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Article

Effects of Water-Deficit Stress on the Growth and Physiological Characteristics of Chloranthus spicatus Seedlings

by
Yangjuan Shang
1,2,*,
Zhibing Wan
1,2,*,
Changheng Zhao
1,2,
Xing Su
2 and
Xinyi Liu
1
1
College of Life and Environmental Science, Huangshan University, Huangshan 245041, China
2
Huangshan Inter-Cloud Ecological Agriculture Development Co., Ltd., Huangshan 245041, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2024, 10(10), 1054; https://doi.org/10.3390/horticulturae10101054
Submission received: 25 August 2024 / Revised: 18 September 2024 / Accepted: 1 October 2024 / Published: 3 October 2024
(This article belongs to the Section Biotic and Abiotic Stress)
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Abstract

:
Chloranthus spicatus is one of the main scented tea varieties cultivated mainly in the Huangshan region, and dried flowers of these plants are mainly used for imparting a characteristic aroma to the tea. However, climatic variations in Huangshan limit its cultivation, with water deficit (WD) being the main limiting factor. The present study evaluated the effects of different WD intensities on the growth and physiological parameters of C. spicatus seedlings to determine the optimal soil moisture content for their large-scale cultivation. The experimental design comprised a control group (95–100%) and three treatment groups, namely mild WD (75–80%), moderate WD (55–60%), and severe WD (35–40%). Each treatment lasted 45 days and was given to 10 potted C. spicatus seedlings, with 3 replicates. Measurements were conducted for the shoot length and diameter; biomass; photosynthesis parameter; activities of antioxidant enzymes, namely superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT); and soluble protein (SP) and malonylaldehyde (MDA) contents of the seedlings. The results indicated that mild and moderate WD positively affected C. spicatus seedlings in terms of shoot length, diameter, biomass, root/shoot ratio, photosynthesis rate, intercellular CO2 concentration, water use efficiency, and carboxylation efficiency. Moreover, the growth and photosynthesis were increased in the mild and moderate WD seedlings but decreased in the severe WD seedlings. Mild and moderate WD also led to a significant decrease in the antioxidant enzyme activities and the MDA content of seedlings (p < 0.05), all of which exhibited higher levels in severe WD seedlings. These results indicated that severe water stress restricted the growth of seedlings, while mild and moderate stress proved beneficial. SP content of the seedlings increased under mild and moderate WD but decreased under severe WD. We infer that the strong hydrophilicity of SPs in the seedlings results in the accumulation of water in plant cells, thus conferring resistance to drought stress. In conclusion, reducing the soil moisture content by 55–80% in the Huangshan region may be a promising strategy for boosting its cultivation.

1. Introduction

Chloranthus spicatus (Thunb.) Makino, a perennial evergreen vine subshrub plant, belongs to the family Chloranthaceae and the genus Chloranthus, also known as Chloranthus. Traditionally, the plants have been termed pearl orchids, fish roe orchids, and chicken claw orchids because of the resemblance of their flowers with pearls and fish eggs and of their inflorescence with chicken feet [1]. Cloranthus species are mainly grown in Anhui, Zhejiang, Jiangsu, Fujian, and Sichuan provinces. These species are commonly distributed on mountain slopes, gullies, and thick forests at altitudes of 150–990 m [2]. The plants are well-known for their floral fragrance that endures longer, leading to the flowers being ranked equivalent to White orchid and Jasmine flowers, which together constitute “Three Flowers of Shexian County” [3]. Chloranthus flowers and leaves impart characteristic scents to tea in China [4]. Chloranthus plants are delicate and grow preferentially in moist regions, but are sensitive to flooding, prefer shade and are sensitive to direct sunlight, and exhibit high sensitivity to cold and torrid conditions [5]. Therefore, the plants’ growth region is extremely limited. Presently, the plants are known to be distributed in Nanyuankou Village, located in Shexian County, Huangshan City, Anhui Province. The plants in the region have been mainly artificially cultivated [1].
Moisture is one of the key survival factors for plant growth. Southern mountainous regions of Anhui Province are characterized by a subtropical monsoon and humid climate zone, which involves four distinct seasons, mild and rainy in spring, with both heat and rain occurring in summer, cool and dry in autumn, and cold and moisture in winter [6]. These climatic factors result in droughts and floods during the summer and autumn, respectively. Typically, plants adapt to the environment through self-regulation under drought or floods; however, extreme conditions inhibit plant growth and even cause the death of plants [7]. A study reported the inhibitory effect of water deficit (WD) on the growth, carbon metabolism, and yield of cowpeas (Vigna unguiculata (L). Walp.) [8]. A soil moisture deficit was reported to cause a decrease in the stomatal conductance (Gs) of lime (Citrus latifolia Tan.), with the Gs values (non-irrigated) under open-field conditions 35% lower than that under shaded conditions [9]. The yield and size of the fruits were also observed to be the maximum under shaded conditions. Water shortage significantly increased the photosynthesis and transpiration in the seedlings of Rhinanthus alectorolophus, although survival, biomass, and gas exchange were altogether decreased [10]. Studies investigating plant response to WD stress have focused mainly on growth and physiology, such as photosynthesis on the leaves [11], osmolyte content [12], enzymatic activity [13], and accumulated biomass [8,14]. High water efficiency in seedlings could improve the quality and yield [15]. However, fewer studies have examined water regulation efficiency in Chloranthus. To address this gap, the present study examined water regulation in 1-year-old potted C. spicatus seedlings by monitoring growth, photosynthesis, and physiological indicators under different soil moisture contents. This study evaluated C. spicatus seedlings’ tolerance to WD, providing a reference for the plantation and management of Chloranthus species in the southern mountains of Anhui Province.

2. Materials and Methods

2.1. Study Materials

Annual potted seedlings of C. spicatus, obtained from Huangshan Inter-Cloud Ecological Agriculture Development Co., Ltd. (Huangshan, China), were used as the study materials. Healthy seedlings with highly consistent and similar growth parameters were selected. In early May 2022, these seedlings were transplanted into pots (diameter 19 cm × height 17 cm) containing a mixture of nutrient soil and garden soil in a 1:3 ratio. The nutrient soil was purchased from Jiangsu Sweeper Biotechnology Co., Ltd. (Nanjing, China). and contained organic matter ≥30% and NPK ≥ 3%. The garden soil contained 65 mg/kg of available nitrogen, 0.4 mg/kg of Olsen-P, 60 mg/kg of available phosphorus, and a pH of less than 4.5 [16]. These seedlings were placed in the Nursery Base of Tiandu Garden at Huangshan University in China and provided with homogenous light and water conditions. The average temperature during the experiment was 24.4 °C, and the day/night temperature was 28.3/18.1 °C, recorded with a thermometer in the garden. The seedlings were managed by the same styles to extirpate weeds and insects. The management method was to conduct manual weeding every half month and spray 10% imidacloprid every month. After 3 months, the seedlings with good and highly consistent growth patterns were selected for experimental studies.

2.2. Experimental Set-Up

The experimental design involved 120 pots and 4 WD stress treatments (Table 1), namely control, mild, moderate, and severe WD. The treatments were administered using the pot-weighing method every two days [17], starting from 1 September 2022. For each treatment, 10 pots were used, with 3 replicates, and a randomized block design was used for the experiments. The study was stopped on day 45 after severe WD seedlings experienced significant wilting. Before the study, field capacity was computed as 0.23 g/g from soil below 1 m of the experimental site.

2.3. Measurements

2.3.1. Growth Parameters

Shoot length and diameter
Shoot length was measured using a steel tape (0.1 cm) on day 1 (1 September) and day 45 (17 October) of WD treatment. The shoot diameter was measured using a vernier caliper (0.02 mm) at both time points. Based on differences in the measured values between the two time points, the relative increments in shoot length and diameter were calculated for all seedlings, except dead ones, from each treatment.
The relative increment in shoot length (H, cm) was calculated as follows: H = H2 − H1 (where H is the relative increment in shoot length, H2 is the shoot length on day 45, and H1 is the shoot length on day 1).
The relative increment in shoot diameter (D, mm) was calculated as follows: D = D2 − D1 (where D is the relative increment in shoot diameter, D2 is the shoot diameter on day 45, and D1 is the shoot diameter on day 1).
Biomass
Using the same D-value method [18], wet and dry biomass of the root, shoot, and leaves were weighed using an electronic balance (0.0001 g). Before weighing, three seedlings from each treatment group were selected and rinsed with tap water. The surface of the seedlings was air-dried to remove moisture. Then, the seedlings were cut into three parts, namely root, shoot, and leaves, by using secateurs. The wet biomass of the root, shoot, and leaves was weighed. The samples were then placed into individual envelopes, which were labeled with the corresponding weight. These envelopes were dried in an oven. The oven was kept at 105 °C for 30 min, and then the temperature was lowered to 80 °C until dryness, and the samples were weighed and recorded.
The relative water content (RWC, %) was calculated as follows: RWC = (WW − DW) × 100/WW (WW and DW denote the wet and dry mass, respectively, of the root, shoot, and leaves).
The root/shoot ratio (RS, %) of the seedlings was calculated as follows: RS = RM × 100/(SM + LM) (RM, SM, and LM are the dry mass of the root, shoot, and leaves, respectively).

2.3.2. Photosynthesis Parameters

Gas exchange in the C. spicatus leaves was measured using a portable photosynthesis system (Model LI-6800, LI-COR, Lincoln, NE, USA) between 9 a.m. and 11 a.m. on a sunny day. The photosynthesis rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and intercellular CO2 concentration (Ci) were the main gas exchange parameters evaluated in this study. Based on these four parameters, the water use efficiency (WUE, Pn/Tr) and instantaneous carboxylation efficiency (CE, Pn/Ci) were calculated [19]. The leaf chamber of LI-6800 was set at a constant light intensity (1500 µmol m−2 s−1), with the red and blue LED sources. The temperature and CO2 concentration were adjusted to 26 °C and 400 µmol mol−1, respectively, and the airflow rate inside was maintained at 500 µmol s−1. A broad-leaf chamber (2 cm2) was used for the measurements. Before recording the gas exchange parameters, the leaf to be tested was kept in the leaf chamber for 5 min to ensure the stability of the parameters. For measurements, three leaves were selected from the middle and upper parts of a seedling from each treatment group, and the three values were averaged to determine the final value.

2.3.3. Physiological Indicators

Leaf samples were collected on the last day of WD stress treatment. Ten leaves of C. spicatus seedlings were randomly sampled from each treatment group and immediately placed in an ice box. One leaf was selected from the middle part of each seedling. Each sampled leaf used in the experiments was fully grown and healthy. In the laboratory, the leaves were rinsed with tap water, air-dried, and subsequently stored at −40 °C until further analysis.
Physiological and biochemical indicators in the leaves were quantified using the protocol of Dong Jianmei [20]. Among them, the activity of superoxide dismutase (SOD) was determined using the nitroblue tetrazolium photoreduction method, and the activity of peroxidase (POD) was determined using the guaiacol method. The activity of catalase (CAT) was determined using ultraviolet spectrophotometry. The soluble protein (SP) content was determined using the Coomassie Brilliant Blue colorimetric method, and the content of malondialdehyde (MDA) was determined using the thiobarbituric acid colorimetric method. For each treatment, 10 leaves were used, each with 3 replicates.

2.4. Statistics and Data Analysis

Data are expressed as the mean ± standard deviation. First, the data were preprocessed using Excel 2016. Repeated-measures analysis of variance (ANOVA) and Duncan’s new multiple range test (p < 0.05) were conducted in SPSS 23.0 to determine significant differences among the groups. Further analysis and graphing were performed using Origin Pro 9.1 software.

3. Results

3.1. Shoot Length and Diameter Differed among C. spicatus Seedlings Subjected to Different WD Stress Treatments

The shoot length and diameter of C. spicatus seedlings varied significantly under different WD stress treatments after 45 days (Figure 1 and Table 2). The overall shoot length (H) under mild, moderate, and severe WD increased by 52%, 47%, and 22%, respectively, with increments of 22.92, 22.19, and 18.35 cm, respectively. The differences among each treatment group were significant (p < 0.05). Similarly, the net increase in diameter (D) under mild, moderate, and severe WD was 5.11, 5.13, and 4.36 mm, respectively (Table 2), corresponding to increases of 44%, 45%, and 23%, respectively, compared with the control, and the differences were significant (p < 0.05). The overall length and diameter of C. spicatus seedlings first showed an increasing trend, followed by a decrease, with an increase in the intensity of WD stress. The overall length and diameter were the highest under moderate, followed by mild WD. These results indicated that appropriately reducing the soil moisture content could significantly increase the shoot length and diameter of C. spicatus seedlings.

3.2. C. spicatus Seedling Biomass Varied in Response to WD Stress Treatments

C. spicatus seedling biomass first increased and then decreased (Figure 2), with differences in the biomass among the treatment groups being significant (p < 0.05). The biomass of seedlings treated with mild, moderate, and severe WD was higher than that of the control seedlings. The wet weight of the roots, shoots, and leaves in mild WD seedlings reached 23.8, 22.3, and 27.2 g/plant, respectively, which were 106%, 81%, and 106% higher than those in control. However, in mild and moderate WD, the wet weight decreased compared with mild WD; however, it remained higher than that in the control. The dry weight of the roots, shoots, and leaves showed the same trend as wet weight, reaching 5.6, 4.5, and 4.3 g/plant in mild WD, respectively, which were 103%, 75%, and 95% higher than those in control.
The water content in the seedlings also exhibited the same trend, with no significant difference (p > 0.05) among the groups. Specifically, the water content of the roots reached 54% in moderate WD; the water content of the shoots reached 67% in severe WD; and the water content of the leaves reached 69% in mild WD. The water contents of the roots, shoots, and leaves in the treatment groups, except for severe WD, were higher than those in the control. These results indicated that the shoots and leaves were the main water storage in C. spicatus seedlings under water scarcity.
With the increase in WD intensity, the root/shoot ratio of C. spicatus seedlings exhibited the same trend as water content; it first increased and then decreased. The root/shoot ratios in all treatments were higher than in control, with the highest root/shoot ratio of 74% noted in moderate WD.
Taken together, these results indicated that mild and moderate WD could stimulate the accumulation of seedling biomass, whereas severe WD causes growth inhibition.

3.3. Changes in Photosynthesis Parameters of C. spicatus in Response to WD Stress

Soil moisture content regulates stomatal conductance in plant leaves [21,22], and this phenomenon is particularly prominent under moderate water stress [18]. The stomatal conductance of leaves plays a key role in balancing water evaporation and biomass accumulation during photosynthesis in plants [10].
As shown in Figure 3, Pn, Ci, WUE, and CE first increased and then decreased in C. spicatus seedlings, whereas Tr and Gs showed an opposite trend. Specifically, in moderate WD, Pn, WUE, and CE increased to 17.31 µmol m−2 s−1, 34,211.60 µmol mol−1, and 0.04 mol m−2 s−1, respectively, which were 28%, 81%, and 26% higher, respectively, than those in the control. However, Tr decreased to a minimum of 0.00051 mol m−2 s−1 in moderate WD, which was 30% lower than that in control. In mild WD, Gs reached a minimum of 3.88 mol m−2 s−1, which was 5% lower than that in control. Ci increased to a maximum of 397.5 mol m−2 s−1, showing a 2% increase compared with control. A comparative analysis showed that moderate water stress in C. spicatus seedlings led to leaf stomatal closure, lowered the water transpiration rate, and slightly increased the intercellular CO2 concentration, thereby improving the WUE and instantaneous CE, eventually increasing the net photosynthesis rate.
The comprehensive analysis showed that mild and moderate WD were beneficial to C. spicatus seedlings, indicating that C. spicatus seedlings are resistant to mild and moderate decreases in the soil moisture content.

3.4. Activity of Antioxidant Enzymes of C. spicatus Seedlings Varied in Response to Different WD Stress Treatments

Water scarcity is deleterious to plants, and to combat the damage induced by WD, plants have produced a physiological mechanism. This mechanism involves the release of some antioxidant enzymes by plants to remove harmful substances such as free radicals [23], followed by the secretion of some substances to protect the cell membrane [24]. These substances help plant cells resist the induced damage.
As shown in Figure 4, the activity of all antioxidant enzymes, namely SOD, POD, and CAT, first decreased and then increased with an increase in the WD stress intensity. Enzyme activities in the seedlings subjected to mild and moderate WD were lower than those in control. Specifically, SOD, POD, and CAT activities in moderate WD seedlings were 156.74 U/g FW, 1561.22 U/g·min FW, and 22,933.82 U/g FW, respectively, which were 39%, 27%, and 47% lower than those in control plants. The values of these indicators showed that mild and moderate water stress did not cause damage to the C. spicatus seedlings. However, in severe WD, a great fluctuation was noted in SOD, POD, and CAT indicators, which showed that severe water stress is deleterious to the seedlings and that the seedlings need to adjust their antioxidant enzyme activity to resist WD-triggered damage.

3.5. SP and MDA Contents of C. spicatus Seedlings Changed in Response to WD Stress

Under soil moisture stress, plants need to protect their cells. On the one hand, plants accumulate osmoregulatory molecules, such as SPs, to regulate the osmotic pressure of their cells. On the other hand, they produce MDA through membrane lipid peroxidation reactions for cellular use [25]. Thus, the SP and MDA contents are often used as indicators of the degree of stress-induced damage in plants [7,26].
In moderate WD, the SP content of the seedlings first increased and then decreased (Figure 5), reaching a peak of 16.45 mg/g, which was 60% higher than that in the control. Each treatment group differed significantly in their SP content (p < 0.05). However, the MDA content showed an opposite trend, reaching a minimum of 289 µmol/g FW in mild WD, which was 27% lower than that in control. The MDA content also differed significantly between the groups (p < 0.05). Interestingly, in severe WD seedlings, SP content decreased, but the MDA content increased significantly, indicating that the severe water stress caused damage to C. spicatus seedlings to a higher degree.
Overall, the results indicated that mild and moderate water stress did not cause damage to C. spicatus seedlings, but rather proved beneficial to seedlings. This result was consistent with those for the growth, biomass accumulation, and photosynthesis parameters of C. spicatus seedlings under water stress.

4. Discussion

4.1. Plant Growth and Biomass under WD Stress

The net growth values were the direct assessment indicators of water stress. It is believed that the effect of different WD stress intensities on the growth status of plants varies to a great extent. Moderate and severe hardening could significantly affect the root/shoot ratio of holm oak (Quercus ilex) seedlings after a 3-month period [27]. The duration of drought stress significantly reduced the aboveground biomass of the treated seedlings; however, it minimally affected the nonstructural carbohydrate (NSC) content. Another study showed that WD treatment caused a significant decrease in the shoot length, leaf area, and biomass, but not the diameter, of Eucalyptus globulus seedlings [28]. Field experiments showed that the deficit irrigation strategy could significantly improve the WUE of silage maize (Zea mays) in arid and semi-arid climates during the growing season [29], with significant effects on the biomass, leaf area, and root/shoot ratio of the plants.
In this study, the net growth and biomass of C. spicatus seedlings increased significantly under mild and moderate WD stress treatments in which the relative soil moisture content was adjusted to 55–80%. These results showed that soil moisture deficit stimulated the growth of C. spicatus seedlings by improving their WUE. In the Huangshan region, the soil is yellow-brown in color, with heavy clay and a compacted, highly leaching sedimentary layer. This soil type exhibits poor air permeability. The strategy of reducing the soil moisture content might be effective in improving soil breathability, thereby promoting seedling growth. This opinion was in line with those of Wenying, Y. et al. [30].
A comparison of seedling biomass under different moisture contents revealed that the plants subjected to mild and moderate water stress retained higher water content in the roots, shoots, and leaves than the control plants. Accordingly, the seedlings subjected to mild and moderate treatments accumulated higher biomass than the control seedlings, despite the soil moisture content being low in the mild and moderate treatments. We believe that the increase in biomass might be related to the water-storing characteristics of plants, attributed to their increased WUE. Thus, under limited water availability, the plants’ high WUE allowed them to store moisture, consistent with the viewpoint of O’Brien [31].
In addition, the root/shoot ratios of the treated seedlings were found to be higher than those of the control. This indicated that soil moisture deficit caused the leaves of the seedlings to turn yellow and wilt. Thus, we infer that to maintain growth, the seedlings need to expand their root area and absorb water, consistent with the view of Hu Yanping et al. [15].

4.2. Plant Photosynthesis Differences under WD Stress

Plants use photosynthesis to maintain their growth and development. Stomatal conductance and other indicators are indispensable to the smooth completion of photosynthesis, and it is regulated by soil moisture, which, thus, affects photosynthesis [25]. Regulation of stomatal conductance involves two pathways, namely stomatal limitation and non-stomatal limitation [21]. Under stomatal limitation, the stomatal conductance of plants decreases, causing a decrease in both the intercellular CO2 concentration and transpiration rate, eventually resulting in a decrease in the net photosynthesis rate of leaves. Under non-stomatal limitation, the intercellular CO2 concentration either increases or remains stable, decreasing the net photosynthesis rate of leaves.
In this study, the trend in the net photosynthesis rate was highly consistent with that of the intercellular CO2 concentration. However, an opposite trend was noted in the stomatal conductance and transpiration rate of the seedlings. The result showed that C. spicatus seedlings exhibited non-stomatal limitation under mild and moderate treatments. Specifically, under moderate treatment, Pn, WUE, and CE were 28%, 81%, and 26% higher than control, and Tr was 30% lower than control. The phenomenon of decreased Tr and increased Pn may be caused by the self-regulation of plants. The regulation originated from the drought sensitivity of C. spicatus seedlings. Conversely, under severe WD, stomatal conductance of the C. spicatus seedlings decreased, resulting in a significant decrease in both intercellular CO2 concentration and net photosynthetic rate. These results suggest that appropriately reducing the soil moisture content can be a promising strategy for improving the WUE and instantaneous CE of C. spicatus seedlings.
An experimental study in Rhinanthus alectorolophus plants under water stress showed that low-irrigated plants exhibited significantly higher rates of photosynthesis and transpiration; still, seedling survival, biomass production, and gas exchange in these plants were significantly reduced under WD [10]. Petropoulos et al. reported that the WD stress increased the leaf density of parsley (Petroselinum crispum [Mill.] Nym. ex A.W. Hill) (plain-leafed or curly leafed parsley varieties), thereby further increasing the yield of oil per square meter [32]. In mulberry (Morus alba) seedlings subjected to three levels of drought stress for a month, stomatal conductance and transpiration rate decreased, whereas seedling growth was inhibited; however, the root/shoot ratio and abscisic acid content of the seedlings increased, indicating that mulberry seedlings resist drought stress by increasing the root absorption area and improving their water-retention capacity. Furthermore, drought hardening increased the seedling’s drought resistance [33]. Under water-deficit conditions, the leaves of Vitis vinifera L. cv. Assyrtiko contained abundant Calcium Oxalate (CaOx) crystals as internal carbon pools providing CO2 to maintain the balance of photosynthesis, despite closed stomata, namely a mechanism of “alarm photosynthesis” to improve the endurance of Vitis under water-deficit conditions [34].

4.3. Antioxidant Enzyme Activities Differ among Plants Treated with Varying WD Intensities

Under water stress, plants secrete harmful substances such as free radicals, clearance of which is essential for sustained growth of plants and facilitated by the synergistic action of SOD, POD, and CAT [7]. In this study, the activities of SOD, POD, and CAT in C. spicatus seedlings under mild and moderate WD were lower than those in control; however, these enzymatic activities were high in the seedlings subjected to severe WD. This indicated that fewer harmful substances, such as oxygen species and free radicals, accumulated under mild and moderate WD, which did not induce acute toxicity in the seedlings. Under severe WD, the enzymatic activity remained high, indicating that more harmful substances accumulated in the C. spicatus seedlings subjected to severe WD. In addition, the result indicated that the control treatment provided excess water, tending to cause the submergence of seedlings. Thus, the clearance of harmful substances in the seedlings subjected to control treatment led to a considerable increase in the antioxidant enzyme activity. The results were consistent with those reported in Magnolia wufengensis [7], Silage maize [29], tomato (Solanum lycopersicum) [35], and Switchgrass (Panicum virgatum L.) [36].

4.4. Osmoregulatory Substances of Plants under WD Stress

SPs and MDA play a pivotal role in maintaining the osmotic pressure in plant cells, and are therefore regarded as the main osmoregulatory substances in plants [25]. Under stress conditions, the SPs and MDA contents of plants reflect the stability of cell protoplasmic colloid, which is crucial in avoiding plant death due to WD induced by changes in osmotic pressure. Therefore, the SPs and MDA contents of plants are considered to be directly correlated with their stress resistance ability [26]. However, in some plants, the increased SP content serves as a mechanism to store water and resist drought. This is because SPs exhibit strong hydrophilicity under WD. SPs have been extensively applied in crop cultivation to promote water retention in plants [28,37,38]. In this study, the SP content of the seedlings first increased and then decreased. Interestingly, the SP content increased significantly in plants subjected to mild and moderate stress. The difference was attributable to the change in water stress levels under mild and moderate treatments. C. spicatus seedlings responded to the change in water stress and maintained growth by increasing the content of SPs. This growth pattern was not inhibited even with stomatal closure. Furthermore, the changing trend in the MDA content aligned with that of the activity of antioxidant enzymes. The seedlings subjected to mild and moderate stress exhibited lower MDA levels and those subjected to severe stress showed higher MDA levels than the control group. The change is possibly due to higher antioxidant activity in plants under mild and moderate stress, coupled with prompt response and accumulation of various protective, stress-related metabolites.
In this study, the treated seedlings could maintain growth, and the growth was not limited by stomatal closure. Specifically, SP content in the shoot tissues played a predominant role in conferring severe drought resistance. As suggested by Mamnouie, the effect of the difference in soil water levels by 50–100% is minimal; however, severe WD causes a remarkable decrease in the yield and 1000 seed weight [39]. The aforementioned views align closely with the results of the present study, indicating that the increased SP content in the C. spicatus seedlings enabled them to effectively resist water stress.

5. Conclusions

In this study, C. spicatus seedlings were subjected to varying intensities of WD to determine the optimal soil moisture content for plant growth. The results revealed that mild and moderate WDs are beneficial to the growth and photosynthesis of C. spicatus plants. However, severe WD decreased both growth and photosynthesis in the seedlings. Under mild and moderate WD, the C. spicatus seedlings exhibited significant increases in their shoot length, diameter, biomass, root/shoot ratio, Pn, WUE, CE, and SP content; however, these indicators exhibited decreased values in the seedlings subjected to severe WD. In terms of physiological indicators, our experimental results indicated that the activities of SOD, POD, and CAT and the MDA content of the seedlings subjected to mild and moderate water stress significantly decreased, unlike the seedlings subjected to severe stress, which maintained higher enzymatic activities and MDA content. Based on these findings, we conclude that 55–80% soil moisture content is optimal for C. spicatus cultivation, and reducing the soil moisture content is a viable strategy for enabling the adaptation of C. spicatus plants to water stress in areas with heavy clay soil such as the Huangshan region.

Author Contributions

Y.S.: Conceptualization and Writing—Original Draft. Z.W.: Methodology. C.Z.: Resources. X.S.: Investigation. X.L.: Software. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Key R&D Program of Anhui Province, China (201904a06020033), and the Research Project of Huang Shan University (hxkt2022068).

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors upon request.

Acknowledgments

We thank our fraternity for reviewing our manuscript. The specific information of the reviewer is as follows: Zhuangzhuang Liu (zzliu91@163.com), Jiangsu Key Laboratory for the Research and Utilization of Plant Resources, Institute of Botany, Jiangsu Province, and Chinese Academy of Sciences.

Conflicts of Interest

Authors Yangjuan Shang, Zhibing Wan, Changheng Zhao and Xing Su were employed by the company Huangshan Inter-Cloud Ecological Agriculture Development Co., Ltd. Authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Phenotypic changes in Chloranthus spicatus seedlings under different water-deficit stress intensities after 45 days.
Figure 1. Phenotypic changes in Chloranthus spicatus seedlings under different water-deficit stress intensities after 45 days.
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Figure 2. Biomass of Chloranthus spicatus seedlings under water-deficit (WD) stress after 45 days. Columns with different small letters indicate significant differences (Duncan’s multiple range test, p < 0.05).
Figure 2. Biomass of Chloranthus spicatus seedlings under water-deficit (WD) stress after 45 days. Columns with different small letters indicate significant differences (Duncan’s multiple range test, p < 0.05).
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Figure 3. Variations in photosynthesis parameters (Pn, Tr, Ci, Gs, WUE, and CE) in the leaves of Chloranthus spicatus seedlings under water-deficit (WD) stress after 45 days. Dots with different small letters indicate significant differences (Duncan’s multiple range test, p < 0.05).
Figure 3. Variations in photosynthesis parameters (Pn, Tr, Ci, Gs, WUE, and CE) in the leaves of Chloranthus spicatus seedlings under water-deficit (WD) stress after 45 days. Dots with different small letters indicate significant differences (Duncan’s multiple range test, p < 0.05).
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Figure 4. Changes in superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities in the leaves of Chloranthus spicatus seedlings under different water-deficit (WD) treatments after 45 days. Dots with different small letters indicate significant differences (Duncan’s multiple range test, p < 0.05).
Figure 4. Changes in superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities in the leaves of Chloranthus spicatus seedlings under different water-deficit (WD) treatments after 45 days. Dots with different small letters indicate significant differences (Duncan’s multiple range test, p < 0.05).
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Figure 5. Soluble protein and malonylaldehyde contents in the leaves of Chloranthus spicatus seedlings under different water-deficit (WD) treatments after 45 days. Dots with different small letters indicate significant differences (Duncan’s multiple range test, p < 0.05).
Figure 5. Soluble protein and malonylaldehyde contents in the leaves of Chloranthus spicatus seedlings under different water-deficit (WD) treatments after 45 days. Dots with different small letters indicate significant differences (Duncan’s multiple range test, p < 0.05).
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Table 1. Details of water-deficit treatments given to annual potted seedlings of Chloranthus spicatus.
Table 1. Details of water-deficit treatments given to annual potted seedlings of Chloranthus spicatus.
TreatmentsRelative Water Content of SoilStress Intension
control95–100%Control WD
mild75–80%Mild WD
moderate55–60%Moderate WD
severe35–40%Severe WD
Table 2. Growth of Chloranthus spicatus seedlings under different water-deficit intensities after 45 days.
Table 2. Growth of Chloranthus spicatus seedlings under different water-deficit intensities after 45 days.
TreatmentsShoot Length (cm)Shoot Diameter (mm)
H1H2HD1D2D
control25.91 ± 6.26 a40.97 ± 7.11 c15.06 ± 5.52 c3.05 ± 0.58 a3.10 ± 0.46 a0.35 ± 0.38 a
mild25.94 ± 6.34 a48.86 ± 9.93 ab22.92 ± 9.90 a3.02 ± 0.65 a3.33 ± 0.73 a0.51 ± 0.39 a
moderate27.31 ± 5.69 a49.50 ± 8.79 a22.19 ± 8.28 ab2.96 ± 0.66 a3.24 ± 0.50 a0.51 ± 0.36 a
severe26.42 ± 4.59 a44.87 ± 7.99 bc18.35 ± 8.19 bc2.90 ± 0.45 a3.08 ± 0.49 a0.44 ± 0.32 a
H1 and H2 denote the shoot length on day 1 and day 45, respectively. H (H2 − H1) denotes the relative increment in shoot length. D1 and D2 denote the shoot diameter on day 1 and day 45, respectively. D (D2 − D1) denotes the relative increment in shoot diameter. Different lower-case letters indicate significant differences between treatments (Duncan’s multiple range test, p < 0.05).
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MDPI and ACS Style

Shang, Y.; Wan, Z.; Zhao, C.; Su, X.; Liu, X. Effects of Water-Deficit Stress on the Growth and Physiological Characteristics of Chloranthus spicatus Seedlings. Horticulturae 2024, 10, 1054. https://doi.org/10.3390/horticulturae10101054

AMA Style

Shang Y, Wan Z, Zhao C, Su X, Liu X. Effects of Water-Deficit Stress on the Growth and Physiological Characteristics of Chloranthus spicatus Seedlings. Horticulturae. 2024; 10(10):1054. https://doi.org/10.3390/horticulturae10101054

Chicago/Turabian Style

Shang, Yangjuan, Zhibing Wan, Changheng Zhao, Xing Su, and Xinyi Liu. 2024. "Effects of Water-Deficit Stress on the Growth and Physiological Characteristics of Chloranthus spicatus Seedlings" Horticulturae 10, no. 10: 1054. https://doi.org/10.3390/horticulturae10101054

APA Style

Shang, Y., Wan, Z., Zhao, C., Su, X., & Liu, X. (2024). Effects of Water-Deficit Stress on the Growth and Physiological Characteristics of Chloranthus spicatus Seedlings. Horticulturae, 10(10), 1054. https://doi.org/10.3390/horticulturae10101054

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