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Article

Changes in the Platycodin Content and Physiological Characteristics during the Fruiting Stage of Platycodon grandiflorum under Drought Stress

by
Min Li
1,2,
Meng Zhang
1,
Lin Cheng
1,
Limin Yang
1,* and
Mei Han
1,*
1
College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun 130119, China
2
College of Traditional Chinese Medicine, JiLin Agricultural Science and Technology University, Jilin 132101, China
*
Authors to whom correspondence should be addressed.
Sustainability 2022, 14(10), 6285; https://0-doi-org.brum.beds.ac.uk/10.3390/su14106285
Submission received: 17 April 2022 / Revised: 18 May 2022 / Accepted: 18 May 2022 / Published: 21 May 2022
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Abstract

:
Medicinal plants are affected by drought stress, mainly reflected in the growth process and secondary metabolite synthesis. Platycodon grandiflorum (Jacq.) A. DC. is a traditional Chinese herbal medicine. The yield of Platycodon grandiflorum cannot meet the market demand, while its yield and quality are limited by the plant growth conditions. We assessed relevant indicators of growth during the fruiting stage of Platycodon grandiflorum under drought stress. The results showed that the fresh root weight (FW), photosynthesis, and chlorophyll fluorescence parameters were significantly reduced after withholding water (AW), but total superoxide dismutase (T-SOD), peroxidase (POD), and catalase (CAT) activities and the contents of soluble protein (SP), proline (PRO), and malondialdehyde (MDA) were significantly increased. The contents of platycodin D (PD) and platycodin D3 (PD3) did not change obviously after withholding water (AW), but in the autumn period, the values increased by 8.95% and 11.67%, respectively. The content of total platycodin increased significantly under drought stress, during the after rewatering (AR) and in the autumn period. The different physiological stress indicators exhibited strong correlations, had synergistic effects of mutual promotion and restriction, and responded to changes in the soil water content. These results suggest that during the fruiting stage, Platycodon grandiflorum encounters drought stress and may resist oxidative damage by increasing protective enzyme activity and osmoregulatory materials to ensure normal plant growth. According to the effect of drought stress on dry weight, the yield of Platycodon grandiflorum was not affected by drought stress, but the total platycodin content in Platycodon grandiflorum roots increased significantly. Therefore, in agricultural production, short-term drought stress should be conducted in the fruiting stage of Platycodon grandiflorum, which can both guarantee the yield and improve the quality of medicinal materials.

1. Introduction

Platycodon grandiflorum (Jacq.) A. DC. (PG) is a member of the Campanulaceae family [1]. Its dried root is used as a traditional Chinese herbal medicine. In the root of PG, oleanane-type triterpenoid saponins of the pentacyclic-type family are the main chemical active components [2]. The platycodin D (PD) is the most abundant and the main bioactive component [3]. At present, the yield of PG cannot meet the market demand, and its yield and quality are limited by the plant growth conditions. PG is widely cultivated in the arid and semi-arid regions of China. To date, the physiological mechanism of PG adaptation to drought is not clear, which has an impact on the improved quality of medicinal materials. Controlling the soil water content at an appropriate time to find a perfect balance between medicinal plant production and quality is essential to improve the quality and yield of medicines.
Drought stress is the physiological response of a plant to a water deficit [4]. Drought can affect plant photosynthesis, water-use efficiency (WUE), maximum photochemical efficiency (Fv/Fm), and actual photochemical efficiency (φPSII), causing a series of physiological changes in medicinal plants, such as protoplasm dehydration, lipid membrane system damage, protective enzyme activity changes, and metabolic disorders. Therefore, drought is a multi-dimensional stress [5]. Drought stress can cause cell membrane peroxidative damage in plants, and malondialdehyde (MDA) is an important product of cell membrane peroxidative damage [6]. To cope with drought conditions, plants respond and adapt to stress by complex molecular responses, including changes in catalase (CAT) and peroxidase (POX) and some organic compounds including osmoprotectants [7]. Moreover, drought also causes plants to produce large amounts of reactive oxygen species (ROS), and high concentrations of ROS are highly toxic to plant cells [8]. Studies showed that Atractylodes chinensis (DC.) Koid had a higher protective enzyme activity under mild water stress but decreased superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities with increasing water stress [9]. In addition, some researchers found an increase in the soluble sugar (SS), soluble protein (SP), and proline (PRO) contents under water stress [10,11,12]. In order to avoid the deleterious effects of abiotic stress, these compounds play an important role in the osmoregulation of subcellular structures [13].
Secondary metabolites are the main active components of medicinal plants, and their synthesis and accumulation are closely related to the growth environment. It is generally believed that during the evolution of medicinal plants, the physiology and secondary metabolism of medicinal plants allowed them to adapt to various adverse conditions [14]. In particular, stress conditions exert strong effects on metabolic pathways. Affected by drought stress, medicinal plants usually produce higher levels of secondary metabolites, including triterpenoids [15]. In recent years, many studies have shown that the active components of medicinal plants can be regulated by changing soil moisture conditions [16]. The accumulation of total platycodin, tripterine, and baicalin increased under mild drought stress [14,17,18], but baicalin was inhibited under severe drought stress. Therefore, secondary metabolites of medicinal plants can help them to adapt to altered physical and chemical environments and mediate synergistic and competitive effects among plants [19]. Understanding the underlying regulatory mechanisms of the biosynthesis of secondary metabolites in medicinal plants under drought stress may contribute to the improved quality of medicinal plants.
The mechanisms of drought tolerance in different medicinal plants are currently being extensively studied. We speculate that the fruiting stage is a critical period that affects the yield and quality of medicinal plants. Feasible anthropogenic ecological regulation may be beneficial to improve the yield and quality of medicinal plants. This work was planned to regulate the soil water content in PG and analyze physiological drought indexes such as root growth, antioxidant enzymes, osmotic molecules, and secondary metabolites. We aimed to clarify the physiological and biochemical response mechanisms of PG to drought stress and provide theoretical guidance for the cultivation of PG with a high platycodin content.

2. Materials and Methods

2.1. Experimental Materials

The potted plant experiment was carried out at the Medicinal Plant Practical Base of Jilin Agricultural University (Changchun, China, 43°48′ N, 125°25′ E). The annual average temperature is 4.8 °C, with a maximum of 39.5 °C and a minimum of −39.8 °C. The average sunlight duration was 194.5 h and 214.1 h, respectively, in September and October in 2019. The average annual precipitation is 568.5 mm and is mainly concentrated from June to August, accounting for approximately 70% of the annual precipitation. The average humidity duration was 71% and 57%, respectively, in September and October in 2019.
The plant material used was biennial PG. The dried and mature seeds of PG were obtained from the Pharmaceutical Botanical Garden of Jilin Agricultural University in October 2017. The same-sized full seeds were sown in the basin on 25 November 2017. The pots were 25.00 cm deep, with an upper surface diameter of 32.0 cm and a basal base diameter of 28.0 cm. Each pot was loaded with 5 kg of organically fertilized soil. The bottom of the pot was covered by a water membrane, completely buried beneath the soil and kept at the same temperature as the soil. The organically fertilized soil used in the experiment was loose sandy loam with good permeability and drainage, with the following characteristics: organic matter 18.42 g·kg−1, available phosphorus 39.86 mg·kg−1, available potassium 98.65 mg·kg−1, and alkali nitrogen 108.15 mg·kg−1. When the seeds of PG germinated in May 2018, 10 seedlings were retained in each pot.

2.2. Experimental Design

The experiment began in August 2019. Forty basins (5 plants per basin) of well-growing and uniform PG plants were randomly divided into two groups, i.e., a drought group (DG) and a control group (CK), before the drought stress was imposed. Two weeks before the drought stress experiment, the PG pots were watered every 2 days to ensure consistent water conditions. This experiment was based on the data from the China Statistical Yearbook that reported the average rainfall from September (the rainfall in September is 43 mm) to October (the rainfall in October is 25 mm) during the past 20 years in Changchun. In this study, the water supply of CK was calculated according to the pot’s upper surface area, and water was supplied 15 times. We calculated the water applied in the CK as approximately 175 mL in September and 100 mL in October. A rain-proof canopy was constructed during the experiment; the film was rolled up on sunny days but laid down at night and on rainy days. Other ecological factors in the control and drought groups, such as the photoperiod and light intensity, were kept close to natural conditions by artificial maintenance. During the experiment, pests and weeds were regularly removed from pots, and other field management was conducted normally. The drought stress experiment of PG was divided into four stages:
(1)
Prior to withholding water (0 d): The drought group was saturated with water at one time; the soil water content was about 33%. The CK was under normal management; water was supplied by simulated artificial precipitation between 5:00 p.m. and 6:00 p.m. every 2 days to ensure the normal growth and development of PG. The water supply scheme is shown in Table 1.
(2)
After withholding water (AW): The drought group was no longer watered after the drought stress experiment began until two-thirds of the leaves of PG had withered.
(3)
After rewatering (AR): When two-thirds of the PG leaves had withered, water was supplied to the drought group again between 5:00 p.m. and 6:00 p.m. that day.
(4)
Autumn period (AP): The water resupply in the drought group was managed the same as that in CK until a unified harvest on 20 October 2019.

2.3. Experimental Treatment

In the experimental stages, samples were collected between 7:00 a.m. and 8:00 a.m. Three basins were randomly selected for sampling. The soil water content was measured by an HH2 soil humidity tester (Delta-t Devices Ltd., Burwell, UK) before each sampling. Samples were collected on days 0, 15, and 18 after the experiment and in the autumn period according to the changes in soil moisture and plant growth (Table 2). Day 0 was before the drought, day 15 was during severe drought (two-thirds of the leaves had withered), and day 18 was the 3rd day after water was resupplied in the drought group; in the autumn period, all plants had withered on 20 October 2019, and the harvest began. The sample material was transported to the laboratory, where it was cleaned and stored at −80 °C until analyses.

2.4. Determination of Fresh and Dry Root Weight

The sediment from PG was first removed with tap water and then washed several times with distilled water. Then, filter paper was used to remove the surface moisture of the PG roots. The fresh root weight (FW) was measured and recorded. Afterward, the PG roots were oven-dried at 60 °C for 72 h. The dry root weight (DW) was measured and recorded.

2.5. Determination of Photosynthesis Parameters

Five leaves of five plants in the drought group and control group were randomly selected before measurements. The photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), intercellular CO2 concentration (Ci), and atmospheric CO2 concentration (Ca) were recorded by a portable photosynthesis device (LCpro+, ADC BioScientifc Ltd., Hoddesdon, UK) from 10:00 a.m. to 12:00 a.m. [20]. The stomatal limit (Ls) was computed as 1 − Ci/Ca, and the instantaneous water-use efficiency (WUE) was calculated as Pn/Tr [21].

2.6. Determination of Chlorophyll Fluorescence Parameters

While determining the photosynthesis parameters, the same leaves were selected to determine the chlorophyll fluorescence parameters by a chlorophyll fluorescent device (OS-5P+, OptiSciences, Hudson, NH, USA). To allow the leaves to adapt to dark conditions, we clamped the leaves for 30 min and then measured them. The measurement indicators included the Fv/Fm value, φPSII, photochemical quenching coefficient (qP), and nonphotochemical quenching coefficient (NPQ).

2.7. Contents of Osmoregulatory Substances and Malondialdehyde

Fresh root tissues (0.5 g) were ground into a homogenate in an ice bath in 5 mL of phosphate-buffered saline (PBS) (0.05 mol/L, pH 7.8). The contents of SS, SP, PRO, and MDA in the root tissue homogenate of PG were determined by using detection kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The absorbance of the reaction solution was measured by an enzyme labeling device (SpectraMax 190, Molecular Devices, San Jose, CA, USA) at wavelengths of 595, 520, 620, and 530 nm. The contents of SS, SP, PRO, and MDA were calculated according to the absorbance value.

2.8. Antioxidant Enzyme Activities

In an ice bath, the homogenate of fresh PG root samples was prepared with PBS and then transferred to centrifuge tubes, after which a buffer was added to a final volume of 5 mL. Then, the samples were centrifuged at 4 °C at 10,000 r·min−1 for 10 min.
The activity of total superoxide dismutase (T-SOD) was determined by analyzing the ability of the enzyme to inhibit the photoreduction of nitroblue tetrazolium (NBT). The optical density readings were recorded at 550 nm (SpectraMax 190, USA Molecular Devices). One unit of SOD activity (U) was defined as the amount of enzyme required to inhibit NBT photoreduction by 50% under assay conditions, expressed in U·mgprot−1 (mg of protein).
The activity of CAT was determined through the ammonium molybdate method. The optical density readings were recorded at 405 nm (SpectraMax 190, USA Molecular Devices). One unit of CAT activity (U) was defined as the amount of enzyme needed to degrade 1 μmol of H2O2 per minute, expressed in U·mL−1.
The activity of POD was determined by analyzing the ability of the enzyme to catalyze hydrogen peroxide. The optical density readings were recorded at 420 nm (SpectraMax 190, USA Molecular Devices). One unit of POD activity (U) was defined as the amount of enzyme needed to catalyze 1 µg of substrate per milligram of tissue protein at 37 °C, expressed in U·mgprot−1 (mg of protein).

2.9. Measurement of Platycodin Content

The weighed PG root (40 mesh; 200 mg) was extracted three times with 80% (v/v) methanol at 80 °C for 3 h each time. The extract was filtered, combined, and evaporated using a solvent evaporator. Finally, the dried extracts were dissolved and diluted with 80% methanol to 10.0 mL [2]. The solution was filtered through a syringe filter (0.45 μm). The volume of injection was 10 μL for each run.
Analysis of the samples was performed on an Agilent 1260 liquid chromatograph system (Agilent, Santa Clara, CA, USA) equipped with a 1260 Infinity II High-Temperature Evaporative Light Scattering (Agilent, Santa Clara, CA, USA). An Agilent Promasil C18 column (4.6 mm × 250 mm, 5 μm) was used, and the column temperature was set at 30 °C. The binary gradient elution system consisted of acetonitrile (eluent A) and 0.2% formic acid (eluent B), and separation was achieved using the following gradient program: 0–15 min, 20% eluent A; 15–60 min, 22% eluent A; 60–65 min, 30% eluent A; 65–70 min, 20% eluent A. The column was ultimately reconditioned with 20% eluent A isocratic for 5 min. The flow rate was 1.0 mL·min−1, and the injection volume was 20 μL. The detection parameters of the evaporative light-scattering detector were as follows: the drift tube temperature was 75 °C, and the carrier gas (N2) flow rate was 2.5 L·min−1. Seven concentrations of primary methanol solutions containing the standard substances PD (LOT: Z08J9L52294), deapioplatycodin (DPD) (LOT: P28D6F8192), and platycodin D3 (PD3) (LOT: P23N8T48914) were prepared. Each concentration of solution was injected in triplicate. Standard curves were constructed by plotting the peak area versus the concentration. The regression equations of the platycodin standard are shown in Table 3.

2.10. Measurement of the Total Platycodin Content

The weight of the PG root (40 mesh) was about 5 g (M); this was supplemented with 40 mL of methanol, sonicated for 50 min, centrifuged at 5000 r·min−1 for 10 min, and filtered. Samples were extracted three times, and the filtrate was combined and concentrated to 15–20 mL. The concentrate was extracted with ether, and the resulting precipitate was dissolved with methanol, filtered, and concentrated to 15–20 mL. Ether extraction was performed again according to the method presented above [22]. Methanol liquid was combined, placed in a constant evaporation dish (M1), and evaporated to dryness. Residues were weighed after a constant weight (M2) was reached. The content of total platycodin (SPD) (SPD (mg·g−1) = (M2 − M1)/M) was calculated based on the measured data.

2.11. Statistical Analysis

Excel 2016 was used to arrange the raw data. SPSS 21.0 was used for Pearson correlations. Origin 9.0 was used for principal component analysis. R version 3.6.1 was used for drawing the correlation heatmap. GraphPad Prism 8 was used to produce plots. Data are expressed as the mean ± standard deviation (n = 5).

3. Results

3.1. Soil Water Content

In this study, the soil water content of PG in the drought group and control group was determined and adjusted at 6 p.m. every 3 days. The soil water content under drought stress of the 2-year potted PG is shown in Figure 1a. During the experiment, the average soil water content of the CK was 17.48%. Before drought stress, the soil water content in the drought group was 33.86%; under drought stress, the soil water content decreased to 9.22% and was restored to 17.98% after water was resupplied. The soil water content change in the CK was consistent with the external environmental temperature; thus, the CK had sufficient water to ensure growth and development. However, due to the difference in the water supply scheme, the soil water content in the drought group was significantly reduced, which produced a pronounced drought stress effect. Therefore, from the perspective of the soil water content, this experiment achieved drought stress, allowing analysis of the physiological and biochemical index changes of PG.

3.2. Root Growth

Root growth is an important indicator that affects the quality and yield of PG. The effect of drought stress on the fresh and dry root weight of PG at the fruiting stage is shown in Figure 1b,c. In the AW, the weight of fresh PG root in the drought group decreased by 22.11% and its dry weight changes were not obvious. In the fruiting stage of PG, drought stress affected the fresh weight more than the dry weight of the root. After the AW, the drought group was resupplied with water, and its root growth was somewhat restored. To understand the long-term effects of drought stress on fruiting-stage PG, we measured the root weight at the AP. In the AP, the weight of dry PG root was significantly increased by 16.35% in the drought group (p < 0.05). We speculated that because of diminished metabolic and physiological activity, part of the dry material of fruiting-stage PG was transported to fruit tissues for reproduction. When the fruiting-stage PG encountered drought stress, this may have attenuated reproductive growth and the accumulation of more dry material to maintain survival. Thus, drought stress may be more favorable for the accumulation of dry matter during the fruiting stage.

3.3. Physiological and Biochemical Indexes

Under drought stress, photosynthetic physiology and metabolic pathways of plants will be regulated accordingly; thus, cells can continue to absorb water from the environment and maintain cell morphology and physiological characteristics [23]. In this study, the photosynthetic physiology, chlorophyll fluorescence parameters, osmoregulatory material, and protective enzyme system under drought stress were analyzed to reveal the physiological and ecological response mechanism of fruiting-stage PG under drought stress.

3.3.1. Photosynthesis

The effect of drought stress on photosynthesis in fruiting-stage PG leaves is shown in Figure 2a–d. During the AW, drought stress significantly reduced the Pn, WUE, and Ci levels in PG leaves at the fruiting stage. After the drought group was resupplied with water, the three indicators recovered to levels similar to those measured in the CK. Drought stress significantly increased Ls, which indicated that the fruiting-stage PG may prevent damage to photosynthetic organs by reducing stomatal opening to control water loss. Therefore, drought stress transiently inhibited photosynthesis in fruiting-stage PG leaves and their physiological function.

3.3.2. Chlorophyll Fluorescent Parameters

The effect of drought stress on the chlorophyll fluorescence parameters in leaves of fruiting-stage PG is shown in Figure 2e–h. Drought stress had significant effects on Fv/Fm, φPSII, and qP, especially at 12 d and 15 d, when Fv/Fm was significantly reduced by 9.3% and 6.3%, respectively. φPSII was significantly reduced at 3 d and 9 d and showed a significant increase after the drought group was resupplied with water (21 d). NPQ was significantly increased at 3 d and 6 d, while at 9 d and 12 d, it seemed to be somewhat adapted to drought conditions, and there was no difference between the drought group and the CK. However, NPQ showed a significant increase at 15 d, 1.38 times that of the CK. Even if the drought group was resupplied with water, NPQ failed to improve. In conclusion, the fluorescence parameters Fv/Fm, φPSII, and qP were significantly decreased in leaves of fruiting-stage PG due to drought stress, but changes were observed at different times.

3.3.3. Osmotic Substances and Malondialdehyde

The effect of drought stress on osmotic substances and the malondialdehyde content in fruiting-stage PG is shown in Figure 3a–d. Drought stress significantly increased the contents of SP, PRO, and MDA in fruiting-stage PG. After the drought group was resupplied with water, the content of PRO was still significantly higher than that of CK, which was as high as 80.2 μg·g−1. The content of SS was not significantly affected by drought stress and was only significantly higher than that of the CK after the drought group was resupplied with water. It was seen that with the aggravation of drought stress, the high production of MDA in the roots of fruiting-stage PG may have been due to increased lipid peroxidation of cell membranes. Moreover, the fruiting-stage PG roots may have maintained the turgor pressure and life activity through the continuous accumulation of osmotic substances such as SP and PRO.

3.3.4. Protective Enzyme

The effect of drought stress on the protective enzyme activity in fruiting-stage PG is shown in Figure 3e–g. The activities of T-SOD, POD, and CAT were significantly higher in the drought group than in the CK in the AW, and the T-SOD activity was higher in the AP. The above indicated that the fruiting-stage PG may protect itself against cellular oxidative damage caused by drought through the plant protective enzyme system.

3.4. Platycodins

When plants are subjected to stress, growth slows and plants produce more secondary metabolites for defense. The effect of drought stress on platycodins in roots of fruiting-stage PG is shown in Figure 4. The effect of drought stress on the PD content was mainly in the AR and AP. In the AR, the PD content of the drought group was significantly lower than that of CK, but in the AP, the PD content reached 277.72 μg·g−1 in the drought group, an increase of 8.95%. The effect of drought stress on the PD3 content was mainly in the AP, when the PD3 content reached 213.28 μg·g−1, increasing by 11.67% compared with that of CK. In the AW, the DPD content exceeded that in the CK. After the drought group was resupplied with water, the DPD content was significantly affected in the AP, reaching 124 μg·g−1, i.e., an increase of 12.54%. Drought stress significantly increased the SPD content in three stages (AW, AR, AP), with values of 62.54 mg·g−1, 59.59 mg·g−1, and 68.56 mg·g−1, respectively. The above results showed that PG may actively respond to adverse environmental conditions through the accumulation of secondary metabolites. In conclusion, drought stress significantly increased the platycodin content in the AP, which promoted the quality of PG. However, there were differences in the biosynthesis of PD, PD3, DPD, and SPD in fruiting-stage PG root after experiencing drought stress. Perhaps, due to the influence of soil water changes, the manner and degree of the structural modification of the sugar group during platycodin synthesis of the fruiting-stage PG differed, and then, the accumulation of PD, PD3, DPD, and other platycodins differed. However, the mechanism of platycodin synthesis, transformation, and structural modification remains to be further analyzed.

3.5. Principal Component Analysis and Correlation Analysis

The results of the principal component analysis of each index in fruiting-stage PG under drought stress are shown in Figure 5a. The variance contribution was greatest for PC1 and PC2, explaining 36.0% and 30.8%, respectively, of the total variance and cumulatively explaining 66.8% of the total variance. The effects of drought stress did not overlap in terms of spatial distribution in the four stages (0 d, AW, AR, AP). The AW and AP stages were located on the positive end of the PC1 axis, the AW and AR stages were located on the positive end of the PC2 axis, and the 0 d stage was located on the negative ends of the PC1 and PC2 axes, which indicated the difference between the four stages. Notably, the two clusters of the 0 d and AW were separated, implying significant differences between the two stages. From each observed index, Ci and qP were negatively associated with PC1 and PC2 at 0 d. SPD, DPD, SS, SP, MDA, T-SOD, POD, CAT, and NPQ were all positively associated with PC1 and PC2 at the AW. PRO and Ls were positively correlated with PC2 in the AR stage but negatively correlated with PC1. PD3, PD, FW, DW, WUE, Pn, Fv/Fm, and φPSII were positively correlated with PC1 in the AP. However, they showed a negative correlation with PC2.
Based on the principal component analysis, we used the correlation cluster heatmap to further analyze the observed indicators in fruiting-stage PG under drought stress, and the results are shown in Figure 5b. We found that under drought stress, PD was significantly positively correlated with DW, SP, and WUE but was significantly negatively correlated with PRO. DPD was significantly positively correlated with T-SOD, CAT, and SP. PD3 was significantly positively correlated with SP, WUE, and CAT. SPD was significantly positively associated with T-SOD, CAT, and SS. A significant positive correlation between PD, DPD, and PD3 indicated that these three monomeric platycodins contributed significantly to the accumulation of total platycodin.

4. Discussion

Water is one of the important environmental factors needed for the growth and survival of medicinal plants. Drought may occur in various growth stages of medicinal plants, which often respond to external drought conditions through their own complex physiological and biochemical reactions. Photosynthesis is one of the most sensitive physiological indicators of plant response to drought stress [24,25]. We found that insufficient soil water can have a significant impact on photosynthesis and chlorophyll fluorescence parameters. The Fv/Fm, WUE, φPSII, and Pn values were lower in plants with drought treatment, reinforcing that drought stress reduced the photosynthetic efficiency of PG. Fv/Fm is a sensitive indicator of the plant photosynthetic performance [26]. An Fv/Fm reduction can be indicative of photoinhibitory damage [27]. The Fv/Fm, WUE, and φPSII values in the drought group resupplied with water returned to normal levels. It was possible to verify that the photoinhibition damage in these plants was transient and did not compromise their physiological performance. Results of the correlation analysis showed that both WUE and φPSII had a significant positive correlation with the yield (DW) and quality (PD, PD3) of the PG medicinal materials. We also observed that PG root growth was affected. In the AW, the PG root dry weight was not significant. However, in the comparison of the autumn PG yield, we found a significant increase of 16.35% in the weight of PG dry roots. We speculated that when the fruiting-stage PG encountered drought stress, reproductive growth may decrease with the accumulation of more desiccation material to maintain survival. Thus, drought stress in fruiting-stage PG may be more beneficial to the accumulation of dry material.
Medicinal plants with an insufficient water supply produce a high concentration of ROS and MDA, which are highly toxic to medicinal plants [28]. In this study, we sought to understand how PG plants resist drought using physiological and productive mechanisms. The results showed that drought stress increased the lipid peroxidation of root cells of fruiting-stage PG and produced a large amount of MDA. The activities of T-SOD, POD, and CAT were also significantly higher, which is consistent with previously published studies [29,30,31]. The above results indicated that oxidative damage occurred in PG plants. To maintain normal growth and development, plants may resist oxidative damage through the synergistic action of the three enzymes. The three enzymes responded consistently to drought stress, but the increases in their activity were different. In addition, we found that SP and PRO accumulated significantly in fruiting-stage PG under drought stress. These synthesized osmotic substances can maintain the balance between water and osmotic pressure in plant cells and can establish a suitable reaction environment for other enzymatic reactions such that plants can adapt to arid environments. Interestingly, a variety of osmotic substances maintain turgor pressure and life activities through continuous accumulation, but not all kinds of osmotic substances respond consistently to drought stress. SP and PRO may play a major role in resisting drought stress in fruiting-stage PG.
In complex and variable environments, multiple secondary metabolites are synthesized in medicinal plants that are pharmaceutically active ingredients [32]. Our previous study showed that the platycodin content of vegetative-period PG can increase under drought conditions [33], but it did not focus on fruiting-stage PG. We found that drought stress during the fruiting stage promoted the accumulation of platycodin components during the autumn harvest. This result is consistent with that presented by other studies [34,35,36]. Drought stress during the fruiting stage of PG may be an effective way to improve the quality of medicinal materials. It is worth noting that the biosynthetic capacity of PD, PD3, DPD, and SPD in fruiting-stage PG roots differed under drought stress. Perhaps, due to the influence of soil water changes, the manner and degree of the structural modification of the sugar group during platycodin synthesis of the fruiting-stage PG differed, and then, the accumulation of PD, PD3, DPD, and other platycodins differed. However, the mechanism of synthesis, transformation, and structural modification of platycodin remains to be further analyzed. Next, we further analyzed the relationship between physiological indicators and metabolites. We found that there were strong correlations between different physiological stress indicators. Platycodin and the root dry weight, T-SOD, CAT, SP, and SS were significantly positively correlated in fruiting-stage PG. SPD had a significant positive correlation with three monomic saponins (PD, DPD, PD3), which indicated that these three monomer saponins contribute greatly to the accumulation of SPD. These indices had synergistic effects of mutual promotion and restriction, responding to changes in the soil water content. In conclusion, subject to short-term drought stress, fruiting-stage PG resisted oxidative damage caused by drought by increasing the protective enzyme activity. Drought stress during the fruiting stage had little effect on the yield of PG during the autumn harvest, but the content of platycodins increased significantly. It is suggested that agricultural production can regulate the quality of PG through short-term drought stress in the fruiting period, so as to promote the quality of medicinal plants and increase the medicinal value of PG.

5. Conclusions

All environmental factors can have an impact on the synthesis and distribution of secondary metabolites in plants [37]. In this study, by developing short-term drought stress in fruiting-stage PG, we found that PG production during the autumn harvest period was not affected by drought stress, but drought stress significantly increased the platycodin content in PG roots. The results are in line with the optimal defense hypothesis [38]. The optimal defense hypothesis holds that under environmental stress, plants produce more secondary metabolites if the plants compensate for the damage poorly and the defense benefits of secondary metabolites increase. Therefore, in agricultural production, the fruiting stage under short-term drought stress can not only ensure the yield of PG but can also improve the quality of medicinal materials. We found that the physiological drought resistance mechanism of PG may protect against oxidative damage by increasing protective enzyme activity and osmoregulatory materials to ensure normal plant growth. However, protective enzyme activity and osmoregulatory material in PG are not always important indicators of their resistance to drought; thus, how drought influences endogenous hormones and contributes to the development of stress resistance would be an important research topic in the future.

Author Contributions

This experimental study was designed by L.Y., M.H. and M.L. Material preparation, data collection, and analysis were performed by M.L., M.Z. and L.C. First author M.L. completed the initial manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

Partial financial support was received from the China Agriculture Research System (Grant number CARS-21).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare that there is no conflict of interests regarding the publication of this article.

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Figure 1. Changes in the soil water content and root growth. (a) Changes in the soil water content. (b) Changes in the weight of fresh root. (c) Changes in the weight of dry root. Data are expressed as the mean ± standard deviation (SD) (n = 5). *, p < 0.05 compared with CK.
Figure 1. Changes in the soil water content and root growth. (a) Changes in the soil water content. (b) Changes in the weight of fresh root. (c) Changes in the weight of dry root. Data are expressed as the mean ± standard deviation (SD) (n = 5). *, p < 0.05 compared with CK.
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Figure 2. Changes in photosynthetic indicators and chlorophyll fluorescence parameters of fruiting-stage Platycodon grandiflorum under drought stress. (a) Changes in the photosynthetic rate (Pn); (b) changes in the water-use efficiency (WUE); (c) changes in the stomatal limit (Ls); (d) changes in the intercellular CO2 concentration (Ci); (e) changes in the maximum photochemical efficiency (Fv/Fm); (f) changes in the actual photochemical efficiency (φPSII); (g) changes in the photochemical quenching coefficient (qP); (h) changes in the nonphotochemical quenching coefficient (NPQ). Data are expressed as the mean ± SD (n = 5). *, p < 0.05 compared with CK.
Figure 2. Changes in photosynthetic indicators and chlorophyll fluorescence parameters of fruiting-stage Platycodon grandiflorum under drought stress. (a) Changes in the photosynthetic rate (Pn); (b) changes in the water-use efficiency (WUE); (c) changes in the stomatal limit (Ls); (d) changes in the intercellular CO2 concentration (Ci); (e) changes in the maximum photochemical efficiency (Fv/Fm); (f) changes in the actual photochemical efficiency (φPSII); (g) changes in the photochemical quenching coefficient (qP); (h) changes in the nonphotochemical quenching coefficient (NPQ). Data are expressed as the mean ± SD (n = 5). *, p < 0.05 compared with CK.
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Figure 3. Changes in osmoregulatory substances, malondialdehyde content, and protective enzyme activities of fruiting-stage Platycodon grandiflorum under drought stress. (a) Changes in the malondialdehyde (MDA); (b) changes in the proline (PRO); (c) changes in the soluble sugar (SS); (d) changes in the soluble protein (SP); (e) changes in the total superoxide dismutase (T-SOD) activity; (f) changes in the peroxidase (POD) activity; (g) changes in the catalase (CAT) activity. Data are expressed as the mean ± SD (n = 5). *, p < 0.05 compared with CK.
Figure 3. Changes in osmoregulatory substances, malondialdehyde content, and protective enzyme activities of fruiting-stage Platycodon grandiflorum under drought stress. (a) Changes in the malondialdehyde (MDA); (b) changes in the proline (PRO); (c) changes in the soluble sugar (SS); (d) changes in the soluble protein (SP); (e) changes in the total superoxide dismutase (T-SOD) activity; (f) changes in the peroxidase (POD) activity; (g) changes in the catalase (CAT) activity. Data are expressed as the mean ± SD (n = 5). *, p < 0.05 compared with CK.
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Figure 4. Changes in the platycodin content of fruiting-stage Platycodon grandiflorum under drought stress. (a) Changes in the platycodin D (PD) contents; (b) changes in the platycodin D3 (PD3) contents; (c) changes in the deapioplatycodin (DPD) contents; (d) changes in SPD contents. Data are expressed as the mean ± SD (n = 5). *, p < 0.05 compared with CK.
Figure 4. Changes in the platycodin content of fruiting-stage Platycodon grandiflorum under drought stress. (a) Changes in the platycodin D (PD) contents; (b) changes in the platycodin D3 (PD3) contents; (c) changes in the deapioplatycodin (DPD) contents; (d) changes in SPD contents. Data are expressed as the mean ± SD (n = 5). *, p < 0.05 compared with CK.
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Figure 5. Principal component analysis (PCA) and correlation analysis of detection indexes in fruiting-stage Platycodon grandiflorum under drought stress. (a) Heatmap of the correlation analysis of the detection indicators; (b) PCA of detection indexes. *, p < 0.05 compared with CK; **, p < 0.01 compared with CK.
Figure 5. Principal component analysis (PCA) and correlation analysis of detection indexes in fruiting-stage Platycodon grandiflorum under drought stress. (a) Heatmap of the correlation analysis of the detection indicators; (b) PCA of detection indexes. *, p < 0.05 compared with CK; **, p < 0.01 compared with CK.
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Table
Table 1. Supply water scheme during the fruiting stage of Platycodon grandiflorum.
Table 1. Supply water scheme during the fruiting stage of Platycodon grandiflorum.
TreatmentPrior to Withholding Water (0 d)After Withholding Water (AW)After Rewatering
(AR)		Autumn Period (AP)
(4 September 2019)
(1 Day)		(4 September 2019–19 September 2019)
(15 Days)		(19 September 2019–30 September 2019)
(12 Days)		(1 October 2019–20 October 2019)
(20 Days)
Control group
(CK)		176 mL176 mL176 mL100 mL
Drought group
(DG)		The saturated soil water content (33%) —176 mL100 mL
Table
Table 2. The sample collection time of Platycodon grandiflorum.
Table 2. The sample collection time of Platycodon grandiflorum.
Collection Time5 September 201919
September 2019		22
September 2019		20
October 2019
Control group
(CK)		(0 d)Day 15
(Day 15 after withholding water)		Day 18
(Day 3 after rewatering)		Autumn harvest period of Platycodon grandiflorum medicinal materials
Drought group
(DG)
Table
Table 3. The results of regression analysis of platycodin.
Table 3. The results of regression analysis of platycodin.
StandardRegression EquationR2Linear Range (µg·mL−1)
Platycodin D (PD)logY = 0.6115 logX − 3.32460.99995~200
deapioplatycodin (DPD)logY = 0.6004 logX − 3.34110.99995~200
Platycodin D3 (PD3)logY = 0.5969 logX − 3.26910.99985~200
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Li, M.; Zhang, M.; Cheng, L.; Yang, L.; Han, M. Changes in the Platycodin Content and Physiological Characteristics during the Fruiting Stage of Platycodon grandiflorum under Drought Stress. Sustainability 2022, 14, 6285. https://0-doi-org.brum.beds.ac.uk/10.3390/su14106285

AMA Style

Li M, Zhang M, Cheng L, Yang L, Han M. Changes in the Platycodin Content and Physiological Characteristics during the Fruiting Stage of Platycodon grandiflorum under Drought Stress. Sustainability. 2022; 14(10):6285. https://0-doi-org.brum.beds.ac.uk/10.3390/su14106285

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Li, Min, Meng Zhang, Lin Cheng, Limin Yang, and Mei Han. 2022. "Changes in the Platycodin Content and Physiological Characteristics during the Fruiting Stage of Platycodon grandiflorum under Drought Stress" Sustainability 14, no. 10: 6285. https://0-doi-org.brum.beds.ac.uk/10.3390/su14106285

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