Effects of 6-Benzylaminopurine Combined with Prohexadione-Ca on Yield and Quality of Chrysanthemum morifolium Ramat cv. Hangbaiju

Abstract

Increasing shoot branch numbers of Chrysanthemum morifolium Ramat cv. Hangbaiju (Hangbaiju) is crucial for producing high flower yields. Pot experiments were designed to evaluate the effects of foliar application of 6-benzylaminopurine (6-BA) combined with prohexadione-Ca (Pro-Ca) on the yield and quality of Hangbaiju flowers. Foliar application of 6-BA combined with Pro-Ca typically increased leaf chlorophyll content and decreased leaf soluble sugar and soluble protein contents throughout the floral organ growth phase. At the bud formation phenophase stage (August), the contents of gibberellin (GA), indole-3-acetic acid (IAA), and zeatin (ZA) decreased (except for GA content in the 10 mg L⁻¹ 6-BA combined with 100 mg L⁻¹ Pro-Ca treatment), but abscisic acid (ABA) content increased. The yield of Hangbaiju flowers was found higher in plants treated with foliar application of 6-BA combined with Pro-Ca. Higher yields were found in the 6-BA₅ + Ca₁₀₀ and 6-BA₁₀ + Ca₁₀₀ treatments than in the 6-BA₅ + Ca₅₀ and 6-BA₁₀ + Ca₅₀ treatments, and the highest yield was observed in the 6-Ba₅ + Ca₁₀₀ treatment both in 2019 and 2020. Nutritional indices such as soluble sugar, soluble protein, total amino acid, and water extract increased, and medicinal indices such as flavonoid, total phenolics, chlorogenic acid, cynaroside, and 3,5-dicaffeoyl quinic acid slightly decreased after foliar application of 6-BA combined with Pro-Ca; however, they were all higher than the standards recorded in the Chinese Pharmacopeia. Overall, foliar application of 6-BA combined with Pro-Ca could increase the yield and nutritional quality of Hangbaiju flowers, enhancing its tea quality.

1. Introduction

The chrysanthemum is one of the most popular flowers from family Asteraceae and have been cultivated for more than 1400 years in China and for at least 1200 years in Japan. Chrysanthemum morifolium Ramat cv. Hangbaiju (Hangbaiju) is a complex hybrid and its popularity and long history of cultivation have led to the introduction of many cultivars showing great diversity of flower form, color and display [1]. It is an economically important species due to medicinal and ornamental value, such as liver function improvement, inflammation reduction, vision enhancement and cardiovascular disease prevention [2]. However, Hangbaiju cultivation is labor-extensive, and therefore, its economic benefit is restrained.

Hangbaiju is a perennial, short-day plant, which flowers when the night length is longer than a critical minimum. This coincides with autumn in a temperate climate with four seasons. In culture, an entire technology is tailored to achieve the desirable plant habit. In addition, Hangbaiju flowers grow at the top of branches, and the number of shoot branches is crucial for producing a high yield of Hangbaiju flowers [3]. Traditionally, the only approach to increasing branch numbers of Hangbaiju plants is frequent manual decapitation, which is both labor-intensive and expensive [4]. Moreover, manual decapitation increases the occurrence of diseases, including viral diseases. Approaches to solving this problem are the focus of our study.

Plant hormones play critical roles in the regulation of shoot branching and flowering. Exogenous plant hormones have been applied in agronomic and horticultural crops to regulate shoot numbers and growth states. 6-benzylaminopurine (6-BA) possesses the ability to induce bud formation and promote lateral bud growth in Lupinus angustifolius L. [5] and Camellia sinensis [6]. Foliar application of 6-BA enhances floral bud differentiation, and its effects have been observed in C. morifolium [7], Dendrobium [8], and Fagopyrum esculentum Moench [9]. The number of lateral buds of C. sinensis [6] and Malus domestica Borkh [10] was also increased by 6-BA application. Furthermore, 6-BA application could retard chlorophyll decomposition and enhance photosynthesis-related enzymes, thereby increasing the photosynthesis rate. Moreover, 6-BA heightens the carbohydrate transport rate from leaves to floral organs, consequently enhancing the yield of Zea mays L. [11] and Gossypium hirsutum L. [12].

Prohexadione-Ca (Pro-Ca) is a gibberellin (GA) biosynthesis inhibitor that has low toxicity and persistence in plants [13]. It has also been proven to be a useful tool for controlling excessive vegetative growth [14], such as inhibiting shoot extension [15] and decreasing internode length of Prunus avium L. [15] and Rubus idaeus Linn. [16,17]. Foliar application of Pro-Ca has been successfully applied to increase the yield and quality of Brassica rapa L. ssp. Pekinensisp [14], Rubus idaeus Linn. [17] and Chrysanthemum morifolium R. cv Monalisa White [18]. Since Pro-Ca has a low potential for bioaccumulation in the environment and has negligible toxicological effects on mammals, it has been used extensively in crops.

Increasing shoot branches and decreasing shoot length are key processes in increasing the yield of Hangbaiju flowers [19]. Increasing shoot branch numbers is complex and finely tuned by endogenous and exogenous hormones. Reducing shoot length can create better conditions for reproductive growth because this approach can reduce the nutrients consumed during vegetative growth [17]. Studies primarily demonstrated foliar application of 6-BA or Pro-Ca separately, and there is a lack of research concerning the effects of 6-BA combined with Pro-Ca on shoot branch and shoot length of Hangbaiju plants. This information will provide a labor-saving approach and is more effective compared to manual decapitation in the cultivation of Hangbaiju. In this study, 6-BA and Pro-Ca were cooperatively used for Hangbaiju plants. The main objectives were to study the effects of foliar application of 6-BA combined with Pro-Ca to Hangbaiju flowers, namely: (1) the yield and quality of Hangbaiju flowers; (2) the changes in leaf physiological index of Hangbaiju; (3) the endogenous hormone contents of Hangbaiju flowers; and (4) the effects on the key enzyme activity in Hangbaiju flowers.

2. Materials and Methods

2.1. Site Description

Pot experiments were conducted at the experimental station of Shandong Agricultural University, Taian, China (lat. 36° N, long. 117° W) in 2019–2020. The area has a temperate continental monsoon climate with an annual mean temperature of 13 °C and annual mean precipitation of 697.3 mm. Seasonal rainfall is concentrated in hot summers, especially in July and August. The properties of soils used in this experiment were as follows in 2019: pH 7.2, 29.3 g kg⁻¹ organic matter, 9 g kg⁻¹ total N, 119 mg kg⁻¹ available nitrogen, 32 mg kg⁻¹ available phosphorus, and 95 mg kg⁻¹ available potassium. They were recorded as follows for 2020: 7.1, 29.8 g kg⁻¹, 8.7 g kg⁻¹, 115 mg kg⁻¹, 35 mg kg⁻¹, and 91.2 mg kg⁻¹, respectively.

2.2. Experiment Design

Each black plastic pot (height, 25 cm; top diameter, 25 cm; bottom diameter, 21 cm) was filled with 10 kg of soil. Five grams of compound fertilizer (15-8-20), 1.2 g controlled-release nitrogen fertilizer (42% N, 60 days release longevity), 1.01 g urea (46% N), and 2 g potassium sulfate (51% K₂O) were incorporated uniformly with soil before planting. Two healthy Hangbaiju seedlings of uniform size and similar growth state were planted in each pot on 20 April 2019, and 22 April 2020. Designs of foliar application of 6-BA combined with Pro-Ca are presented in

Table 1. Designs of foliar application of 6-BA combined with Pro-Ca (mg L⁻¹).

Treatments	Control	6-BA5 + Ca50	6-BA5 + Ca100	6-BA10 + Ca50	6-BA10 + Ca100
6-BA	water decapitation	5	5	10	10
Pro-Ca		50	100	50	100

. Foliar application of 6-BA was performed on 11 June 2019, and 13 June 2020, and repeated three times with an interval of 14 days. Foliar application of Pro-Ca was carried out on 25 July 2019, and 27 July 2020, and repeated once 14 days after the initial applications. The control plants were decapitated three times. In addition, the same volume of water was sprayed on the control plants when the Pro-Ca solution was applied to the other treatments. A total of 50 pots were allocated to each treatment, of which 20 pots (5 pots at each of 4 sampling occasions) were used for leaf sampling and 10 pots were used for the analysis of flowers. Pots allocated to the various treatments and samplings were arranged in a completely random scheme. Twenty pots per treatment were kept as reserves and not used for any analysis.

2.3. Sampling

The fully expanded leaves from the upper zone of the plants were sampled every 30 days from 14 August 2019 and 17 August 2020, for a total of four times. At each sampling, each treatment was randomly set for five replicates (five pots). The collected leaves were stored at −80 °C for physiological analyses. The leave samples collected on 14 August 2019 and 17 August 2020 were also used to measure the endogenous hormone content. Fresh flowers with 70% tubiform-flower flowering were harvested on 10 and 17 November in 2019 and 2020, respectively. The flower number per plant, the diameter and weight of single fresh flowers, and the total weight of fresh flowers per pot were measured. Partial fresh flowers were frozen in liquid nitrogen and stored at −80 °C to determine the phenylalanine ammonia-lyase (PAL), chalcone isomerase (CHI), and polyphenol oxidase (PPO) content. The other flowers were dried at 50 °C to a constant weight and then finely powdered for subsequent analysis. Each treatment was determined for five replicates in all quantitative analysis.

2.4. Physiological Index and Enzyme Activity Analysis

Chlorophyll content was determined using the ethanol extraction method. The soluble sugar and soluble protein contents were measured by Coomassie Brilliant Blue and anthrone colorimetry, respectively. PPO activity was determined using the catechol method [20]. High-performance liquid chromatography (HPLC) was performed to determine the endogenous hormones contents. Fresh flower samples of Hangbaiju (0.25 g) were covered with 2.5 mL boric acid buffer (containing 0.25 g PVP and 0.15 g quartz sand) at pH 8.8. PAL and CHI activities were measured using the colorimetric method at 290 nm, as described by Sun [21].

2.5. Quality Index Analysis

The flavonoid and total phenol contents were measured using the aluminum nitrate colorimetric method [21] and Folin–Ciocalteu reagent [17], respectively. The protein, soluble sugar, and total amino acid contents were assayed using the biurea method, anthrone colorimetry, and ninhydrin colorimetry methods, respectively [20]. The water extract content was determined using the cold maceration method according to the Chinese Pharmacopeia [22], and the contents of chlorogenic acid, cynaroside, and 3,5-dicaffeoyl quinic acid were measured by HPLC [22] and detected at 348 nm with an Acquity UPLC^®BEH-C18 column (2.1 mm × 100 mm × 1.7 μm). The mobile phase used was (A) acetonitrile and (B) 0.05% H₃PO₄ for gradient elution (0–11 min: 10–18% A, 11–30 min: 18–20% A) at 20 °C with a flow rate of 0.3 mL min⁻¹ and sample volume of 4 μL.

2.6. Statistical Analysis

All results were subjected to statistical analysis using data processing system software. The analysis of variance (ANOVA) and least significance difference (LSD) tests were used for significant differences among treatments at a 5% level of significance (p < 0.05) in SPSS Statistics 20.0 (Chicago, IL, USA). The data were sorted and depicted with tables and figures using Microsoft Excel 2010 software (Microsoft Corp., Redmond, WA, USA).

3. Results

3.1. Effects of 6-BA Combined with Pro-Ca on Yield of Hangbaiju Flowers

Significant effects of 6-BA and Pro-Ca in yield parameters and yield of Hangbaiju flowers existed, both in 2019 and 2020. Foliar applications of 6-BA combined with Pro-Ca increased yield and the associated components in Hangbaiju flowers, with the exception of the 6-Ba₁₀ + Ca₅₀ treatment (

Table 2. Changes of yield and yield components of Hangbaiju flowers.

Year	Treatments	Single Flower Fresh Weight (g)	Single Flower Diameter (cm)	Open Flower Numbers per Pot (num.)	Yield (g pot−1)
2019	Control	1.62 ± 0.10 bc	4.84 ± 0.14 bc	70.25 ± 3.59 b	113.81 ± 5.82 c
	6-Ba5 + Ca50	1.90 ± 0.21 a	4.95 ± 0.26 b	63.67 ± 3.79 c	120.97 ± 7.19 bc
	6-Ba5 + Ca100	1.87 ± 0.12 a	5.20 ± 0.15 a	90.67 ± 3.79 a	169.55 ± 7.08 a
	6-Ba10 + Ca50	1.58 ± 0.15 c	4.69 ± 0.16 c	62.67 ± 2.52 c	99.01 ± 3.98 d
	6-Ba10 + Ca100	1.73 ± 0.08 b	4.90 ± 0.13 b	74.50 ± 5.97 b	128.89 ± 10.33 b
2020	Control	1.65 ± 0.10 bc	4.82 ± 0.15 bc	71.23 ± 3.41 b	112.34 ± 6.04 c
	6-Ba5 + Ca50	1.89 ± 0.17 a	4.98 ± 0.22 b	62.89 ± 3.25 c	118.98 ± 8.32 bc
	6-Ba5 + Ca100	1.88 ± 0.11 a	5.32 ± 0.17 a	91.47 ± 3.48 a	179.72 ± 6.43 a
	6-Ba10 + Ca50	1.56 ± 0.15 c	4.63 ± 0.13 c	61.46 ± 2.33 c	97.35 ± 3.24 d
	6-Ba10 + Ca100	1.70 ± 0.08 b	4.93 ± 0.17 b	73.26 ± 4.67 b	130.26 ± 11.15 b

Control: water + decapitation; 6-BA₅ + Ca₅₀: 5 mg/L 6-BA + 50 mg/L Pro-Ca; 6-BA₅ + Ca₁₀₀: 5 mg/L 6-BA + 100 mg/L Pro-Ca; 6-BA₁₀ + Ca₅₀: 10 mg/L 6-BA + 50 mg/L Pro-Ca; 6-BA₁₀ + Ca₁₀₀: 10 mg/L 6-BA + 100 mg/L Pro-Ca. Different letters indicate significant differences (p < 0.05) according to the Tukey’s test. The errors represent the standard error of the mean (n = 5).

). When the 6-BA level was the same, the yield and its components in Hangbaiju flowers increased with increasing concentrations of Pro-Ca, irrespective of the 6-BA level being 5 mg L⁻¹ or 10 mg L⁻¹. The highest yield was recorded in the 6-Ba₅ + Ca₁₀₀ treatment (an increase of 48.98% in 2019 and 59.98% in 2020, respectively, compared with the control), followed by the 6-Ba₁₀ + Ca₁₀₀ treatment (which increased by 13.25% in 2019 and 15.95% in 2020, respectively, compared with the control).

3.2. Effects of 6-BA Combined with Pro-Ca on Quality of Hangbaiju Flowers

3.2.1. Effect on Nutritional Quality Attributes

The addition of 6-BA and Pro-Ca had no significant effects on any of the nutritional indices considered, both in 2019 and 2020 (

Table 3. Changes of nutritional quality index of Hangbaiju flowers (%).

Year	Treatments	Soluble Sugar	Soluble Protein	Total Amino Acid	Water Extract
2019	Control	22.39 ± 1.57 c	9.92 ± 0.25 b	0.91 ± 0.01 ab	35.57 ± 1.13 b
	6-Ba5 + Ca50	25.54 ± 0.25 a	10.06 ± 0.24 ab	0.86 ± 0.04 b	39.29 ± 0.53 a
	6-Ba5 + Ca100	22.57 ± 0.78 c	10.87 ± 0.61 a	0.95 ± 0.11 ab	34.96 ± 5.46 b
	6-Ba10 + Ca50	24.72 ± 0.76 ab	10.43 ± 0.14 a	0.86 ± 0.04 b	37.71 ± 1.18 ab
	6-Ba10 + Ca100	23.42 ± 0.94 b	10.65 ± 1.08 a	1.02 ± 0.05 a	35.42 ± 3.27 b
2020	Control	21.78 ± 1.52 c	9.86 ± 0.35 b	0.90 ± 0.03 ab	35.13 ± 1.53 b
	6-Ba5 + Ca50	25.68 ± 0.42 a	10.01 ± 0.22 ab	0.85 ± 0.04 b	39.84 ± 0.74 a
	6-Ba5 + Ca100	22.13 ± 0.39 c	10.88 ± 0.42 a	0.92 ± 0.11 ab	34.56 ± 3.13 b
	6-Ba10 + Ca50	24.89 ± 0.56 ab	10.51 ± 0.45 a	0.84 ± 0.04 b	38.02 ± 1.42 ab
	6-Ba10 + Ca100	23.38 ± 0.86 b	10.67 ± 0.47 a	0.98 ± 0.06 a	34.92 ± 3.41 b

Control: water + decapitation; 6-BA₅ + Ca₅₀: 5 mg/L 6-BA + 50 mg/L Pro-Ca; 6-BA₅ + Ca₁₀₀: 5 mg/L 6-BA + 100 mg/L Pro-Ca; 6-BA₁₀ + Ca₅₀: 10 mg/L 6-BA + 50 mg/L Pro-Ca; 6-BA₁₀ + Ca₁₀₀: 10 mg/L 6-BA + 100 mg/L Pro-Ca. Different letters indicate significant differences (p < 0.05) according to the Tukey’s test. The errors represent the standard error of the mean (n = 5).

). Foliar application of 6-BA combined with Pro-Ca consistently increased soluble sugar and soluble protein contents as compared to the control. When the 6-BA level was the same, the soluble sugar content decreased as the combined concentration of Pro-Ca increased. In contrast, the soluble protein content increased. The total amino acid content decreased in the 6-Ba₅ + Ca₅₀ and 6-Ba₁₀ + Ca₅₀ treatments, but increased in the 6-Ba₅ + Ca₁₀₀ and 6-Ba₁₀ + Ca₁₀₀ treatments, which increased by 4.40% and 12.09% in 2019 and 2.22% and 8.89%, respectively, in 2020. The water extract content exhibited the opposite tendency to that of the total amino acid content. Comparatively, the 6-Ba₅ + Ca₁₀₀ treatment had relatively lower contents of soluble sugar and water extract, and relatively higher protein and total amino acid contents than the other treatments.

3.2.2. Effects on Medicinal Quality Attributes

The addition of 6-BA and Pro-Ca significantly affected all medicinal indices, except for flavonoid content (p < 0.01) (

Table 4. Changes of medicinal quality index of Hangbaiju flowers (%).

Year	Treatment	Flavonoid	Total Phenolics	Chlorogenic Acid	Cynaroside	3,5-Dicaffeoyl Quinic Acid
2019	Control	10.04 ± 1.15 a	2.67 ± 0.11 ab	0.79 ± 0.02 b	0.25 ± 0.01 bc	1.94 ± 0.03 b
	6-Ba5 + Ca50	10.01 ± 0.43 a	2.81 ± 0.08 a	0.83 ± 0.04 a	0.26 ± 0.00 b	2.02 ± 0.01 a
	6-Ba5 + Ca100	9.56 ± 0.11 b	2.68 ± 0.09 ab	0.71 ± 0.02 d	0.24 ± 0.01 c	1.84 ± 0.02 c
	6-Ba10 + Ca50	10.04 ± 0.59 a	2.55 ± 0.02 b	0.75 ± 0.02 c	0.26 ± 0.00 b	1.92 ± 0.04 b
	6-Ba10 + Ca100	10.10 ± 0.81 a	2.76 ± 0.08 a	0.81 ± 0.01 ab	0.30 ± 0.01 a	1.90 ± 0.03 bc
2020	Control	10.05 ± 0.87 a	2.63 ± 0.11 ab	0.78 ± 0.03 b	0.24 ± 0.02 bc	1.96 ± 0.03 b
	6-Ba5 + Ca50	10.00 ± 0.21 a	2.93 ± 0.11 a	0.85 ± 0.03 a	0.27 ± 0.00 b	2.12 ± 0.01 a
	6-Ba5 + Ca100	9.43 ± 0.07 b	2.66 ± 0.09 ab	0.69 ± 0.01 d	0.22 ± 0.02 c	1.82 ± 0.01 c
	6-Ba10 + Ca50	10.05 ± 0.35 a	2.34 ± 0.05 b	0.74 ± 0.01 c	0.27 ± 0.00 b	1.94 ± 0.03 b
	6-Ba10 + Ca100	10.13 ± 0.31 a	2.79 ± 0.09 a	0.82 ± 0.02 ab	0.32 ± 0.01 a	1.93 ± 0.04 bc

Control: water + decapitation; 6-BA₅ + Ca₅₀: 5 mg/L 6-BA + 50 mg/L Pro-Ca; 6-BA₅ + Ca₁₀₀: 5 mg/L 6-BA + 100 mg/L Pro-Ca; 6-BA₁₀ + Ca₅₀: 10 mg/L 6-BA + 50 mg/L Pro-Ca; 6-BA₁₀ + Ca₁₀₀: 10 mg/L 6-BA + 100 mg/L Pro-Ca. Different letters indicate significant differences (p < 0.05) according to the Tukey’s test. The errors represent the standard error of the mean (n = 5).

). Foliar application of 6-BA combined with Pro-Ca typically decreased flower flavonoid content, except for the 6-Ba₁₀ + Ca₁₀₀ treatment. The total phenolic content was increased by foliar application of 6-BA combined with Pro-Ca, except for in the 6-Ba₁₀ + Ca₅₀ treatment. The chlorogenic acid content in flowers in the 6-Ba₅ + Ca₅₀ and 6-Ba₁₀ + Ca₁₀₀ treatments was higher than that in the control treatment, whereas it was lower in flowers in the 6-Ba₅ + Ca₁₀₀ and 6-Ba₁₀ + Ca₅₀ treatments. The cynaroside and 3,5-dicaffeoyl quinic acid contents in flowers treated with 6-BA combined with Pro-Ca were higher than those in the control treatment, except for in the 6-Ba₅ + Ca₁₀₀ treatment. All parameters in flowers treated with 6-BA combined with Pro-Ca exhibited a similar tendency in both 2019 and 2020. When the 6-BA level was 5 mg L⁻¹, they consistently decreased as the combined concentration of Pro-Ca increased; when the 6-BA level was 10 mg L⁻¹, they consistently increased. Compared with the control, the 6-Ba₅ + Ca₁₀₀ treatment slightly decreased the contents of flavonoids, total phenolics, chlorogenic acid, cynaroside, and 3,5-dicaffeoyl quinic acid, but they were all higher than the standards recorded in the Chinese Pharmacopeia (Chinese Pharmacopoeia Committee, 2020). A similar trend for all medicinal indices considered was detected in 2019 and 2020.

3.3. Effects of 6-BA Combined with Pro-Ca on Leaf Physiological Index of Hangbaiju

The changes in the leaf physiological index were similar in 2019 and 2020, and we only analyzed these changes in 2020. Figure 1 shows the changes in the leaf physiological index as well as evolution over time. The behavior of each physiological index was significantly different. The chlorophyll content in the control and 6-Ba₁₀ + Ca₁₀₀ treatments decreased sharply from August to October, and the chlorophyll content in other treatments exhibited a tendency to decrease with a slight peak in September (Figure 1A). From October onwards, the chlorophyll content in all treatments increased, with the exception of the 6-Ba₁₀ + Ca₅₀ treatment.

The changes in soluble sugar content in leaves in all treatments behaved similarly from August to October (Figure 1B). They decreased markedly and reached the lowest values in September, then increased drastically, and reached the highest values in October. Thereafter, the soluble sugar content varied among treatments. It decreased slightly in the 6-Ba₅ + Ca₅₀ and control treatments and decreased markedly in the 6-Ba₅ + Ca₁₀₀ and 6-Ba₁₀ + Ca₁₀₀ treatments, whereas it increased continuously in the 6-Ba₁₀ + Ca₅₀ treatment.

The soluble protein content in all treatments showed a similar pattern (Figure 1C). It gradually increased and reached maximum values in October and then decreased with time. During the whole growth period, soluble protein content was always lower in the 6-Ba₅ + Ca₁₀₀ and 6-Ba₁₀ + Ca₁₀₀ treatments than in the 6-Ba₅ + Ca₅₀ and 6-Ba₁₀ + Ca₅₀ treatments, and the lowest values were always recorded in the 6-Ba₅ + Ca₁₀₀ treatment.

3.4. Effects of 6-BA Combined with Pro-Ca on the Contents of Leaf-Endogenous Hormones in Hangbaiju

The changes in endogenous hormone contents were similar in 2019 and 2020; thus, we analyzed these changes in 2020 only. Figure 2 shows the changes in endogenous hormones of Hangbaiju leaves in August. The contents of GA, indole-3-acetic acid (IAA), and zeatin (ZA) were significantly decreased by foliar application of 6-BA combined with Pro-Ca, with the exception of the 6-Ba₅ + Ca₁₀₀ treatment. When 6-BA levels were the same, GA content significantly decreased as the concentration of Pro-Ca was increased, and a more prominent decrease was found between the 6-Ba₅ + Ca₅₀ and 6-Ba₅ + Ca₁₀₀ treatments than between the 6-Ba₁₀ + Ca₅₀ and 6-Ba₁₀ + Ca₁₀₀ treatments (Figure 2A). When the 6-BA level was 5 mg L⁻¹, IAA and ZA contents markedly increased with increasing concentrations of Pro-Ca; when the 6-BA level was 10 mg L⁻¹, the opposite tendency was observed (Figure 2B,C). Abscisic acid (ABA) content was increased by foliar application of 6-BA combined with Pro-Ca, especially in the 6-Ba₅ + Ca₁₀₀ treatment which was 1.98 times higher than that in the control treatment (Figure 2D).

3.5. Effects of 6-BA Combined with Pro-Ca on Activity of Key Enzymes in Hangbaiju Flowers

The changes in key enzyme activity of Hangbaiju flowers were similar in 2019 and 2020, and thus we analyzed these changes in 2020 only. There were significant differences in the activities of PAL, CHI, and PPO. Foliar application of 6-BA combined with Pro-Ca enhanced PAL activity, but markedly decreased CHI activity (Figure 3). When the 6-BA level was 5 mg L⁻¹, PAL activity increased as the concentration of Pro-Ca increased, but the tendency reversed when the 6-BA level was 10 mg L⁻¹ (Figure 3A). CHI activity in plants treated with 6-BA combined with Pro-Ca exhibited a similar tendency to that of PAL activity (Figure 3B). Foliar application of 6-BA combined with Pro-Ca enhanced PPO activity, except for in the 6-Ba₅ + Ca₁₀₀ treatment (Figure 3C). The tendency of PPO activity in 6-BA combined with Pro-Ca treatments was contrary to that of PAL activity.

4. Discussion

4.1. Regulation of 6-BA Combined with Pro-Ca on Yield of Hangbaiju Flowers

The close relationship between reproductive and vegetative organs has been extensively investigated and demonstrated. Floral bud formation and growth are strongly affected by leaf physiological traits because the growth of newly formed floral buds requires a mass of carbohydrates derived from leaves [23]. Yang et al. [24] reported that the soluble sugar content in Bougainvillea glabra leaves decreased during floral organ growth. Fang et al. [12] found that a decrease in sucrose and starch content in cotton leaves was consistently associated with an increase in the carbohydrate content of its floral buds. Floral organ growth in Hangbaiju is a continuous process that is concentrated from mid-August to mid-September [4]. In this study, a decrease in chlorophyll content also occurred during this period. Thus, we inferred that the soluble sugar in Hangbaiju leaves might be transported to floral organs to satisfy the requirements of floral organs. Changes in chlorophyll, soluble sugar, and soluble protein content in leaves offered a good indicator of leaf physiological status. The chlorophyll content decreased from September to October but increased from October to November. Meanwhile, an opposite tendency was observed in the soluble sugar and soluble protein contents. These changes suggest that the transport rate of soluble sugars from leaves to floral organs is not consistent.

From October onwards, the chlorophyll content was higher in leaves in the 6-BA₅ + Ca₁₀₀ and 6-BA₁₀ + Ca₁₀₀ treatments than that in the 6-BA₅ + Ca₅₀ and 6-BA₁₀ + Ca₅₀ treatments, whereas the soluble sugar and soluble protein contents were lower than those in leaves in the 6-BA₅ + Ca₅₀ and 6-BA₁₀ + Ca₅₀ treatments (Figure 1). Moreover, the yield of Hangbaiju flowers in these treatments exhibited a similar tendency to that of the chlorophyll content. Thus, we inferred that 6-BA₅ + Ca₁₀₀ and 6-BA₁₀ + Ca₁₀₀ treatments had a greater ability to transport soluble sugar from leaves to floral organs than the 6-BA₅ + Ca₅₀ and 6-BA₁₀ + Ca₅₀ treatments, consequently resulting in less leaf vigor and accelerated senescence. This inference was supported by a decrease in GA content and an increase in ABA content in leaves. Li et al. [25] indicated that 6-BA enhanced the transport rate of soluble sugars from leaves to flowers, in agreement with our results.

Li et al. [10] suggested that 6-BA inhibited excessive vegetative growth and promoted reproductive growth. Jin et al. [9] reported that spraying 6-BA increased the yield of F. esculentum Moench by accelerating floral bud formation. The greater flower numbers of Hangbaiju treated with 6-BA combined with Pro-Ca indicated that foliar application of 6-BA combined with Pro-Ca was beneficial to floral organ growth. In addition, higher ABA content and lower contents of IAA and GA were observed concurrently in leaves treated with 6-BA combined with Pro-Ca. Zheng et al. [26] reported that IAA and GA suppressed floral bud formation, but ABA accelerated this process. The content and balance of endogenous hormones are tightly linked to floral bud formation [27]. Kang et al. [14] proved that Pro-Ca influenced the metabolism of endogenous hormones. A lower GA content is beneficial for floral bud formation in Hangbaiju flowers [3] and Pro-Ca inhibits GA biosynthesis [14]. Wang et al. [28] found that the application of 6-BA increased the number of Chrysanthemum flowers. We speculated that the increased number of Hangbaiju flowers might be attributed to the effects of 6-BA and/or Pro-Ca on the regulation of endogenous hormone levels. The decrease in GA content and the increase in ABA content were more prominent in plants in the 6-BA₅ + Ca₁₀₀ and 6-BA₁₀ + Ca₁₀₀ treatments than in plants in the 6-BA₅ + Ca₅₀ and 6-BA₁₀ + Ca₅₀ treatments. In addition, the flower number and yield of Hangbaiju showed a similar trend. Therefore, we inferred that spraying 6-BA in June could increase the branch number of Hangbaiju and spraying Pro-Ca in late July and early August could increase flower number, thereby increasing the flower yield of Hangbaiju.

4.2. Regulation of 6-BA Combined with Pro-Ca on Quality of Hangbaiju Flowers

Treatment with 6-BA caused the transportation rate of nutrients to spike and improved soluble sugar and sucrose contents in wheat [25]. Our results also indicated that foliar application of 6-BA combined with Pro-Ca obviously increased the nutritional quality of Hangbaiju flowers. This could be observed through the elevated contents of soluble sugar, soluble protein, total amino acids, and water extracts, consequently enhancing its tea quality. Simultaneously, a slight decrease in the medicinal indices of Hangbaiju flowers was found based on the relatively lower contents of chlorogenic acid, cynaroside, 3,5-dicaffeoyl quinic acid, and total flavonoids. Since carbohydrates are the common precursors of primary and secondary metabolic pathways, competition between primary and secondary metabolic pathways always exists. The elevated contents of soluble sugar, soluble protein, total amino acid, and water extracts suggested that a majority of carbohydrates were allocated to the primary metabolic pathway, and consequently, a minority of these carbohydrates were allocated to the secondary metabolic pathway. Thus, the lower contents of chlorogenic acid, cynaroside, 3,5-dicaffeoyl quinic acid, total phenolics, and flavonoids were not surprising. Puhl et al. [29] reported that the application of Pro-Ca reduced flavonol content in Vitis vinifera L. leaves, which agreed with our results.

Chlorogenic acid, cynaroside, 3,5-dicaffeoyl quinic acid, and flavonoids are all derived from the phenylpropanoid pathway, and the synthesis of these ingredients is a very complicated process and catalyzed by many enzymes. PAL is the first rate-limiting enzyme of the phenylpropanoid pathway. The effects of PAL activity on the content of these ingredients are controversial in the literature. Some studies have shown that PAL activity is closely related to the total flavonoid, chlorogenic acid, and 3,5-dicaffeoyl quinic acid in Scutellaria baicalensis [30] and C. morifolium [31]. In contrast, some studies have reported that flavonoid content is negatively correlated with PAL activity [32,33]. CHI is a key enzyme involved in flavonoid metabolism [34]. It facilitates flavonoid biosynthesis and enhances flavonoid content in Ginkgo biloba [35], Glycyrrhiza uralensis Fisch [36], and Scutellaria baicalensis [37]. PPO is another key enzyme in the metabolism of phenols, which catalyzes the synthesis of other flavonoids and decreases flavonoid content [38]. Schmitzer et al. [39] observed that spraying Pro-Ca decreased flavonoid content and strengthened PPO activity in rose flowers. Thus, foliar application of 6-BA combined with Pro-Ca increased PAL activity, however the decreased content of these medicinal ingredients in Hangbaiju flowers might be attributed to the complex synergistic interaction of PAL, CHI, and PPO. Our findings are consistent with the report of Roemmelt et al. [40], who demonstrated that spraying Pro-Ca could alter flavonoid metabolism.

In addition to their medicinal importance, Hangbaiju flowers have become attractive as common materials for functional tea because of their unique flavor, color, and health benefits [41]. The quality evaluation of Hangbaiju tea is dependent on its taste and quality of nutrition and health benefits. Foliar application of 6-BA combined with Pro-Ca significantly increased the nutritional quality of Hangbaiju tea but slightly decreased its health-related qualities (Table 3 and Table 4). The health-related ingredients of Hangbaiju tea are secondary metabolites, and most secondary metabolites have some unique pungent taste. For example, the taste of flavonoids is bitter. High contents of secondary metabolites such as chlorogenic acid, cynaroside, 3,5-dicaffeoyl quinic acid, phenolics, and flavonoids in Hangbaiju generate a somewhat terrible taste, which is not desired in tea. Foliar application of 6-BA combined with Pro-Ca slightly lowered the contents of its health-related ingredients, favoring the promotion of market acceptance. Overall, foliar application of 6-BA combined with Pro-Ca enhanced flower yield and tea quality in Hangbaiju. Moreover, the 6-BA₅ + Ca₁₀₀ treatment exhibited the best comprehensive effect.

5. Conclusions

Foliar application of 6-BA combined with Pro-Ca enhanced the yield and nutritional quality of Hangbaiju flowers, and its health-related quality was more beneficial for enhancing the tea quality of Hangbaiju flowers. Higher yield and quality of Hangbaiju flowers were found in the 6-BA₅ + Ca₁₀₀ and 6-BA₁₀ + Ca₁₀₀ treatments than in the 6-BA₅ + Ca₅₀ and 6-BA₁₀ + Ca₅₀ treatments. A similar trend was observed in 2019 and 2020. During the growth stages, the contents of soluble sugar and soluble protein in leaves were lower in the 6-BA₅ + Ca₁₀₀ and 6-BA₁₀ + Ca₁₀₀ treatments than those in the 6-BA₅ + Ca₅₀ and 6-BA₁₀ + Ca₅₀ treatments. In contrast, the chlorophyll content exhibited a reversed trend. The high yield of Hangbaiju flowers in the 6-BA₅ + Ca₁₀₀ and 6-BA₁₀ + Ca₁₀₀ treatments may be attributed to the fact that the 6-BA₅ + Ca₁₀₀ and 6-BA₁₀ + Ca₁₀₀ treatments had a greater ability to transport soluble sugars from leaves to floral organs than the 6-BA₅ + Ca₅₀ and 6-BA₁₀ + Ca₅₀ treatments. Similarly, 6-BA₅ + Ca₁₀₀ and 6-BA₁₀ + Ca₁₀₀ treatments resulted in higher nutritional quality than 6-BA₅ + Ca₅₀ and 6-BA₁₀ + Ca₅₀ treatments. When the 6-BA level was 5 mg L⁻¹, the health index of Hangbaiju flowers consistently decreased as the combined concentration of Pro-Ca increased; when the 6-BA level was 10 mg L⁻¹, they consistently increased. These results indicate that a synergistic effect exists between 6-BA and Pro-Ca, and that the concentration of Pro-Ca has a prominent effect. Thus, the 6-BA₅ + Ca₁₀₀ treatment was especially effective. The results of this study could provide a labor-saving and worth popularizing method for the cultivation of Hangbaiju.