the crops to minimize the CPPU content. The results showed that CPPU at a concentration of 5 mg L⁻¹ plus 14-hydroxylated brassinosteroid (14-OH BR) at a concentration of 0.15 mg L⁻¹ has a nearly equal effect to CPPU at a concentration of 10 mg L⁻¹; it maintains the kiwifruit yields and quality as well as increases the postharvest time. Transcriptome sequencing data revealed that the regulation of 14-OH BR on kiwifruit growth acts mainly by activating Brassinosteroid (BR) signaling to synergistically and antagonistically stimulate the signaling of other endogenous growth regulators, including auxin (IAA), abscisic acid (ABA), cytokinin (CK), gibberellin (GA), jasmonic acid (JA) and ethylene (ET).

1. Introduction

Being of Chinese origin, kiwifruit (Actinidia chinensis) is an edible berry rich in vitamins, minerals, and amino acids. The red-fleshed cultivars have an attractive appeal and unique flavor, approved by consumers. As the predominant cultivar, ‘Hongyang’ was first released from Cangxi County of Sichuan Province in China [1], but it is susceptible to Pseudomonas syringae pv. Actinidiae (Psa); it is a bacterial pathogen which can cause huge economic losses to kiwifruit plants [2]. Kiwifruit ‘Donghong’ is a red-fleshed kiwifruit cultivar selected from the F1 offspring of open-pollinated kiwifruit ‘Hongyang’ at Wuhan Botanical Garden, Chinese Academy of Sciences [3]. Maturation of ‘Donghong’ kiwifruit accompanied by a complicated bioactive component [4]. A non-destructive device called Kiwi-Meter™, was recently developed to assess its maturation and ripening [5]. ‘Donghong’ is popular because of its disease resistance and longer shelf life, as well as the particularly sweet taste. However, like ‘Hongyang’, the small fruit size of the red-fleshed kiwifruit limits its commercial benefit, whose average fruit weight is only 65~75 g.

To increase the fruit size and yield, 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), a plant growth regulator (PGR), has been widely applied in the initial stages of red-fleshed kiwifruit development [6,7], it was found that a dose-dependent CPPU regulatory model exists in ‘Hongyang’, and 5 mg L⁻¹ was the optimal concentration for CPPU to enhance fruit enlargement by stimulating cell division and cell expansion [8]. The underlying CPPU regulation mechanism for red-fleshed ‘Hongyang’ kiwifruit development was found to involve influencing the biosynthesis of gibberellin (GA), cytokinin (CK), abscisic acid (ABA) and auxin (IAA) and enhancing the energy supply [7]. As a Phenylurea-derived cytokinin, CPPU has also been applied to various horticultural crops, such as grape [9], apple [10] and peach [11], to enhance the fruit set and improve the fruit size, yield and quality.

In fact, so far, a number of PGRs are being widely applied in a variety of plant species to induce a particular plant response. For example, with the proper application of CKs and GAs in the cultivation of Zantedeschia, PGRs can improve the flowering intensity, extend the postharvest life and enhance the leaves’ greenness index and protein quantity [12,13,14,15]. A study of gibberellic acid (GA₃) and arbuscular mycorrhizal fungi (AMF) on the flowering and quality of Zantedeschia albomaculata (Hook.) Baill ‘Albomaculata’ plants indicated that AMF and GA₃ had a favorable effect on the quality of inflorescences [16]. Janowska et al. also reported that benzyladenine (BA) and GA₃ affect the elongation of inflorescences and spike, respectively, as well as the content of micro- and macronutrients in its leaves in the Gladiolus hybridus cv. Black Velvet [17,18]. The study conducted on the influence of gibberellic acid (GA₃) on the content of biologically active substances in Crocosmia × crocosmiiflora ‘Lucifer’ tubers suggested that the antioxidant activity of the extracts from tubers increased consistently with the GA₃ concentration [19]. Andrzejak et al. assessed the influence of the Trichoderma spp. on the flowering and nutritional status of Begonia × tuberhybrida Voss. ‘Picotee Sunburst’ and Gladiolus hybridus L. ‘Advances Red’ plants. These results displayed that Trichoderma spp. enhanced the flowering of the ‘Picotee Sunburst’ cultivar by 2.7–8.7 days, encouraged the development of buds and flowers and altered their size [20], improved the nutrient uptake, chlorophyll a + b, carotenoids and flowering in the plant of Gladiolus hybridus L. ‘Advances Red’ [21].

Even though the dosage-controlled agricultural use of CPPU is legal, the potential toxicity associated with CPPU is still unclear [22]; a repeated-dose toxicity study of CPPU in rats indicated the potential danger of CPPU on the ovaries and steroid production [23] and animal studies in zebrafish also indicated that CPPU adversely affects cardiac morphology and function [24]. Many environmental and animal-friendly PGRs were reported to have growth-promoting functions, such as 14-hydroxylated brassinosteroid (14-OH BR); a brassinolide analog derived from natural extracts [25], Zeatin [26], GA₄₊₇ [27] and Jianda 990.

In the present study, we combine low-dosage CPPU (5 mg L⁻¹) with different biological PGRs for fruit enlargement testing via a standard fruit dipping method. We measured the fruit characteristics of each treatment, trying to minimize the CPPU application contents in kiwifruit ‘Donghong’ without detectable yield trade-offs.

2. Materials and Methods

2.1. Plant Materials

The fruits examined were from five-year-old mature Donghong (Actinidia chinensis) vines grown at the “8S” kiwifruit demonstration garden in Daxing Town, Pujiang County, Chengdu city, China.

2.2. Experimental PGRs

In this study, apart from CPPU (AS 0.1%, Sichuan Lanyue Technology Co., Ltd., Chengdu, China), 4 other biological PGRs were exploited, including 14-hydroxylated brassinosteroid (14-OH BR, AS, 0.007 5%, Chengdu Newsun Crop Science Co., Ltd., Chengdu, China), Jianda 990 (SG, 39.998% GA₃ + 0.002% 14-OH BR, Chengdu Newsun Crop Science Co., Ltd., Chengdu, China), Zeatin (WP, 0.1%, Zhengzhou Zhengshi Chemical Products Co., Ltd., Zhengzhou, China), GA₄₊₇ (AS, 40%, Jiangxi New Reyphon Biochemical Co., Ltd., Ji’an, China). In addition, sugar, alcohol, and a range of minerals (calcium and magnesium, each at a concentration of 150 g L⁻¹, iron, manganese, copper, zinc and boron, each at a concentration of 2–6 g L⁻¹, AS, Chengdu Newsun Crop Science Co., Ltd., Chengdu, China) were used as an extra supplement of all treatments.

2.3. Experimental Treatments

There were twelve treatments performed 14 days after anthesis (DAA, on 2 May): CPPU at a concentration of 10 mg L⁻¹ (control), CPPU at a concentration of 5 mg L⁻¹ + 14-OH BR at a concentration of 0.15, 0.075 and 0.0375 mg L⁻¹ (T1, T2 and T3, respectively), CPPU at a concentration of 5 mg L⁻¹ + Jianda 990 diluted by 3 × 10³, 6 × 10³ and 9 × 10³ (T4, T5 and T6, respectively), CPPU at a concentration of 5 mg L⁻¹ + Zeatin at a concentration of 1, 0.1 and 0.01 mg L⁻¹ (T7, T8 and T9, respectively), CPPU at a concentration of 5 mg L⁻¹ + GA₄₊₇ at a concentration of 30, 60 and 120 mg L⁻¹ (as T10,T11 and T12, respectively), a 500-fold dilution of sugar, alcohol, calcium and magnesium was also applied for each treatment.

In all the treatments, the test solution was applied as a 3s fruit dip. Each treatment is repeated 3 times, and each replication includes 2 kiwi trees. Except for the different experimental treatments, the fertilization, watering and pest control during the entire growth period are all consistent.

2.4. Evaluation of Fruit Quality

One hundred fruits were randomly selected for each treatment after the standard harvesting period (20 September for ‘Donghong’). The fruits were first measured to determine the horizontal and vertical diameter (for shape index) and single fruit weight, then 50 fruits were randomly selected to determine the hardness; the remaining 50 fruits were tested for the Vc content, total solubility solids (TSS), Titratable acids (TA) and TSS/TA ratio, as well as the mineral and trace element content. The vitamin C concentration was determined by High-Performance Liquid Chromatography [28], the TSS was measured with a digital refractometer (PAL-1, Atago, Tokyo, Japan) with juice drops, the TA were determined by using an acid-base titration method based on CNS GB/T 12456-2008 [29], and mineral and trace element content were determined by the national standard GB 5009.268-2016 [30].

2.5. Fruit Postharvest Storage Quality

For postharvest quality, 150 kiwifruits for each treatment were randomly harvested at the standard harvesting period; the covered bags were taken off, and fruits were packed in plastic baskets and placed in the refrigerator (temperature 0.5–1 °C, humidity 90–95%) for 3 months. Afterwards, 50 fruits were randomly picked to test hardness, soluble solids and dry matter on days 3, 7 and 12.

2.6. Transcriptome Profiling

Transcriptome profiling was carried out by Wuhan Metware Biotechnology Co., Ltd. (Wuhan, China) (19 October 2020; http://www.metware.cn/), following their standard procedures.

2.6.1. RNA Extraction and RNA-Seq

The total RNA of the samples was extracted with an RNA prep Pure Plant kit (DP441, Tiangen, China). Illumina RNA-Seq was performed by Metware Biotechnology Co., Ltd. (Wuhan, China). The RNA quality was detected by a NanoPhotometer spectrophotometer (IMPLEN, Westlake Village, CA, USA), Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), and Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). The poly(A) mRNA was enriched by magnetic beads with oligo (dT). The mRNA was randomly fragmented. First-strand cDNA was synthesized using the M-MuLV reverse transcriptase system. The RNA strand was then degraded by RNase H, and the second-strand cDNA was synthesized using DNA polymerase. The double-stranded cDNAs were ligated to sequencing adapters. The cDNAs (~200 bp) were screened using AMPure XP beads. After amplification and purification, cDNA libraries were obtained and sequenced using the Illumina Nova seq6000 system.

2.6.2. Sequence Data Processing

The raw reads were transformed from the sequencing raw image data by CASAVA base recognition. To obtain high-quality data, adapters of sequences were cut, and low-quality reads with ≥5 uncertain bases or with over 50% Qphred ≤ 20 bases were removed using fastp. The GC content of clean reads was calculated. The Q20 and Q30 values were also produced by FastQC to evaluate the base quality. Then, the clean reads were mapped to the kiwifruit reference genome using HISAT2 with default parameters. Gene expression levels were determined using the FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) method.

Differentially expressed genes (DEGs) were defined with a threshold of |log₂fold change| ≥1 and FDR ≤ 0.05. The identified DEGs were subjected to enrichment analysis of KEGG pathway analysis.

2.7. Statistical Analysis

Data analysis was performed with Graph Prism software. The Student’s t-test was used to compare the means of average fruit weight between CPPU concentrations at 10 and 5 mg L⁻¹ (p ≤ 0.01). One-way ANOVA was performed for each treatment to obtain a statistical assessment. The Tukey’s test was used for mean comparisons of twelve treatments on fruit quality during kiwifruit (Actinidia chinensis) ripening (p ≤ 0.05).

3. Results

3.1. The Effect of Different Treatments on Fruit Development of A. chinensis ‘Donghong’

The optimal concentration of CPPU to enhance ‘Donghong’ fruit enlargement is 10 mg L⁻¹ in our experience; halving the concentration would cause a severe reduction in the average fruit weight (Figure 1). To find alternative kiwifruit enlargement stimuli packages, we took CPPU at a concentration of 10 mg L⁻¹ as the control. We tested different biological PGRs plus CPPU at a concentration of 5 mg L⁻¹ to assess whether any other PGRs can partially replace CPPU’s function and compensate for the yield loss.

Among the twelve treatments, our results showed that, apart from T3 and T4, which demonstrated a significant weight decrease in comparison to the control (

Table 1. The effect of different treatments on fruit quality during kiwifruit (Actinidia chinensis) ‘Donghong’ ripening.

Treatment	Fruit Weight (g)	Shape index	Hardness (N/cm)	TSS (%)	Titratable Acids	TSS/TA Ratio	Vitamine C (mg/100 g)
Control	90.27 ± 4.62 a	1.22 ± 0.11 a	36.27 ± 4.56 b	9.4 ± 0.28 a	1.1 ± 0.02 a	8.54 ± 0.03 a	87.36 ± 2.34 a
T1	96.82 ± 3.28 a	1.38 ± 0.12 a	42.73 ± 3.04 a	9.1 ± 0.35 a	1.06 ± 0.03 a	8.49 ± 0.58 a	84.05 ± 4.82 a
T2	92.17 ± 18.36 a	1.2 ± 0.02 a	47.04 ± 2.89 a	8.5 ± 1.55 a	1.11 ± 0.07 a	7.65 ± 1.76 a	77.23 ± 4.15 b
T3	66.21 ± 10.55 b	1.22 ± 0.11 a	51.13 ± 7.13 a	6.3 ± 1.97 b	1.22 ± 0.01 a	5.16 ± 1.71 b	71.36 ± 3.18 b
T4	81.13 ± 11.53 b	1.21 ± 0.01 a	41.04 ± 1.47 a	9.1 ± 0.71 a	1.2 ± 0.07 a	7.58 ± 0.21 a	75.86 ± 7.08 b
T5	97.44 ± 10.49 a	1.23 ± 0.01 a	43.12 ± 0.26 a	8.1 ± 0.56 a	1.1 ± 0.07 a	7.27 ± 0.1 a	85.88 ± 6.18 a
T6	98.14 ± 4.7 a	1.23 ± 0.03 a	42.74 ± 1.14 a	8.9 ± 0.42 a	1.2 ± 0.11 a	7.41 ± 0.39 a	77.14 ± 3.52 b
T7	91.48 ± 1.61 a	1.27 ± 0.04 a	44.36 ± 2.22 a	8.3 ± 0.35 a	1.04 ± 0.06 a	7.98 ± 0.13 a	72.15 ± 7.51 b
T8	93.77 ± 8.85 a	1.21 ± 0.01 a	41.22 ± 2.01 a	8.8 ± 1.34 a	1.13 ± 0.02 a	7.78 ± 1.32 a	82.76 ± 0.37 a
T9	92.56 ± 5.81 a	1.22 ± 0.01 a	44.07 ± 1.02 a	6.9 ± 0.63 b	1.16 ± 0.11 a	5.44 ± 0.02 b	83.29 ± 6.21 a
T10	84.35 ± 3.35 a	1.21 ± 0.01 a	42.62 ± 1.53 a	7.8 ± 0.42 b	1.32 ± 0.05 a	5.96 ± 0.61 b	92.08 ± 5.09 a
T11	89.09 ± 5.3 a	1.19 ± 0.01 a	40.45 ± 0.96 a	8.4 ± 0.56 a	1.24 ± 0.03 a	6.77 ± 0.25 b	84.87 ± 6.01 a
T12	89.51 ± 6.24 a	1.18 ± 0.02 a	39.09 ± 1.05 a	9.2 ± 0.35 a	1.29 ± 0.01 a	7.13 ± 0.62 a	93.38 ± 6.32 a

The same letters at the end of each column mean they are not significantly different by Dunnett’s test at p ≤ 0.05.

), all other treatments recovered the weight loss caused by reduced CPPU usage and maintained a similar shape index as the control, suggesting that the application of a correct concentration of different PGRs can reduce the content of CPPU needed for the kiwifruit ‘Donghong’. Besides, all twelve treatments considerably increased the fruit hardness while they tended to decrease the TSS at the same time, implying that the reduction of CPPU can prolong fruit storage time further. Specifically, we found that T1, T4 and T12 have the smallest varying degrees in terms of TSS, which resulted in a relatively higher TSS/TA ratio. Further data showed that these three treatments have insignificant Vitamin C content loss.

We further investigated the effects of different treatment on the mineral and trace element content. As shown in the

Table 2. The effect of different treatments on the mineral and trace element content during kiwifruit (Actinidia chinensis) ‘Donghong’ ripening.

Treatment	Ca	Mg	B	Cu	Fe	Mn	Mo	Zn
	mg/kg
Control	117.9 ± 13.69 b	95.6 ± 11.11 b	2.57 ± 0.11 a	2.1 ± 0.49 a	2.42 ± 1.4 b	0.015 ± 0.002 b	0.94 ± 0.1 a	0.74 ± 0.07 b
T1	250.4 ± 15.48 a	139.6 ± 13.09 a	2.41 ± 0.24 a	1.3 ± 0.15 b	0.44 ± 1.27 c	0.011 ± 0.002 b	1.09 ± 0.06 a	0.84 ± 0.04 b
T2	129.5 ± 13.43 b	92.8 ± 11.31 b	2.07 ± 0.27 a	1.52 ± 0.02 b	2.24 ± 0.68 b	0.015 ± 0.002 b	1.02 ± 0.24 a	0.77 ± 0.24 b
T3	110.5 ± 8.48 b	108.8 ± 3.46 b	2.46 ± 0.02 a	1.48 ± 0.13 b	3.21 ± 0.81 ab	0.019 ± 0.004 b	1.35 ± 0.19 a	1.12 ± 0.36 b
T4	122.5 ± 13.5 b	103.9 ± 5.02 b	2.5 ± 0.01 a	1.29 ± 0.02 a	2.06 ± 0.05 b	0.013 ± 0.003 b	1.08 ± 0.04 a	0.61 ± 0.19 b
T5	141.6 ± 2.26 b	111 ± 8.7 b	2.51 ± 0.26 a	1.33 ± 0.14 b	2.14 ± 0.7 b	0.008 ± 0.002 b	1.02 ± 0.06 a	0.88 ± 0.19 b
T6	138.4 ± 13.15 b	110 ± 8.06 b	2.13 ± 0.12 a	1.12 ± 0.02 b	3.13 ± 0.86 ab	0.008 ± 0.003 b	0.93 ± 0.09 a	0.6 ± 0.11 b
T7	119.8 ± 11.59 b	111.5 ± 6.97 b	1.96 ± 0.32 b	1.15 ± 0.28 b	1.91 ± 0.62 b	0.013 ± 0.001 b	1.07 ± 0.01 a	0.75 ± 0.02 b
T8	136.2 ± 16.16 b	114.3 ± 14.01 b	2.42 ± 0.12 a	1.56 ± 0.16 b	2.79 ± 0.03 b	0.012 ± 0.002 b	1.07 ± 0.03 a	0.79 ± 0.02 b
T9	99.2 ± 2.89 b	94.5 ± 4.84 b	2.25 ± 0.05 a	1.32 ± 0.16 b	2.84 ± 0.86 b	0.012 ± 0.001 b	1.12 ± 0.11 a	0.82 ± 0.01 b
T10	103.3 ± 12.09 b	95.7 ± 12.02 b	2.17 ± 0.06 a	1.56 ± 0.31 b	4.06 ± 1.19 a	0.014 ± 0.002 b	1.28 ± 0.11 a	0.84 ± 0.16 b
T11	120.4 ± 4.38 b	112.7 ± 4.45 b	2.08 ± 0.04 a	1.12 ± 0.58 b	2.37 ± 0.12 b	0.011 ± 0.014 b	1.12 ± 0.14 a	0.61 ± 0.44 b
T12	126.6 ± 9.28 b	119 ± 10.12 b	2.14 ± 0.03 a	1.95 ± 0.61 b	2.55 ± 0.49 b	0.031 ± 0.01 a	1.33 ± 0.12 a	1.24 ± 0.21 a

The same letters at the end of each column mean they are not significantly different by Dunnett’s test at p ≤ 0.05.

, T1 was found to have an amazing boost effect on the calcium and magnesium synthesis during kiwifruit ripening, suggested its promising function in fruit mineral content improvement. Whereas, other treatments did not show noticeable variation. Besides, all treatments declined the copper content because of the lower CPPU, meanwhile, T10 and T12 were found to have a stimulating effect on the iron and zinc generation, respectively.

3.2. Evaluation of Fruit Postharvest Quality of Different Treatments

Concerning the effect of different treatments on the postharvest quality of the fruit under cold storage, as storage time was prolonged, all treatments showed a tendency of decreased firmness of fruits; we found T3 and T7 possessed the highest and lowest hardness degree, respectively, compared to the non-treated control at day 12 (

Table 3. Effect of different treatments on the kiwifruit (Actinidia chinensis) ‘Donghong’ postharvest quality under cold storage.

Treatment	Hardness (N/cm)			TSS (%)	Dry Matter (%)
	Day 3	Day 7	Day 12
Control	12.36 ± 0.68 b	6.04 ± 0.54 b	4.37 ± 0.72 b	12.76 ± 0.56 a	13.97 ± 1.18 a
T1	13.85 ± 0.23 b	7.52 ± 0.43 b	5.27 ± 0.56 b	13.56 ± 0.26 a	16.81 ± 0.24 a
T2	15.69 ± 0.12 b	8.46 ± 0.28 b	6.58 ± 0.04 b	13.56 ± 1.78 a	14.41 ± 0.47 a
T3	17.08 ± 0.90 a	11.32 ± 1.10 a	8.92 ± 1.3 a	11.03 ± 0.91 a	14.64 ± 0.36 a
T4	12.34 ± 1.10 b	6.89 ± 0.13 b	4.88 ± 0.09 b	12.33 ± 0.22 a	15.27 ± 0.96 a
T5	13.87 ± 1.02 b	7.24 ± 0.12 b	5.35 ± 0.09 b	12.65 ± 0.14 a	15.04 ± 0.46 a
T6	15.98 ± 1.12 b	9.35 ± 1.09 b	6.41 ± 1.08 b	12.45 ± 0.7 a	15.89 ± 1.65 a
T7	17.55 ± 0.77 a	11.23 ± 0.87 a	8.91 ± 0.37 a	13.45 ± 0.39 a	15.41 ± 1.03 a
T8	14.23 ± 0.94 b	7.68 ± 0.78 b	5.29 ± 0.34 b	14.01 ± 0.62 a	14.99 ± 0.45 a
T9	15.02 ± 1.20 b	7.02 ± 0.80 b	4.69 ± 1.80 b	13.13 ± 0.33 a	15.01 ± 0.09 a
T10	15.59 ± 1.13 b	8.69 ± 0.69 b	6.35 ± 0.93 b	12.66 ± 0.70 a	16.66 ± 0.08 a
T11	11.45 ± 0.85 b	5.76 ± 0.12 b	3.56 ± 0.65 b	13.65 ± 0.19 a	13.84 ± 0.39 a
T12	12.32 ± 1.21 b	5.88 ± 1.02 b	3.47 ± 1.40 b	13.37 ± 0.41 a	14.61 ± 1.18 a

The same letters at the end of each column mean they are not significantly different by Dunnett’s test at p ≤ 0.05.

). The other treatments did not show a significant difference in the fruit-softening level. Regarding the TSS and dry matter, no significant variation was obtained when storage was finished (Table 3).

3.3. Comprehensive Evaluation of Different PGRs on Regulation of Fruits Characteristics during Substitution of CPPU in Kiwifruit ‘Donghong’

To screen out the optimal PGR in maintaining fruit quality when the substitution of CPPU is carried out, a method of modified Z-score, z’ = (x − μ)/σ was used to standardize all tested terms, where x is the raw value of T1–T12, μ is the control value, and σ is the population standard deviation. Except for the negative contributor of Titratable acids, all corresponding standardized data of each term were added to represent the total score of each treatment; the higher the value, the better the treatment could be.

As shown in Figure 2, T1 and T10 clearly have the greatest potential to improve both harvest and postharvest fruit quality. Assuming that they could be applied in the field to minimize the application content of CPPU in kiwifruit ‘Donghong’, we selected T1, which has the highest z’ score, to clarify the underlying mechanism for its successful substitution in more detail. T1 consists of CPPU at a concentration of 5 mg L⁻¹ and 14-OH BR at a concentration of 0.15 mg L⁻¹, which means a PGR of 14-OH BR plays a critical role in the promotion of fruit enlargement (Figure 3) as well as maintains the impressive fruit characteristics. 14-OH BR is a natural brassinolide analogue derived from rape pollen; it plays an important role in accelerating cell division and growth, nutrient absorption and transportation, as well as improving stress resistance; it also proved to be highly safe [31,32]. To elucidate the mechanisms underlying the role of 14-OH BR in the regulation of kiwifruit development further, RNA-seq was conducted.

3.4. Transcriptional Enrichment Analysis of 14-OH BR Regulation of Kiwifruit Development

The flesh of 3 groups of fruit samples 6 days after dipping (on 15 May) were used: fruits induced by water (6DCK), fruits induced by CPPU at a concentration of 5 mg L⁻¹ (6DUP) and fruits induced by CPPU at a concentration of 5 mg L⁻¹ and 14-OH BR at a concentration of 0.15 mg L⁻¹ (6D3). Using the Illumina HiSeq sequencing platform, a total of 58.19 Gb of paired-end short read data (after quality control and removal of reads containing adapter, reads containing ploy-N, and low-quality reads from raw data) were obtained. The percentages of Q30 and GC were 95.35–95.79% and 46.13–46.46%, respectively, indicating that the quality of transcriptome sequencing data is high.

A total of 31,308 genes were functionally annotated in the databases (Figure 4A, Supplementary file S1). Moreover, 920 (701 up and 219 down-regulated), 829 (503 up and 326 down-regulated) and 955 (374 up and 581 down-regulated) DEGs (|log2Fold Change| ≥ 1, and FDR < 0.05) were identified in the pairwise comparison of 6DCK versus 6DUP, 6DCK versus 6D3 and 6DUP versus. 6D3, respectively (Figure 4B, Supplementary file S2). The results indicated that 14-OH BR could induce the changes in transcripts at the very beginning of kiwifruit enlargement.

To dissect how 14-OH BR affects kiwifruit development further, we focused on the enrichment analysis of KEGG between 6D3 and 6DUP. GO and KEGG enrichment analysis was performed on the differential genes. The GO results showed that the differential genes were mainly concentrated in plant-activated biological processes, including abscisic acid, auxin and the ethylene-activated signaling pathway (Figure 5).

The results of KEGG enrichment analysis also showed that hormone transduction activation plays an important role in this process (Figure 6), suggesting that 14-OH BR regulation of kiwifruit development occurs mainly through changes to endogenous phytohormone signaling.

3.5. Candidate Genes Related to Phytohormone Signaling Transduction Involved in 14-OH BR-Regulated Kiwifruit Development

To unravel the molecular details regarding gene functioning in the regulation of phytohormone biosynthesis and signaling, we screened our RNA-seq datasets. The results exhibited induced gene downregulation of auxin signaling transduction in kiwifruits due to 14-OH BR treatment (

Table 4. List of DEGs involved in phytohormone biosynthetic and signaling in kiwifruit (Actinidia chinensis) ‘Donghong’ in response to 14-OH BR treatment.

ID	Functional Description	log2FoldChange (6D3 vs. 6DUP)	p-Value	Regulated
Ethylene signaling
gene-CEY00_Acc29732	ethylene receptor	1.139	0.000275	up
Gibberellin signaling
gene-CEY00_Acc00196	transcription factor PIF3	1.857	1.86 × 10−5	up
gene-CEY00_Acc31645	gibberellin receptor GID1	3.242	1.72 × 10−8	up
gene-CEY00_Acc00595	DELLA protein SLR1	−2.127	3.50 × 10−5	down
gene-CEY00_Acc16443	DELLA protein GA	−1.072	2.18 × 10−9	down
gene-CEY00_Acc31074	DELLA protein	−1.128	9.64 × 10−8	down
Abscisic acid signaling
gene-CEY00_Acc01626	protein phosphatase 2C 37-like	−2.358	4.32 × 10−13	down
gene-CEY00_Acc25841	protein phosphatase 2C	−2.137	1.51 × 10−6	down
gene-CEY00_Acc03739	protein phosphatase 2C 37-lik	−1.143	2.16 × 10−5	down
jasmonate acid biosynthesis and signaling
gene-CEY00_Acc02848	coronatine-insensitive protein 1	1.27	0.001792	up
gene-CEY00_Acc19795	JAR1-like	1.857	0.000702	up
gene-CEY00_Acc29006	transcription factor MYC2	−2.414	2.1 0× 10−20	down
gene-CEY00_Acc28151	transcription factor MYC2	−2.484	9.81 × 10−21	down
gene-CEY00_Acc17977	transcription factor MYC2-like	1.925	0.001716	up
gene-CEY00_Acc12940	JAZ1	−1.778	1.67 × 10−5	down
Auxin signaling
gene-CEY00_Acc07415	auxin synthetase GH3	−5.077	2.61 × 10−77	down
gene-CEY00_Acc19916	auxin-responsive protein SAUR36	−6.22	3.52 × 10−11	down
gene-CEY00_Acc23578	auxin-induced protein 6B-like	1.34	0.000355	up
gene-CEY00_Acc32215	auxin response factor	1.666	0.001074	up
gene-CEY00_Acc30497	AUXIN SIGNALING F-BOX 2-like	1.009	5.28 × 10−5	up
gene-CEY00_Acc25587	auxin-responsive protein IAA4-like	−1.651	2.52 × 10−10	down
gene-CEY00_Acc28907	auxin-responsive protein SAUR36	−7.345	1.62 × 10−7	down
gene-CEY00_Acc25588	auxin-responsive protein IAA4-like	−1.58	2.73 × 10−5	down
gene-CEY00_Acc16761	auxin-responsive protein SAUR36-lik	−6.026	3.32 × 10−10	down
gene-CEY00_Acc08534	auxin response factor 19	1.021	1.43 × 10−6	up
gene-CEY00_Acc09025	auxin-responsive protein SAUR24	−1.758	0.001619	down
gene-CEY00_Acc19500	auxin-induced protein 22D-like	-2.445	4.72 × 10−30	down
gene-CEY00_Acc13744	auxin early response protein GH3.5	−6.588	4.41 × 10−9	down
gene-CEY00_Acc10765	auxin-responsive protein ARG7	−1.323	0.000405	down
Brassinosteriod signaling
gene-CEY00_Acc29651	receptor BRI1	1.229	3.24 × 10−5	up
gene-CEY00_Acc12003	transcription factor BES1/BZR1	1.603	0.000802	up
gene-CEY00_Acc12063	cyclin D3-1	−1.131	0.000403	down
gene-CEY00_Acc15686	BR-signaling kinase	1.5	0.000112	up
Cytokinin signaling
gene-CEY00_Acc29817	ARR5-like isoform X1	−1.931	0.002259	down

). These results were supported by the decreased expression of most auxin-responsive genes, such as auxin-amido synthetases GH3, as well as various auxin-responsive proteins (such as SAUR36, IAA4, SAUR24, 22D and ARG7). Furthermore, 14-OH BR treatment also repressed the expression of the CK signaling pathway positive regulator of ARR5.

For the induced signaling transduction, we found protein phosphatase 2C (PP2C), which is an ABA signaling negative regulator, the GA signaling negative regulator DELLA protein as well as jasmonate acid (JA)signaling repressor JAZ1. They were downregulated at the same time. We also found that receptors of ethylene, GA and JA were induced by 14-OH BR treatment. Besides, the increased expression of JAR1 suggested the biosynthesis of JA was also stimulated.

Besides, as expected, genes involved in brassinosteroid (BR) signaling were upregulated, including the BR receptor BRI1, positive regulator BSK and corn transcription factor BES1/BZR1 (Table 4).

Collectively, these results suggested that 14-OH BR compensate the reduced content of CPPU to promote the kiwifruit ‘Donghong’ enlargement mainly through enhanced signaling transduction of BR, GA, ABA and JA, along with repressed signaling of auxin and CK. Additionally, it looks like the regulation of 14-OH BR treatment also involved with the JA biosynthesis and the involvement of ethylene biosynthesis and signaling are quite limited at the early stage of kiwifruit development.

4. Discussion

Plant growth regulators (PGRs) are natural or synthetic compounds that can positively modify plant growth and development by mimicking naturally occurring plant hormones in very small concentrations [33]. Forchlorfenuron (CPPU, N-(2-Chloro-4-pyridyl)-N’-phenylurea) is a synthetic cytokinin extensively used to increase the berry size of grapes and kiwifruit. It can stimulate the periclinal cell division leading to more round-shaped berries and increasing fruit quality. However, the incidence of CPPU has caused an adverse impact on the kiwifruit industry; its toxicological properties may persist in their transformation products (TPs), which were speculated to have an even higher toxicity than the parent molecule [34]. In this study, we found that the application of 14-OH BR, Jianda 990, Zeatin and GA₄₊₇ in the right concentration (T1, T4 and T12) could further minimize the CPPU content from 10 mg L⁻¹ to 5 mg L⁻¹ without significant fruit quality decreasing in comparison to the control of CPPU at a concentration of 10 mg L⁻¹ use alone (Table 1). Moreover, most treatments increased the hardness of the fruit and decreased the TSS, thus prolonging the kiwifruit ‘Donghong’ storage time (Table 3). GAs constitute a group of plant hormones that control developmental processes such as germination, shoot elongation, tuber formation, flowering, fruit set, and growth in diverse species [35]. It was reported that early growth in tomatoes depends on GAs [36], and GA treatment has the potential to increase fruit firmness and size, and delays the maturity of ‘sweetheart’ sweet cherry [37]. In our data, both GA₄₊₇ and GA₃ (the main component of Jianda 990) were found to stimulate kiwifruit growth. In detail, GA₄₊₇ at a high concentration and GA₃ at a low concentration have the best effects (Table 1 and Table 3). Zeatin and its derivatives occur in many plant extracts and are the active ingredient in coconut milk, which causes plant growth [38]; applications of zeatin riboside stimulated cell growth (cell division or/and cell elongation) in banana fruit [39]. Our data also showed that it has a fruit-prompting function (Table 1). In terms of the mineral and trace element content, we found T1 can increase the calcium and magnesium content of kiwifruit, which might be because 14-OH BR can increase nutrient absorption. At the same time, all twelve treatments displayed a depressed copper content. This could because less CPPU was applied; the underlying mechanism was not clear. Besides, no noticeable changes of fruit quality were found postharvest. Nevertheless, among the 12 treatments, T1, which consisted of CPPU at a concentration of 5 mg L⁻¹ and 14-OH BR at a concentration of 0.15 mg L⁻¹ was finally chosen for further detailed molecular analysis, because it fully maintained the characteristics of kiwifruit ‘Donghong’ when CPPU (10 mg L⁻¹) alone is used (Table 1, Table 2 and Table 3).

BRs have been collectively referred to as ‘pleotropic phytohormones’. They are essential for many biological processes in plants, such as initiation and cessation of flowering, plant canopy architecture, micropropagation, cell division and elongation, vegetative growth, flowering, fruit set, fruit ripening, quality and yield [40]. It was found that the application of BRs can boost the ripening of tomatoes and grapes [41,42]. In early fruit development, the application of exogenous 24-epibrassinolide can induce the expression of cell cycle-related genes in cucumbers [43]. Transcriptomic sequencing was conducted to elucidate the molecular mechanisms underlying 14-OH BR regulation of red flesh kiwifruit development at the initial stage. A total of 1894 DEGs were identified due to CPPU and 14-OH BR treatment. It was reported that both the initial cell division and the later cell expansion synergistically determine the final maximum size of fruits, where different phytohormones play a critical role in these processes [44,45]. In terms of the CPPU regulation of red flesh kiwifruit development, it was recorded that the GA and CKs contents were strongly increased, while the ABA and auxin contents were clearly decreased. Corresponding transcriptomic research has also consistently shown that expression levels of genes encoding GA and CK biosynthetic key enzymes, such as GA20-oxidase 1 (GA20ox1) and cytokinin phosphoribohydrolase ‘Lonely guy’ (LOG3), were significantly increased. In contrast, gene transcripts encoding auxin and ABA biosynthetic critical enzymes including flavin monooxygenase (YUCC2 and YUCCA2-like) and nine-cis-epoxycarotenoid dioxygenase (NCED3) were repressed [7,46]. When extra 14-OH BR was applied, our data showed that BR signaling was initiated as expected, and the expression of most auxin-responsive genes, such as SAUR36, IAA4, SAUR24, 22D and ARG7 decreased further.

In contrast, ABA signaling was enhanced by repressing the negative regulator PP2Cs (Table 4). These data suggested that, apart from the reduced content, the low-intensity response of auxin and, by contrast, increased ABA signaling transduction were indispensable in the 14-OH BR regulation of fruit development during the fastest growth stage. This finding was consistent with the finding that ABA was essential to the fruit size [47], whereas auxin signaling was repressed [7]. Furthermore, we also found that a transcription factor of ARR5 involved in CK signaling was repressed, whereas GA signaling was more stimulated via the regulation of the core components. These data indicate that 14-OH BR regulation of kiwifruits ‘Donghong’ growth is more dependent on the ABA and GA signaling transduction than the auxins and CKs during the early growth phase.

Interestingly, in our case, JA biosynthesis and signaling were also promoted under the stimulation of 14-OH BR. It was reported that, in strawberries, the bioactive JA jasmonoyl-isoleucine (JA-Ile) accumulated at the immature fruit stages and then decreased as the fruit ripens [48]. However, as a stress hormone, the way JA signaling functions in the fruit growth stage was still mostly unclear. Besides, as a climacteric fruit, the ripening of kiwifruit is tightly regulated by ethylene [49]. It has been reported that CPPU treatment increases ethylene biosynthesis [50], which did not agree with our data, possibly because of the different fruit growth testing stages.

5. Conclusions

In the present study, by screening effects of different concentrations of four PGRs plus lowered content (5 mg L⁻¹) of CPPU on the red flesh kiwifruit ‘Donghong’ enlargement, we screened 14-OH BR as a promising safer and effective PGR to minimize the use content of CPPU without detectable kiwifruits yield trade-offs. Moreover, 14-OH BR controls kiwifruit development mainly through the activation of BR signaling transduction, which can further synergistically and antagonistically regulate signaling of other endogenous growth regulators, including auxin, ABA, CKs, GA, JA and ethylene.