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. 2023 Jul 31;29(7):971–983. doi: 10.1007/s12298-023-01339-z

Exogenous spraying of 4-chlorophenoxyacetic acid sodium salt promotes growth and flavonoid biosynthesis of mulberry leaves (Morus alba L.)

Zhen Zhang 1,2,#, Yun Duan 1,5,6,#, Yu Zhang 3,4, Shanying Zhang 2, Xiaoyu Liang 3,4, Jiahao Li 2, Saihao Ren 2, Chenghui Zhang 2,3,4,✉, Meng Wang 3,4,✉
PMCID: PMC10462555  PMID: 37649887

Abstract

Mulberry (Morus alba L.) leaves are known as an ideal vegetable with good antioxidant effect, which can bring delicious taste and multiple health benefits. In the present study, the effects of 4-Chlorophenoxyacetic acid sodium salt (4-CPANa) treatment on growth and content of flavonoid compounds in mulberry leaves were investigated. Moreover, the changes in the expression levels of genes involved in flavonoid biosynthetic pathways, and the accumulation of important secondary metabolites including rutin (Rut), chlorogenic acid (ChA), isoquercitrin (IQ) and astragalin (Ast), were investigated in mulberry leaves. The results showed that 4-CPANa treatment could significantly promote the differentiation and growth of mulberry, increased shoot number, bud number, leaf fresh weight and leaf area of mulberry compared with control. Besides, the contents of ChA, Rut, IQ and Ast were significantly increased after 4-CPANa (5 mg/L) treatment. Further analysis revealed that 5 mg/L 4-CPANa strongly induced the expression of flavonoid biosynthesis-related genes including flavonoid 3-O-glucosyltransferase (F3GT) gene, chalcone synthase (CHS) gene, 4-xoumarate-CoA ligase (4CL) and phenylalanine ammonia lyase (PAL) gene. In conclusion, exogenous spraying of 4-CPANa provides a new way to improve the medicinal quality and development of mulberry leaf food with high value.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-023-01339-z.

Keywords: Mulberry leaf, Flavonoid, 4-chlorophenoxyacetic acid sodium salt, Gene expression

Introduction

In recent years, the development of edible mulberry leaves has attracted more and more attention in Asia and other countries. Mulberry leaf is an ideal vegetable because it not only satisfies people's consumption at the table, but also has health functions (Tu et al. 2019). Mulberry leaf is rich in medicinal active substances, such as flavonoids, phenolic acids, polysaccharides, alkaloids, phytosterols and other medicinal active ingredients (Salcedo et al. 2016). Prenylated flavonoids, the characteristic phytochemicals dominated in Moraceae family, exerting various health benefits like antioxidant activity (Yang et al. 2015), can effectively reduce lipid accumulation in the body and prevent obesity (Chang et al. 2016; Fan et al. 2020; Zhong et al. 2020). In addition, mulberry leaf polyphenol extract can significantly reduce the accumulation of peripheral lipids by inducing apoptosis of mature adipocytes and inhibiting adipocyte differentiation, thus acting as a preventive and therapeutic agent against obesity (Chang et al. 2016). These studies have shown that mulberry leaf is a kind of plant resource with good health care function.

Accordingly, mulberry leaf has been utilized in variety of food, such as mulberry leaf tea (Sheng et al. 2018), mulberry leaf green juice powder and mulberry leaf soybean milk (Kim et al. 2018), with the most popular mulberry bud tea being the most promising mulberry leaf food. The difference between mulberry leaf tea and bud tea is that leaf tea is usually taken from leaves that are not at the top, while the materials of bud tea are derived from one bud and two leaves from the top of mulberry shoots (leaf bud and the two leaves immediately below it). Due to the precise requirements for these rare raw materials, the production of bud tea is limited. As the demand for bud tea has increased, the new bud of mulberry seedlings is seriously lacking, leading to the shortage of market supply (Hu 2020; Li et al. 2019). Therefore, the selection of mulberry leaf raw materials rich in medicinal ingredients by improving the quality of raw materials is crucial for the development of mulberry leaf food as well as for the market demand.

4-Chlorophenoxyacetic acid sodium salt (4-CPANa) is a salt formed from 4-chlorophenoxyacetic acid and sodium ions with a neutral pH. 4-CPANa and 4-chlorophenoxyacetic acid (4-CPA) have similar properties and belong to plant growth regulator. Some studies have shown that 4-CPA can promote the development and maturation of parthenocarpic fruits (Nandwani 2014). For example, Karyagin et al. ( 2020) found that 4-CPA could induce the synthesis of indole-3-acetic acid (IAA) in tomato ovaries to mature parthenocarpic fruits earlier. In addition, the biomass and size of parthenocarpic fruits were larger than that of seed-bearing fruits (Karyagin et al. 2020). A similar study also found that 4-CPA significantly increased the fresh weight, dry weight, size and yield of tomato fruits (Meliha 2006). Many studies have shown that 4-CPA has a good effect on the yield and quality of Solanaceae plants, but there has been no study on the effect of 4-CPA on mulberry leaves, and the mechanism of its effect on mulberry leaves growth and development is still unclear. Therefore, the aim of this study was to clarify the effects of 4-CPANa on the growth of mulberry, the physiological indexes of mulberry leaves, the accumulation of traditional Chinese medicine components in mulberry leaves, and expression levels of related enzyme genes. To develop an efficient, rapid, convenient and safe regulation and control technology for optimizing the medicinal quality of mulberry leaves for edible mulberry industry, and provide theoretical basis and practical guidance.

Material and methods

Plant material and exogenous hormone treatment

The seedlings of one-year-old “Gui sang you 62” (GSY62) mulberry (Morus alba L.) seedings varieties without physiological damage and microbial infection were kindly supplied by Zhejiang Haining Changxin Mulberry planting base, Zhejiang, China. A total of about 3000 mulberry seedlings were planted, and 1500 mulberry seedlings were planted outdoors. Other mulberry seedlings were planted in planting boxes in sunlight greenhouses with an average (minimum–maximum) relative humidity of 62–83%. The distance between seedlings was 20 cm. The ratio of vermiculite to lawn soil in the outdoor field and sunlight greenhouse planting box was 1:5. Before planting mulberry seedlings outdoors and indoors, mix the fertilizer evenly into the soil. The contents of organic matter, urea (total nitrogen ≥ 46%), total phosphorus, total potassium, and available potassium added were 80.00 g/kg, 100 g/kg, 0.5 g/kg, 11.00 g/kg, and 280 g/kg, respectively. The light in the sunlight greenhouse was consistent with the outdoor light, and the air circulation was normal. The average temperature in sunlight greenhouse was 26–35 °C. Some mulberry seedlings planted outdoors were used to screen the concentration of 4-CPANa. Mulberry seedlings planted in sunlight greenhouse were used to analyze the number of new buds and new shoots, physiological indexes, flavonoids, key enzyme gene expression in flavonoid biosynthesis pathway and dietary risk assessment.

The growth cycle of mulberry can be divided into germination period, vigorous growth period, slow growth period and dormancy period. Different concentrations of 4-CPANa solution (0, 5, 10, 15, 20, 25, 30, 300, 900 mg/L) were sprayed at the germination stage (When 1 to 2 new bud leaves grew on the main stem of mulberry seedlings) of one-year-old mulberry seedlings. 4-CPANa (0 mg/L) was distilled water. The amount of spraying for each plant was 5 ± 0.1 mL. Each concentration of 4-CPANa treated 150 mulberry seedlings, and each concentration was sprayed once. According to a completely random design, each treatment was repeated 3 times (Zhang et al. 2021). After treatment, irrigate the seedings once in 2 days with 500 ~ 600 mL of water. After spraying for 10 days, the number of new shoots and buds was determined. Through the comparative analysis of the number of new shoots and new buds of mulberry seedlings treated with different concentrations of 4-CPANa, the best concentration of 4-CPANa was selected.

Preparation of mulberry leaf samples

When mulberry seedlings sprouted, 4-CPANa (5 mg/L) was sprayed according to the above application method. The control seedlings were sprayed with the same amount of distilled water. Mulberry leaves were collected at 0 d, 5 d, 10 d, 15 d, 20 d, 25 d and 30 d after spraying, and the number of buds and shoots, leaf area, fresh weight, physiological index and flavonoid content of mulberry leaves were determined. Each treatment was repeated 3 times according to completely random design.

Determination of shoot number, bud number, fresh leaf weight and leaf area

Ten mulberry seedlings treated with control and 5 mg/L 4-CPANa were selected, and the number of shoots and buds of mulberry seedlings were determined by direct counting method; The leaf fresh weight (g, accurate to 0.001 g) and leaf area (cm2) of the third leaf from the top of mulberry seedlings were measured by direct weighing method and grid counting method (Radzali et al. 2016).

Determination of the chlorophyll content

The chlorophyll content of fresh mulberry leaves was determined by Lichtenthaler method (Lichtenthaler 1987). Fresh mulberry leaves (0.2 g) were ground with a grinder, added to 5 mL of 80% acetone, and then centrifuge at 5000 r/min at 4℃ for 5 min after mixing. The absorbance of the supernatant at 663, 645 and 475 nm was measured with a spectrophotometer. The content of Chl (a + b) was calculated by mg/g fresh weight (FW). The calculation method is as follows:

Chlorophyll a = 12.21 A663 − 2.81 A645

Chlorophyll b = 20.13 A645  − 5.03 A663

Chlorophyll content = (Chlorophyll concentration×Extract volume)/ Fresh weight of sample.

Measurement of the soluble sugar content

The soluble sugar content of fresh mulberry leaves was determined according to the instructions of the kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., Nanjing, China). The absorbance was measured at 620 nm. The soluble sugar content was calculated by mg/g FW.

Determination of the malondialdehyde (MDA) content.

Fresh mulberry leaves were taken for the determination of MDA content. The MDA content kit was provided by Nanjing Jiancheng Institute of Biological Engineering (Nanjing, China). The absorbance of the supernatant was measured at 530 nm according to the manufacturer's instructions. The content of MDA in mulberry leaves was expressed by nmol/g FW.

Measurement of SOD and POD activities

The activities of SOD and POD in fresh mulberry leaves were determined by the kit (Nanjing Jiancheng Biological Engineering Research Institute Co., Ltd., Nanjing, China). The absorbance of the supernatant was measured at 550 nm and 420 nm according to the manufacturer's instructions.

Analysis of flavonoids

The dried mulberry leaves were crushed and sifted through 40 mesh to make mulberry leaf powder. Weighed 0.5 g of mulberry leaf powder into a conical flask and added 10 mL 70% ethanol. Soaked at room temperature for 30 min and then extracted by ultrasonic for 30 min. After the extraction, the supernatant was absorbed and placed in a centrifuge tube, centrifuged at 12,000 rpm for 5 min, and then filtered by 0.22 μm microporous membrane. The supernatant was obtained as the sample solution. 10 µL was injected into a high-performance liquid chromatography (LC-20AD, Shimazu, Japan) on CNWSIL C18 column (4.6 × 250 mm, 5 μm; CNW, Shanghai, China) and UV detector. The test solution (10 µL) was injected into the HPLC system (LC-20AD, Shimazu, Japan) on a CNWSIL C18 column (4.6 × 250 mm, 5 μm; CNW, Shanghai, China) and UV detector.

The mobile phase was acetonitrile (A) − 0.5% phosphoric acid aqueous solution (B). The gradient elution was as follows: 0–5 min was 10% A, 5–10 min was 10–13.5% A, 10–15 min was 13.5–19% A, 15–18 min was 19–22% A, 18–19 min was 22% A, 19–35 min was 22% A, 35–40 min was 30–40% A, 50–55 min was 40–10% A, 55–75 min was 10% A (the next injection below the balanced column). The flow rate was 0.8 mL/min, the injection volume was 10 μL, the detection wavelength was 358 nm, and the column temperature was 25 °C. Calibration curves of pure standards were used to quantify Rut, ChA, IQ and Ast.

Appropriate amounts of Rut, ChA, IQ and Ast (Table S1) were taken and diluted with methanol step by step into a series of concentrations. The peaks of flavonoid standards (Rut, ChA, IQ, and Ast) were distinguished by retention time. The peak area is substituted into the standard curve.

Expression analysis of key genes involved in flavonoid biosynthesis by real-time PCR

Real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to analyze the expression of key enzyme genes in the flavonoid biosynthesis pathway of mulberry buds at 2 h, 3 h, 6 h, 12 h and 24 h after 5 mg/L 4-CPANa treatment. The fresh mulberry leaves were selected for RNA extraction, and then the first strand cDNA was synthesized by reverse transcription. Total RNA extraction kit was provided by Tiangen Biochemical Technology (Beijing) Co., Ltd. RT-qPCR was performed using RhiScript III RT Supermix (Vazyme, Nanjing, China). Relative expression levels of target genes were analyzed using the 2−ΔΔCt method (Schmittgen 2008). All primer pairs used for RT-qPCR analyses of mulberry leaves were listed in Table S2.

Determination of 4-CPANa dietary risk assessment

The residues of 4-CPANa were determined at 5, 7, 10 and 14 d after 5 mg/L 4-CPANa treatment. The sample pretreatment method was referred to the method of Huang et al. (2014), and the HPLC method was optimized on the basis of Huang et al. (2014). High performance liquid chromatography (HPLC) Instrument conditions: Shimadzu High performance liquid chromatography (LC-20AD) with CNWSIL C18 column (4.6 × 150 mm, 5 μm) and UV detector (SPD-20A); The mobile phase consisted of 5 mmol/L ammonium acetate and 0.05% formic acid aqueous solution mixed (A) to acetonitrile (B); The flow rate was 0.5 mL/min. The detection wavelength of 4-CPANa was 228 nm. Column temperature 25 °C; The injection volume was 5 μL. The isocratic elution program: 0–30 min (75%A: 25%b). The 4-CPANa standard was prepared into 100 mg/L solution with methanol, diluted to 3.125, 6.25, 12.5, 25, 50, 100 mg/L with acetonitrile, and quantified by external standard method. Risk assessment of 4-CPANa in mulberry leaves was performed according to the method of Dong et al. (2017).

Statistical analysis

The data was analyzed with IBM-SPSS 25.0 statistical software (IBM Corporation, USA). The differences were examined by two-factor analysis of Variance (ANOVA), followed by the least significant difference (LSD) test (P ≤ 0.05). These values were reported using a three-fold repetition method, and all results had a standard deviation (SD). Origin software package was used for statistical analysis and mapping.

Results

Optimum concentration of 4-CPANa

At 10 d, the effect of new shoots and new buds treated with different concentrations of 4-CPANa was shown in Fig. 1. The number of new shoots and new buds treated with 5 mg/L 4-CPANa was significantly higher than that of other concentrations of 4-CPANa, which was increased by 1.04-fold and 1.35-fold compared to the control (0 mg/L 4-CPANa).

Fig. 1.

Fig. 1

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Effect of the different concentrations of 4-CPANa treatment on the number of new shoots (A) and new buds (B) of mulberry seedlings. Each value represents the mean ± SD of three replicates. Values not sharing a common letter are significantly different at P ≤ 0.05

Dynamic growth regulation of mulberry seedlings by 4-CPANa

In the early field experiments, the effect of different concentrations of 4-CPANa on mulberry trees was compared, and the effect of different concentrations was comprehensively analyzed through a combination of bud and shoot count. The results showed that 5 mg/L 4-CPANa had the most significant effect on promoting the growth of mulberry. For the accuracy of the experiment, the efficacy of 5 mg/L 4-CPANa was verified. In order to evaluate the effect of 4-CPANa on mulberry seedlings, the number of shoots, buds, leaf fresh weight and leaf area were measured (Fig. 2A, B, C and D) and the growth status of mulberry seedlings was recorded at 0, 5, 10, 15, 20 and 25 d after 5 mg/L 4-CPANa treatment (Fig. 2E, F). Significant differences appeared among the plants growing under different treatments. As can be seen from the appearance of mulberry seedlings, the mulberry seedlings treated with 5 mg/L 4-CPANa grow vigorously, with a large number of leaves and large leaf area, while the mulberry seedlings treated with control grow slowly, with fewer leaves and smaller leaf area (Fig. 2E, F). As shown in Fig. 2A and 2B, the number of lateral shoots and buds of mulberry were significantly higher than those of the control within 25 d after foliar spraying of 5 mg/L 4-CPANa (P ≤ 0.05). On the 25 d, the number of new shoots and buds of mulberry was 1.69 and 2.47-fold of that of the control, respectively. After the control treatment and 4-CPANa treatment, the leaf fresh weight and leaf area of mulberry were gradually increased, and from the 5th day after spraying 5 mg/L 4-CPANa, the leaf fresh weight and leaf area of mulberry were significantly higher than that of the control. At 5 d, the leaf fresh weight and leaf area of mulberry leaves were 3.07-fold and 3.26-fold compared to the control (Fig. 2C, D).

Fig. 2.

Fig. 2

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Effect of 5 mg/L 4-CPANa treatment on shoot number (A), bud number (B), leaf fresh weight (C) and leaf area (D) of mulberry seedlings. Seedling growth of control (E) and 5 mg/L 4-CPANa treatment (F) at 0, 5, 10, 15, 20 and 25 d after treatment. Each value is the mean ± SD of three replicates. Values not sharing a common letter are significantly different at P ≤ 0.05

Effect of 4-CPANa on the chlorophyll contents and soluble sugar content

Chlorophyll a and chlorophyll b are the main pigments that absorb and transfer light energy and can improve the efficiency of light capture (Melkozernov and Blankenship 2006). Similarly, the development of new shoots is largely dependent on photosynthesis provided by the leaves below the lateral branches (Ashley 1972). The contents of chlorophyll (a + b) and soluble sugar after 4-CPANa treatment were shown in Fig. 3A and 3B. The content of chlorophyll (a + b) in mulberry leaves in the treatment group was firstly increased and was significantly higher than that of the control within 15 d, and then showed a decreasing trend significantly lower than that of the control after 20 d (Fig. 3A). We observed that the chlorophyll (a + b) content of mulberry trees treated with 5 mg/L 4-CPANa was higher than that of the control in the growth period (0–20 d) (Fig. 3A). Meanwhile, the growth rate of the number of buds and new shoots was faster than that of the control, and there were significant differences in the number of buds and new shoots at 0 d and 20 d (Fig. 2A, B). On the contrary, the chlorophyll content of mulberry trees treated with 5 mg/L 4-CPANa and the control gradually decreased to the initial chlorophyll content at mature stage after 20 d (Fig. 3A), and the growth rate slowed down, and there was no significant difference in the number of new shoots at 20 d and 25 d (Fig. 2A, B). Therefore, we consider that exogenous application of 4-CPANa promoted growth of mulberry, though the optimum effect depends on the concentration and duration of application.

Fig. 3.

Fig. 3

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Effect of 4-CPANa treatment on the contents of chlorophyll (a + b) (A) and soluble sugar (B) in mulberry leaves. Mulberry leaves were treated with 5 mg/L 4-CPANa. Data was measured at 0, 5, 10, 15, 20 and 25 d after treatment. Each value is the mean ± SD of three replicates. Values not sharing a common letter are significantly different at P ≤ 0.05

As shown in Fig. 3B, soluble sugar content in mulberry leaves treated with 5 mg/L 4-CPANa increased with growth. In addition, 5 mg/L 4-CPANa treatment significantly increased the accumulation of soluble sugar within 25 d to 36.55 mg/g FW, which was 1.12-fold that of the control. However, in the vigorous growth stage (0–15 d), the soluble sugar content of 4-CPANa treatment was significantly lower than the control.

Effect of 4-CPANa treatment on the MDA content and SOD and POD activities

MDA is one of the most important products of membrane lipid peroxidation, and its production can also aggravate membrane damage (Wang 2014). The effect of 4-CPANa on the content of MDA in mulberry leaves is shown in Fig. 4A. The content of MDA in mulberry leaves without 4-CPANa was higher, but the content of MDA in mulberry leaves treated with 4-CPANa was significantly decreased, and the content of MDA in mulberry leaves on 20 d was 0.22-fold lower than that after 0 d. The contents of SOD and POD protective enzymes in mulberry leaves were also determined. As shown in Fig. 4B and 4C, the activities of SOD and POD increased gradually after 4-CPANa treatment. In addition, compared with 0 d, SOD activity in 4-CPANa and control increased by 702.27% and 452.80% on 25 d, respectively, and POD activity increased by 32.24% and 17.64%, respectively.

Fig. 4.

Fig. 4

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Effect of 4-CPANa treatment on the contents of MDA (A), SOD (B) and POD (C) in mulberry leaves. Mulberry leaves were treated with 5 mg/L 4-CPANa. Data was measured at 0, 5, 10, 15, 20 and 25 d after treatment. Each value is the mean ± SD of three replicates. Values not sharing a common letter are significantly different at P ≤ 0.05

The effects of 4-CPANa on flavonoids and the expression levels of flavonoid-related genes

Flavonoids, such as chlorogenic acid (ChA), rutin (Rut), isoquercitrin (IQ) and astragalin (Ast), are important phytochemicals in mulberry leaves, with strong free radical scavenging activity, and have been reported to have many beneficial effects on human health, such as lowering blood sugar (Hamidullah et al. 2015; Havsteen et al. 2002; Testai 2015), protecting nervous system (Shakeri et al. 2019), anti-aging (Jo et al. 2009), antitussive and expectorant effects (Mandal et al. 2010). Chlorogenic acid in mulberry leaves is a good source of dietary supplements to prevent diabetic complications. IQ can effectively alleviate paracetamol (APAP)-induced liver injury by inhibiting oxidative stress and nitrosation stress induced by APAP (Xie et al. 2015). Rut can improve blood glucose levels in patients with urinary tract disease by inhibiting intestinal carbohydrate absorption, reducing gluconeogenesis, increasing glucose uptake, and stimulating pancreatic insulin secretion (Ahmed et al. 2010; Jadhav and Puchchakayala 2012; Kappel et al. 2013). Flavonoids in the extract of mulberry leaves can effectively protect erythrocytes from oxidative damage induced by free radicals. Among them, Ast has the greatest protective effect. To examine the effects of 4-CPANa on flavonoids in mulberry leaves, four types of flavonoids: ChA (C16H18O9), Rut (C27H30O16), IQ (C21H20O12) and Ast (C21H20O11) were analyzed. The most abundant flavonoid was ChA, followed by Rut, IQ, Ast. There were significant differences in the content of the four flavonoids within 30 d after different treatments, the content of four flavonoid was significantly different. At 5 d, the ChA content of 4-CPANa treatment reached the highest, which was 1.15-fold that of the control (Fig. 5A). Moreover, under the condition of 5 mg/L 4-CPANa, the content of Rut reached the highest value at 25 d, which was 1.1-fold that of the control (Fig. 5B). Moreover, the contents of IQ and Ast showed a trend of gradual increase. Compared with the control, the contents of IQ and Ast in mulberry leaves at 5 mg/L 4-CPANa were significantly increased, which were 1.31 and 1.24-fold of the control at 30 d, respectively (Fig. 5C, D). In addition, compared with 0 d, the contents of ChA, Rut, IQ and Ast in mulberry leaves treated by control and 4-CPANa increased by 0.04, 2.46, 7.33, 3.44 and 0.18, 3.03, 8.51, 4.08 times, respectively.

Fig. 5.

Fig. 5

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Effect of 4-CPANa treatment on the chlorogenic acid (A), rutin (B), isoquercitrin (C) and astragalin (D) in mulberry leaves. Data was measured at 0, 5, 10, 15, 20, 25 and 30 d after treatment. Each value is the mean ± SD of three replicates. Values not sharing a common letter are significantly different at P ≤ 0.05

To clarify the regulatory mechanism of 4-CPANa on the accumulation of flavonoids, we conducted a preliminary investigation from the perspective of molecular biology. Thus, the effect of 5 mg/L 4-CPANa treatment on the expression levels of flavonoid-related genes over 24 h of mulberry leaf growth was determined. The results showed that after 5 mg/L 4-CPANa treatment, the four key enzyme genes showed a trend of increasing at first and then decreasing. The first gene to develop molecular response after 4-CPANa treatment was MaPAL, an important rate-limiting enzyme gene in the synthetic pathway of flavonoids, and the expression level of MaPAL gene at 2 h was 3.48-fold higher than that at 0 h (Fig. 6A). Secondly, the expression of the key enzyme gene MaF3GT was significantly induced at 3 h, which was 9.82-fold higher than that at 0 h (Fig. 6B). However, the response of Ma4CL gene was the strongest at 6 h, which was upregulated 3.95-fold higher than at 0 h (Fig. 6C). In addition, MaCHS genes, an important intermediate enzyme in the flavonoid synthesis pathway, were significantly up-regulated by 3.64-fold at 12 h (Fig. 6D). In terms of gene expression level, exogenous 4-CPANa significantly up-regulated the expression of genes related to the flavonoid/phenylpropanin pathway in mulberry leaves (P ≤ 0.05).

Fig. 6.

Fig. 6

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The expression of genes related to flavonoid biosynthesis in mulberry leaves treated with 5 mg/L 4-CPANa. The gene expression intensity in A–D was compared with that on the 0th day

Risk assessment of 4-CPANa in mulberry leaves

The residue of 4-CPANa in mulberry leaves was 0.2663 ~ 0.2873 mg/kg after 5 d of application, and the STMR of 4-CPANa was 0.2725 mg/kg. Additionally, the residue of 4-CPANa in mulberry leaves could not be detected after 7 d, 10 d and 14 d, which met the requirements of food safety of mulberry leaves during harvest (Table S3). The acceptable daily intake (ADI) of 4-CPANa is 0.08 mg/kg bw (European Food Safety Authority 2020). According to the average daily vegetable intake and average body weight of different age groups in China (Dong et al. 2017), the EDI and RQ of 4-CPANa were calculated. High RQ means high pesticide residues (Wang et al. 2015), and when RQ is greater than 1, it has a great impact on consumer health (Saini et al. 2015). The dietary risk assessment data of mulberry leaf vegetables in this study showed that the highest RQ value of 4-CPANa was 0.0336, which was much lower than 1 (Table S4). In addition, the highest RQ of 4-CPANa after the dietary risk assessment of mulberry leaf tea was 0.0007, which was much lower than 1 (Table S5). This indicated that the use of 5 mg/L 4-CPANa on mulberry trees did not cause a higher dietary risk, and the recommended safety interval is 7 d. It is safe to consume this mulberry leaf 7 d after application.

Discussion

In recent years, with the continuous strengthening of the development of mulberry leaf food in China, the demand for mulberry leaf raw materials with good quality and high yield has also been increasing day by day. Therefore, the adoption of safe, efficient and convenient way to improve the quality and yield of mulberry leaves has become an urgent problem for the edible mulberry industry. In order to solve the problem of low yield and poor quality of crops, the chemical regulation methods used in the actual agricultural production mainly include plant hormones, plant growth regulators and so on. These methods can effectively increase crop yield and promote the synthesis of different medicinally active components in medicinal plants, such as flavonoids, polyphenols and 1-deoxynojirimycin. Therefore, exogenous 4-CPANa regulation technology was used to promote the growth of mulberry leaves and increase the content of flavonoids in mulberry leaves. In this study, the effects of 8 concentrations (5, 10, 15, 20, 25, 30, 300, 900 mg/L) of 4-CPANa on three aspects of mulberry growth status, number of buds and number of shoots were compared comprehensively. It was found that among the 8 concentrations of 4-CPANa, the number of new shoots and buds of mulberry seedlings treated with 5 mg/L 4-CPANa was the highest (Fig. 1A, B). 5 mg/L 4-CPANa significantly increased shoot number, bud number, leaf fresh weight and leaf area of mulberry seedlings within 25 d (Fig. 2A, B, C, D). The results showed that 5 mg/L 4-CPANa played a significant role in promoting the growth of mulberry, and was used to conduct the next related studies. To explore the effects of 4-CPANa on physiological indexes and flavonoid accumulation in mulberry leaves, chlorophyll (a + b) and soluble sugar content, malondialdehyde (MDA) contents, antioxidant enzymes (POD and SOD) activities, flavonoid content, and related gene expression levels of mulberry leaves were determined. In addition, the dietary risk assessment of 4-CPANa residues in mulberry leaves was also performed.

Plant growth regulators are a class of compounds that can promote or change plant physiological or morphological processes at very low concentrations. 4-CPANa is a synthetic auxin belonging to the auxin class of plant growth regulators. Application of synthetic auxin can induce fruit setting and increased plant yield (Jasmin et al. 2018). Related studies have demonstrated that appropriate concentrations of 4-CPA have positive effects on plant growth. For example, 4-CPA at the recommended concentration could increase tomato fruit size and fruit setting rate, thus increasing tomato yield (Tonder and Combrink 2013). However, 4-CPA could negatively affect plant growth when the appropriate concentration range is exceeded. Previous study has shown that high concentrations of 4-CPA would significantly reduce the yield and plant growth of hydroponic sweet pepper and increased the number of malformed fruits (Martin et al. 2015). Meliha et al. (2006) also found that 4-CPA beyond the recommended concentration could increase the fruit quantity and the incidence of parisexual fruit, but would produce malformed and inferior fruits. The reason for promoting plant growth and development is that the appropriate concentration of 4-CPA stimulates the increase of some endogenous hormones in plants, and then promotes plant growth and development (Lu et al. 2016). However, when the concentration of 4-CPA is too high, it will lead to a sharp decline in plant endogenous hormones after reaching the peak, and when the concentration exceeds the optimum, it will lead to the production of ethylene, the decrease of growth promotion, and even the inhibition, and eventually lead to fruit dysplasia (Meliha 2006). For example, Nitsch's (1955) study reported that the content of IAA was initially proportional to the increase of fruit diameter, but decreased sharply after reaching a certain level, which led to fruit deformity. In this study, it was observed that with the increase of 4-CPANa concentration, when the concentration of 4-CPANa reached 300 or 900 mg/L, it would have herbicidal or ephinastic effect on mulberry seedlings, which lead to wilting and shedding of mulberry buds, a sharp decrease in the number of leaves, leading to highly inhibited growth and development of mulberry seedlings. This may be due to the herbicidal effect of high concentration of 4-CPANa to accelerate plant maturation and shorten plant life cycle.

According to the effect of 5 mg/L 4-CPANa on the chlorophyll content of mulberry leaves. We found that exogenous application of 4-CPANa during mulberry leaf growth period was beneficial to mulberry leaf growth. It has been reported that plant growth regulators (PGRs) can stimulate photosynthesis of leaves and improve carbon supply capacity (Gao et al. 2017; Gene 1993; Xia et al. 2009). As a primary photosynthetic substance, sucrose in soluble sugar can provide carbon and energy for the growth of new buds from source tissues and channel reservoir tissues. In the growth stage of new buds, improving its utilization rate is the basis of high yield. The export rate of sucrose in soluble sugars is related to the rate of photosynthesis (Kaur 2007). Application of 4-CPANa increased chlorophyll content in mulberry leaves, promoted photosynthesis in mulberry leaves, and increased the export rate of sucrose in soluble sugar in mulberry leaves. While chlorophyll increased, soluble sugar remained at a low level. In our study, chlorophyll content of mulberry leaves treated with 4-CPANa was significantly higher than that of the control in the vigorous growth period (0–15 d), thus affecting carbohydrate metabolism and transport, which can be seen from the significantly lower soluble sugar content of mulberry leaves in the same growth period than that of the control (Fig. 3A, B). Moreover, N, N-dimethyl piperidinium chloride (DPC) application increased leaf chlorophyll content (Reddy et al. 1996), but with the increase of leaf carbohydrate content, and net photosynthesis decreased. Application of DPC to increase leaf carbohydrate accumulation may decrease photosynthetic rate (Tung et al. 2018).

Under biological or abiotic stress conditions, a large amount of reactive oxygen species (ROS) will be produced in plants (Chutipaijit 2016), resulting in the peroxidation of cell membrane, and eventually destroy the structure and function of cell membrane (Gill and Tuteja 2010; Henricks and Nijkamp 2001; Siedlinski et al. 2009). MDA is one of the important products of membrane lipid peroxidation, and the content of MDA is a common index to reflect the degree of membrane lipid peroxidation in plant physiology. In order to counteract these damages, plants have evolved a series of protective systems to scavenge these reactive oxygen species and reduce the damage to cell membranes. Both SOD and POD are the main enzymes involved in the protection system. SOD first catalyzes the decomposition of intracellular ROS O2− into H2O2 and then H2O2 is decomposed by intracellular CAT and POD. Therefore, the damage of ROS to the membrane system is minimized, thus the damage and death of plant cells are reduced (Mittler 2002). In this study, 4-CPANa treatment of 5 mg/L did not cause abiotic stress of mulberry seedlings. It increased the activities of SOD and POD, decreased the content of MDA, effectively enhanced the ability of the plant to scavenge ROS, maintained the metabolic balance of ROS, reduced oxidative stress response, and thus promoted the growth of mulberry. However, SOD activity was lower than control in 0–10 d after 5 mg/L 4-CPANa treatment. This may be the reason that MDA content in 0–10 days is higher than that in the control seedlings. Compared with the control, SOD catalyzed ROS O2− with lower efficiency and could not provide H2O2 to POD in time. Therefore, MDA content was still higher than that of the control within 0–10 d even if POD activity was higher than that of the control.

Auxins, including 4-CPA, not only regulate plant growth and development, but also play an important role in regulating the synthesis of plant flavonoids (Sheshglani and Asghari 2020). In this study, it was observed that exogenous 4-CPANa treatment could significantly increase the content of Rut, IQ, Ast and ChA in mulberry leaves. Exogenous application of auxin causes changes in the content of other endogenous hormones in plants, which synergistically regulate the accumulation of flavonoids. It can also affect the synthesis of flavonoids by affecting physiological and biochemical metabolism, expression of structural genes and activity of MBW complex. Similar studies have shown that NAA (1-naphthaleneacetic acid) enhances the production of ethylene and abscisic acid (ABA) by increasing the content of ethyl acetate (ACC) and ABA-glucose ester (ABA-GE), two precursors required for ethylene synthesis in sweet cherries. In addition, NAA treatment improved the transcription levels of anthocyanin accumulation-related synthetic genes, transport genes and regulatory genes. Finally, sweet cherry ripening was induced and anthocyanin content was increased (Cuch et al. 2021). According to the report, lemons were treated with methyl jasmonate (MeJA) before harvest to boost antioxidant compounds such as phenolics in lemon juice (Escolano et al. 2019), MeJA also increased the concentration of total phenolics in the plum (Zapata et al. 2014) and total phenols and anthocyanins in sweet cherry fruits (Gimenez et al. 2017), this is attributed to MeJA increasing the activity of phenylalanine ammonia-lyase (PAL), a key enzyme in the phenolic biosynthesis pathway (García and Plaza 2013). In addition, melatonin regulates key enzymes in phenolic metabolism and maintains high phenolic content in postharvest, such as inducing the expression of PAL and CHS, and increasing the expression levels of genes related to PAL and CHS activity (Ma et al. 2016; Zhang et al. 2018; Zheng et al. 2019). According to the above results, it is necessary to explore the expression of related genes in the pathway of flavonoid synthesis in mulberry leaves by 4-CPANa in order to understand the mechanism of 4-CPANa regulating the accumulation of flavonoids in mulberry leaves.

PAL and 4CL encode key genes that convert phenylalanine into phenylpropanin biosynthesis pathway and flavonoid biosynthesis pathway, linking primary and secondary metabolism (Wang et al. 2020). CHS is the key enzyme gene that provides the basic skeleton for flavonoid compounds, while F3GT is the last key enzyme gene for the formation of stable flavonols. Exogenous 4-CPANa not only increased the accumulation of four kinds of flavonoids in mulberry leaves, but also significantly induced the expression of four key enzyme genes, including PAL, 4CL, CHS and F3GT (P ≤ 0.05). The expression of flavonoid biosynthesis genes induced by exogenous 4-CPANa may be due to the increase of GA content in mulberry leaves induced by endogenous gibberellin GA biosynthesis genes induced by 4-CPANa, while GA can significantly induce the expression of key enzymes in flavonoid biosynthesis pathway such as PAL. Some studies have shown that, Lu et al. (2016) found that during the induction of unisexual grape fruit development, 4-CPA significantly increased the content of endogenous bioactive GA, significantly induced the up-regulation of GA biosynthesis genes and inhibited the expression of GA catabolism genes (Lu et al. 2016). Similiar, Ding et al. (2013) also found that cytokinin increased GA content by up-regulating the expression level of tomato GA biosynthesis gene (Ding et al. 2013). Moreover, Guo et al. (2018) have confirmed that GA can significantly up-regulate the expression of PAL and isoflavonesynthase (IFS) (Guo et al. 2018), and plays an important role in the synthesis of flavonoids (Sivanandhan et al. 2011; Thiruvengadam and Chung 2015). Based on the above results, it is suggested that exogenous 4-CPANa may improve the accumulation of flavonoids in mulberry leaves by inducing the expression of key enzyme genes in the flavonoid biosynthetic pathway.

Conclusion

The effect of 4-CPANa on growth, flavonoid compound content and gene expression levels of key enzymes in flavonoid synthesis pathway of mulberry seedlings was studied, and finally the dietary risk assessment of mulberry leaves was carried out. Overall, the following conclusions can be drawn by analyzing the physiological indexes, molecular biology and metabolites of mulberry leaves: (1) the optimum concentration of 4-CPANa for differentiation and growth of mulberry was 5 mg/L; (2) 4-CPANa (5 mg/L) significantly promoted the differentiation and growth of mulberry, and did not cause abiotic stress of mulberry seedlings; (3) 4-CPANa (5 mg/L) significantly induced the expression of four key enzyme genes F3GT, 4CL, PAL and CHS in the flavonoid biosynthesis pathway of mulberry leaves, and significantly increased the accumulation of four flavonoids in mulberry leaves: Rut, ChA, IQ and Ast; (4) the EDI and RQ calculated according to the final residue of 4-CPANa in mulberry leaves are far less than 1, which is a low risk factor. Moreover, the effect of 4-CPANa on other enzyme genes in the flavonoid synthesis pathway of mulberry leaves remains unclear. Therefore, the regulatory mechanism of 4-CPANa on flavonoid accumulation in mulberry leaves needs to be further studied. In this study, the regulatory mechanism of 4-CPANa on the growth of mulberry leaves and the formation of flavonoids was revealed by combining the physiological indexes, molecular biology and metabolites of mulberry leaves, which will provide theoretical basis and practical guidance for the future cultivation of mulberry leaves, improvement of the quality of mulberry leaves and the development of medicinal products of mulberry leaves. It also provides fresh insights for studying the effects of plant growth regulators on field crops.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file 1 (DOCX 34 KB) (34.1KB, docx)

Acknowledgements

We thank each of the authors of this article for their technical assistance.

Author contributions

ZZ and YD helped in data collection and writing frst draft; ZZ did the statistical analysis; ZZ and SZ conceived idea; CZ and MW reviewed the paper and provide resources; JL and SR helped conduct the experimental portion; and YZ and XL did supervision. All authors read and approved the final manuscript.

Funding

This work was financially supported by National Key Research and development Project of "2020 Science and Technology to Boost Economy", National Agricultural Product Quality and Safety Risk Assessment Project (GJFP2020012) and 2019 Hainan Basic and Applied Basic Research Plan (Natural Science) High-level Talents Project (2019RC058).

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Zhen Zhang, Yun Duan have contributed equally to this work.

Contributor Information

Chenghui Zhang, Email: zchlm@hainanu.edu.cn.

Meng Wang, Email: wangmeng@hainanu.edu.cn.

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