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Article

Drought Stress Drives Sex-Specific Physiological and Biochemical Differences in Female and Male Litsea cubeba

by
Ming Gao
1,2,
Yunxiao Zhao
1,2,
Yicun Chen
1,2,* and
Yangdong Wang
1,2,*
1
State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing 100091, China
2
Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(6), 594; https://doi.org/10.3390/horticulturae11060594 (registering DOI)
Submission received: 1 April 2025 / Revised: 6 May 2025 / Accepted: 14 May 2025 / Published: 27 May 2025
(This article belongs to the Special Issue Germplasm, Genetics and Breeding of Ornamental Plants)
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Abstract

:
Numerous studies have focused on dioecious plants and their sex-specific responses to drought stress. However, sexual dimorphism in drought stress responses between male and female Litsea cubeba, a dioecious species significant to the terrestrial ecosystem in China that is frequently exposed to drought conditions, remains insufficiently characterized. In this study, we examined the sex-specific physiological and biochemical responses of L. cubeba to natural drought stress. The results revealed that natural drought induced significant sexual dimorphism in physiological and biochemical traits of L. cubeba. Females exhibited a higher malondialdehyde (MDA) content than males under prolonged drought conditions; females also exhibited significantly higher catalase (CAT) and peroxidase (POD) activities in both leaves and roots compared to males, with the average CAT and POD activities of all varieties increasing by 104.28% and 23.67% in leaves and 51.17% and 174.57% in roots, respectively. Meanwhile, the dehydrogenase (DHA) activity and chlorophyll (chl) and carotenoid levels of females were higher than those of males. The contents of proline (Pro), soluble sugar (SS), abscisic acid (ABA), and jasmonic acid (JA) in females were significantly higher than those in males. Our results demonstrated that females possess a greater tolerance to natural drought stress than males; this is due to their more efficient antioxidant system, better osmotic adjustment, lower chlorophyll degradation rate, and higher concentrations of ABA and JA, which aid in stomatal closure and facilitate the reactive oxygen species (ROS)-scavenging abilities of females in response to drought stress. Our findings provide evidence that dioecious L. cubeba may adopt distinct survival strategies during natural drought events and enhance our understanding of sexually dimorphic responses to drought stress in L. cubeba.

1. Introduction

Drought is regarded as the most serious source of abiotic stress affecting plants [1]. Projections suggest that global land surface warming may result in longer and more intense droughts [2,3]. The incidence of drought events in China peaked in 2022, during which the highest drought levels since 1961 were experienced. During the summer and autumn of that year, Southern China experienced an unprecedented drought [4,5,6]. In the Yangtze River Basin, the number of drought days reached 77 in 2022, which is 54 days more than the same period in the previous year. As a result, more than 6.09 million hectares of forests and crops were devastated by this persistent drought, leading to direct economic losses amounting to CNY 51.2 billion [4]. Drought stress has the potential to significantly hinder the growth and development of plants by impacting their morphological, physiological, and molecular characteristics. Drought stress can trigger the generation of reactive oxygen species (ROS), which may cause considerable harm to biomacromolecules. This damage can result in a decrease in plant cell growth and photosynthesis, a slowdown in stem elongation and root development, and a diminished flower and fruit yield [7,8,9,10,11]. Plants employ various adaptive and defensive strategies to reduce the harm induced by drought stress. Various enzymatic antioxidants are used to alleviate oxidative damage caused by ROS [12,13]. Furthermore, proline and soluble sugar accumulate to mitigate the effects of drought conditions [14,15]. In addition, abscisic acid (ABA) and jasmonic acid (JA) are crucial in orchestrating the plants’ adaptive responses to drought stress [16,17,18,19,20]. These hormones promote stomatal closure, reduce evapotranspiration and photosynthesis, induce osmolyte accumulation, and facilitate ROS scavenging [17,21]. Additionally, it has been demonstrated that signaling by phytohormones facilitates the development of the secondary cell wall and helps sustain stem water potential by regulating the growth of xylem vessels [22,23].
Although they represent only 5–6% of angiosperms [24], dioecious species play a crucial role in terrestrial ecosystems, as do monoecious plants, and their sexual dimorphism in drought stress responses enables them to serve as unique models for elucidating environmental adaptation mechanisms in angiosperms. These plant species, which encompass distinct male and female individuals, have developed unique adaptations through the processes of natural evolution and environmental pressures. Consequently, dioecious plants display noticeable sexual dimorphisms, which can be observed in their morphology, physiological characteristics, and strategies for resource acquisition. This differentiation not only enhances their survival and reproductive success but also contributes to the overall biodiversity and resilience of the ecosystems they inhabit [25,26,27,28]. Previous studies documented the sexual dimorphisms response to various environmental stresses, including drought, warming, Ultraviolet B (UV-B) radiation, and nutrient deficiency. Among certain types of Populus cathayana and Morus alba, male individuals are more suited to stressful situations than female individuals [29,30]. On the other hand, in other species, including Salix myrsinifolia and S. paraplesia, the opposite results have been observed [31,32,33]. These studies indicate that sexual dimorphism-based performance may differ between species [34].
Mountain pepper (Litsea cubeba (Lour.) Pers.), belonging to the Lauraceae family, is a species indigenous to China that is extensively distributed across Southeast Asia and Japan. L. cubeba is recognized as a key industrial feedstock due to its fruit peel-derived essential oil, which is rich in citral isomers (geranial and neral) and serves as a crucial raw material in the manufacture of spices and medicinal items [35]. L. cubeba is a dioecious plant, and the primary distinction between male and female individuals is manifested in its floral structures. Female flowers contain staminodes, whereas male flowers retain pistillodes. As an anemophilous (wind-pollinated) species, its flowering period extends from February to March, followed by fruit development occurring from March to September. However, the ripening stage and geographic range of L. cubeba has been affected by high summer temperatures and extreme drought conditions in recent decades. Moreover, it is expected that more frequent and intense droughts will occur in the future, which might affect population distribution and numbers. Nevertheless, the sexual dimorphism in the physiological and biochemical characteristics of male and female individuals of L. cubeba have been rarely reported. In this study, physiological and biochemical indexes were assessed in female and male L. cubeba under natural drought stress conditions to clarify sex dimorphism response to drought stress. The aims of this study are as follows: (1) to determine the physiological and biochemical differences between male and female L. cubeba in response to natural drought stress and (2) to evaluate whether male and female L. cubeba plants exhibit distinct response strategies to natural drought stress. This study will further deepen our understanding of sexually dimorphic responses to drought stress in L. cubeba.

2. Materials and Methods

2.1. Description of Site

This study was conducted in diverse test forests situated in Longshan Forest Farm in Anji County, Huzhou City, Zhejiang Province, China (30.8572° N, 119.7195° E), at an altitude of 65 m. The soil type used in this experiment site was red soil, and its pH was 5.69. Furthermore, its organic matter content was 7.75 g/kg, total N was 0.6 g/kg, total P was 0.35 g/kg, and exchangeable K was 44.63 g/kg. The average annual precipitation of the study is 800 mm, and its mean annual temperature is 16.58 °C. In the summer of 2022, Anji County had an extreme drought event, and the average rainfall for the months of May, June, July, and August in 2022 was 44.80% lower than that in 2021 (Figure 1). Additionally, the number of extremely dry days exceeded 30 days, setting a historical record. The rainfall and temperature data for 2021 and 2022 of the experimental site are shown in Figure 1.

2.2. Plant Material

The plant material used in this study is L. cebuba. This plant typically initiates flowering in March, with its peak bloom occurring by mid-March. Following the successful pollination of female flowers, fruit development commences. The key stage for the formation of essential oils and citral in the fruit occurs in July, and these phytochemical constituents undergo progressive stabilization during maturation processes, achieving compositional stability by August [36,37]. In the test forests, four cultivars (No. 6, No. 10, No. 21, and No. 24) of L. cebuba were planted in 2018 with a randomized complete block design, which comprised four distinct blocks to avoid position effects, each containing 40 trees. L. cubeba selections used in this study are shown in Table 1. They were planted with a spacing of 3 × 4 m. All trees underwent uniform horticultural management. On 16 August 2022, after a continuous drought period exceeding 30 days, 80 six-year-old trees per cultivar were selected for analysis, with consistent age and drought exposure criteria applied across all sampled cultivars. Every cultivar comprised 40 male and 40 female trees, with 10 individuals per gender randomly selected from each of four predefined blocks. Intact healthy leaves and root organs were systematically and randomly sampled from each tree for this study.
Soil moisture content (SWC) was determined with the oven-drying method using aluminum containers at 105 °C overnight on 16 August 2022, and the mass lost is expressed as a percentage of the dry weight. Three 20 m × 20 m quadrants were established following the diagonal sampling method. Within each quadrant, three male and female trees were randomly selected. For each tree, soil samples were collected from four directions (east, south, west, north) under the canopy projection from three depth layers (0–10 cm, 10–30 cm, and 30–60 cm). After mixing the 12 soil cores (3 individuals × 4 directions) to form 1 composite sample per depth, the samples were transferred to aluminum containers for soil moisture measurement. Soil moisture content on 20 August 2021 was measured using the same method and used as a control. The selection of 20 August 2021 as the control was based on the following reasons: first, a continuous rainfall event (with a total precipitation of 104 mm) occurred from 11 to 18 August 2021, and the measurement of soil moisture content was conducted one day after rainfall cessation (20 August) to capture stabilized post-precipitation conditions; secondly, August represents both the vigorous growth stage of L. cubeba and the critical period when essential oil accumulation in fruits stabilizes. Measurements were selected from the same calendar month across years to minimize confounding effects due to environmental variables. The soil moisture content data are shown in Table 2. The field capacity of the experiment site was measured using the cutting ring method proposed by Özcan [38]. The field capacity of the experimental site was 31.5%.

2.3. Determination of Indicators of Oxidative Metabolism

The levels of malondialdehyde (MDA), hydrogen peroxide (H2O2), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) were determined using commercial kits (Suzhou Grace Biotechnology Co. Ltd., Suzhou, China). In brief, 0.1 g leaves or roots were ground into a slurry using 1 mL of extraction buffer, followed by centrifugation at 1610 g for 10 min at 4 °C for H2O2, CAT, POD, and SOD, and 716 g rpm for 10 min at 25 °C for MDA. The supernatant was placed on ice for testing and collected to determine enzyme activities using the corresponding assay kits. The activities of MDA, H2O2, CAT, POD, and SOD were evaluated using ELISA kits (M0106, M0107, M0103, M0105, and M0102, respectively).

2.4. Determination of Proline and Soluble Sugar

Proline (Pro) concentration was determined utilizing a Pro assay kit from Suzhou Grace Biotechnology Co. Ltd. (Suzhou, China). A 0.1 g sample from both leaf and root organs was homogenized in 1 mL of extraction buffer to create a slurry, which was then placed in a water bath for 10 min to enhance the extraction process. Following this, the mixture was centrifugated at 1118 g for 10 min at a temperature of 25 °C, after which the supernatant was examined according to the guidelines provided in the kit (M0108). For the measurement of soluble sugar (SS), a soluble sugar assay kit from Suzhou Grace Biotechnology Co. Ltd. (Suzhou, China) was employed. One gram of leaf and root material was combined with 5 mL of 80% ethanol to form a slurry. This mixture was incubated in a water bath at 80 °C for 40 min before being centrifuged at 716 g for 10 min at 25 °C. The resulting supernatant was then analyzed following the kit’s specifications (M1503) [25].

2.5. Analysis of Photosynthetic Pigments

Sampled leaves were collected from canopy positions with uniform irradiance without shaded regions. Leaves at identical developmental stages were sampled from 9:00 to 11:00 AM. Chlorophyll a (chl a), chlorophyll b (chl b), and carotenoids were obtained using 80% (v/v) acetone according to the methods of Metzner [39]. The absorption of chl a, chl b, and carotenoids was determined at 663, 646, and 470 nm [33].

2.6. Phytohormone Content Quantification

Two endogenous phytohormones, abscisic acid (ABA) and jasmonic acid (JA), were quantified in the leaves and roots of L. cubeba using high-performance liquid chromatography [40,41,42]. Initially, the plant material was processed by grinding it into a slurry with 80% methanol, which was pre-cooled in an ice bath. This slurry was subsequently wrapped in plastic and stored in a refrigerator overnight at 4 °C. Subsequently, the mixture was centrifuged at 8000 g for 10 min at 4 °C to separate the supernatant. The residual material was then re-suspended in 8 mL of pre-cooled 80% methanol, and the supernatant was collected again through another round of centrifugation. Subsequently, samples were extracted and decolorized with petroleum ether three times. The pH of the aqueous phase was adjusted to 2–3 with 1 mol/L citric acid solution, followed by extraction with ethyl acetate twice. The resulting upper organic phase was evaporated under nitrogen before being re-dissolved and mixed with methanol. Before the HPLC analysis, the samples were filtered through a 0.45 µm microporous nylon membrane. For the analysis, a Waters 2695 High-Performance Liquid Chromatograph (Waters Corporation, Milford, MA, USA) was used. The detection wavelength was set to 254 nm for ABA and 210 nm for JA. The chromatographic column employed was Compass C18(2) (250 mm × 4.6 mm, 5 µm, Waters, USA). The mobile phase for ABA comprised 1% acetic acid in methanol (A) and 0.1% acetic acid in acetonitrile (B), while 0.1% phosphoric acid in acetonitrile was utilized for JA. A flow rate of 1 mL/min was maintained with an injection volume of 10 µL, and the detection temperature was established at room temperature. The standards used for the determination of ABA and JA were abscisic acid (Shanghai yuanye Bio-Technology Co., Ltd., Shanghai, China, B50724, 100 mg) and methyl jasmonate (Shanghai yuanye Bio-Technology Co., Ltd., V31058, 5 g) dissolved in methanol. Their R2 values were 0.9991 and 0.9986, respectively.

2.7. Dehydrogenase Activity Measurement

The dehydrogenase (DHA) activity was measured using dehydrogenase activity assay kits (Suzhou Grace Biotechnology Co., Ltd., Suzhou, China) according to the kit’s instructions. In brief, 0.1 g roots were ground into a slurry using 1 mL of extraction buffer, followed by centrifugation at 10,000 rpm for 5 min at 25 °C. The supernatant was collected to determine activities using the ELISA kit (M1027).

2.8. Statistical Analyses

All assays were performed in triplicate. The data were expressed as the mean ± the standard error of the mean from independent replicates. The statistical analyses were conducted using IBM SPSS Statistics version 19 (IBM Corp., Armonk, NY, USA). Prior to the analyses, the data were assessed to verify normality and the homogeneity of variances. To identify individual differences in means, Student’s t-test was employed, with a significance threshold set at p < 0.05. To evaluate the effects of sex, organ, variety, and their interactions on various parameters, a three-way analysis of variance (ANOVA) was performed. Furthermore, a two-way ANOVA was utilized to assess the influence of variety, sex, and their interaction on DHA activity, carotenoids, and total chlorophyll levels. A principal component analysis (PCA) was conducted to examine correlations among the studied traits with R 4.3.2 (R Foundation for Statistical Computing, Vienna, Austria).

3. Results

3.1. Sexual Differences in Oxidative Stress and Antioxidants

Under prolonged drought stress, a significant difference in MDA contents between sexes was observed in the four varieties, except for the leaves of variety 10. The MDA contents in all four female varieties, both in leaves and roots, were higher than those in males, except the roots of varieties 6 and 21 (Figure 2a). The average MDA content in the leaves of female plants across the four varieties increased by 26.42% compared that of male plants. The MDA contents in the leaves of the four varieties were significantly higher than those in the roots for both sexes. Additionally, the hydrogen peroxide contents in the leaves and roots of the three female varieties (6, 21 and 24) were significantly lower than those in males when exposed to prolonged drought (Figure 2b). However, no significant differences in hydrogen peroxide contents between sexes were noted in variety 10. Except the roots of variety 21, significantly higher hydrogen peroxide contents were observed in the roots compared to the leaves under drought stress. In addition, there were significant interactive effects of sex, variety, and organ type on MDA (p < 0.001) and hydrogen peroxide (p = 0.003) contents (Table 3).
To explain the observed differences in ROS accumulation between sexes in L. cubeba, we analyzed the difference in ROS-scavenging enzyme activity under drought stress. Results showed that prolonged drought stress led to significant differences in CAT and POD activities between males and females, with CAT and POD activities being significantly higher in females than in males in both leaves and root across all four varieties (Figure 2d,e). The average CAT and POD activities in the leaves and roots of female plants across the four varieties exhibited a significant increase of 104.28% and 51.17% and 23.67% and 174.57%, respectively, compared to those in males. Similarly, the root SOD activities in females were significantly higher than those in males for varieties 6 and 10 under drought stress. However, no significant differences in SOD activities were observed between males and females in the leaves of three varieties under prolonged drought stress (Figure 2f). Additionally, CAT and POD contents in the leaves of all four varieties were markedly higher than those in the roots. To assess the gender differences in root activity under drought conditions, we measured dehydrogenase (DHA) activity. The reduction of 2,3,5-triphenyl tetrazolium chloride (TTC) induced by dehydrogenase in plant roots serves as an indicator of DHA activity reflecting root activity. The results demonstrated that when L. cubeba was subjected to prolonged drought stress, the DHA activities in females were significantly higher than those in males across all varieties (Figure 2c), and the average DHA activity in female plants across the four varieties increased by 42.14% compared to that of male plants. Furthermore, significant interactive effects of sex, variety, and organ type on DHA, CAT, POD, and SOD activities were identified (Table 3 and Table 4).

3.2. Sexual Differences in Photosynthetic Pigments

When the four varieties were exposed to prolonged drought, significant differences in total Chl and carotenoid levels between sexes were observed. The total Chl levels in females across the four varieties exhibited significant increases of 17.06%, 60.22%, 34.98%, and 5.65%, respectively, compared to those of males. Similarly, the carotenoid levels in females across the four varieties increased by 69.30%, 75.48%, 19.97%, and 10.62%, respectively, compared to those in males (Table 5). Additionally, a notable interactive effect of both sex and variety was observed on carotenoid concentrations (p < 0.001) and total chlorophyll (p < 0.001) (refer to Table 4).

3.3. Sexual Differences in Proline and Soluble Sugar Contents

The prolonged drought stress resulted in a notable variation in Pro content between sexes across all varieties, except in the leaves of variety 10, where females exhibited significantly higher Pro content than males (Figure 3a). The average Pro contents in both the leaves and roots of female plants across the four varieties were significantly increased by 33.85% and 41.95%, respectively, compared to those in males. A similar trend was observed for soluble sugar content, except in the leaves of varieties 10 and 21 (Figure 3b). Compared to roots, the content of soluble sugar was notably greater in leaves under drought stress. Furthermore, a significant interactive effect was observed involving sex, organ, and variety on both Pro (p < 0.001) and soluble sugar (p < 0.001) contents (Table 3).

3.4. Sexual Differences in ABA and JA

The prolonged drought stress conditions induced a differential response in JA and ABA levels between the sexes of L. cubeba. As illustrated in Figure 4a, females subjected to drought exhibited significantly higher JA content compared to males, both in leaves and roots. Notably, in the leaves of variety 21, the JA content in females was 2.94 times higher than that in males. Similarly, ABA levels in females were significantly elevated in both leaves and roots compared to males, except in variety 21 and the roots of variety 10 under drought stress (Figure 4b). Particularly, in variety 6, the ABA content in females was found to be 4.72 times higher than that in males. Furthermore, a notable interactive effect involving sex, organ, and variety on JA (p < 0.001) and ABA (p < 0.001) contents was recorded (Table 3).

3.5. Relationships Among All Traits in Each Sex Under Drought Stress

Principal component analysis (PCA) illustrated a distinct delineation regarding the combinations of traits under drought stress (Figure 5, Figure 6 and Figure 7). The two-component PCA models accounted for 62.35%, 77.26%, and 69.03% of the total variance observed in the overall population, female plants, and male plants, respectively (Figure 5, Figure 6 and Figure 7). Under drought stress conditions, a clear separation was observed between females and males along the first and second PCA axes (Figure 5). Furthermore, PC1 was significantly influenced by JA, POD, and MDA, and PC2 was strongly influenced by H2O2, Car, ABA, and Pro.

4. Discussion

4.1. Drought Stress Significantly Affects Sexual Dimorphism in Physiological Responses of L. cubeba

Plant sexual dimorphism refers to differences in traits between male and female individuals, reflecting the distinct reproductive functions driven by sex-specific optima adaptations [43]. In dioecious species, these trait variations predominantly result from the interplay between sexual selection and natural selection concerning resource distribution, with the intensity and orientation of selection differing among species [44]. Recent research has revealed that abiotic factors also play a crucial role in modifying sexual dimorphism across the plant lifecycle, applicable to both animal-pollinated and wind-pollinated species [34,43,45,46,47,48,49]. Meanwhile, some studies have demonstrated sexual dimorphism in biochemical, physiological, and structural responses to drought stress in various plants [12,46,50].
In our study, the mean soil moisture content was 15.35% under the sustained drought conditions of August 2022 (Table 2), accounting for 48.73% of the field capacity of the soil at the experimental site. According to Liu et al. (2010), this value is categorized as a moderate-to-severe water deficit [51]. This mean soil moisture content represents a 32.08% reduction compared to the soil moisture content of 22.60% recorded in August of 2021, which received adequate precipitation (Table 2). Therefore, the soil moisture values of August 2022 support the notion that the sampled trees at the experimental site effectively sustained drought stress. Our study indicates that natural drought stress significantly influences the sexual dimorphism in the physiological index, including MDA, H2O2, and antioxidant enzyme activities (SOD, CAT, POD), photosynthetic pigments (total Chl and carotenoid), JA and ABA, and predominant osmotic regulators (proline and soluble sugar) of L. cubeba. This is further corroborated by the notable interactive effect of sex, variety, and organ on the measured indexes (see Table 3 and Table 4). These sexual differences may explain the female-biased sex ratio observed in the natural populations of L. cubeba, with a female-to-male ratio of 1:0.6–0.8 [52]. Furthermore, female and male L. cubeba growing under the same dried environment exhibited notable sex differences in physiological responses, suggesting that the requirements for growth and reproduction in males and females could be more influenced by sex specialization than by environmental factors [34].
Female L. cubeba demonstrated higher tolerance and more efficient mechanisms compared to males, and this observation aligns with the earlier observations made for L. cubeba subjected to waterlogging stress [25]. However, this finding contradicts conventional theories suggesting environmental stress produces more significant adversely effects in females, leading to higher reproductive costs, particularly in comparison to poplar [12]. This difference might stem from the influence of sex, species, and environmental factors on reproductive investments [53,54]. Additionally, female L. cubeba demonstrates enhanced tolerance to drought compared to male L. cubeba, potentially offsetting the reproductive costs incurred by females [44,55]. During the months of May to August, female L. cubeba can generate a large number of fruits, and July is the key period for the formation of essential oils and citral in fruits [36,37], a time that corresponds with an extended drought period. Increased drought tolerance in females is advantageous for sustaining the population and maintaining stable yields.

4.2. Females Exhibited More Effective Protective Mechanisms than Males Under Drought Condition

MDA and H2O2 are closely linked to membrane lipid peroxidation and oxidative damage [15]. In L. cubeba, the leaves of all female plants displayed higher MDA content than those of males under stress conditions, indicating that females experience more significant damage to their membrane structure. Conversely, the H2O2 levels in males among three varieties exceeded those found in females, suggesting that the stress-mediated generation of high H2O2 levels leads to a substantial ROS-induced oxidative burden, which negatively impacts membrane function in males [25].
Earlier research had demonstrated that in response to environmental stress, the regulation of osmotic regulators, the antioxidant enzyme system, and the phytohormones are altered as a protection mechanism to cope with stress [23,52,56]. The activities of CAT and POD were elevated in females in comparison to males when exposed to drought stress (Figure 2d,e), which suggested that although the MDA levels in females exceed those of males, females exhibit a better ability to regulate ROS homeostasis by enhancing the activities of the CAT and POD antioxidant enzymes, thereby protecting against ROS-related damage under drought stress [17]. Furthermore, female plants of L. cubeba displayed higher concentrations of carotenoids and total chlorophyll when exposed to drought stress (Table 5), which might be attributed to the significant role of elevated activities of antioxidative enzymes in alleviating ROS damage by scavenging free radicals, thereby rendering them less reactive. In our experiment, all sampled leaves were collected from canopy positions with uniform irradiance devoid of shaded regions, demonstrating that the differences in chlorophyll and carotenoid contents were attributable to sex rather than light intensity. Female plants of L. cubeba possess higher concentrations of carotenoids and total chlorophyll when exposed to drought stress, suggesting that the chlorophyll degradation rate in females was lower than that of males under natural drought, which also enhanced the tolerance of female plants to drought stress [57].
Additionally, females exhibited an effective osmotic adjustment strategy to withstand the challenges posed by drought. Proline and soluble sugar, as the primary osmotic regulators, could accumulate rapidly in response to cellular stress, thereby protecting cellular molecules and structures against dehydration [58]. In L. cubeba, females demonstrated higher levels of soluble sugar and proline than males under drought stress, significantly impacting drought tolerance (Figure 4). The increase in soluble sugar levels also contributed to the maintenance of turgor pressure [59,60]. In this study, the leaves exhibited significantly higher soluble sugar content than roots across all varieties. This phenomenon aids leaves in replenishing their moisture in a dry environment by passively absorbing water, compensating for the root system’s long-term lack of oxygen and its inability to transport sufficient moisture from the air to various parts.
ABA signaling, facilitated by phosphorylation through various downstream kinases, regulates plasma membrane transporters by closing the stomata, thereby conserving water for survival and enhancing water-use efficiency [56]. Kong demonstrated that PtoERF15 directly regulated PtoMYC2b, a core transcription factor in JA signal transduction, which, in turn, influences vascular development to facilitate drought adaptation in Populus tomentosa [23]. In this study, ABA levels in the leaves of female plants were notably greater than those observed in male plants in varieties 6, 10, and 24. Additionally, the JA content in all female plants was significantly elevated compared to that in male plants when exposed to drought stress (Figure 4). The higher concentrations of ABA and JA in female plants aid in stomatal closure and facilitate ROS scavenging in response to drought stress. The significant differences in ABA and JA contents among varieties indicate substantial intraspecific variation, suggesting that variety selection should be comprehensively prioritized during the screening of drought-resistant resources. Furthermore, our results indicate that the ABA levels in leaves were significantly higher than those in roots, suggesting a long-distance transport mechanism of ABA from roots to leaves to promote stomatal closure [61,62]. However, this hypothesis requires confirmation through future research.
Sex-specific drought adaptation strategies have been documented in dioecious species. In Juniperus thurifera, female plant trunks showed xylem anatomical traits related to higher hydraulic efficiency over safety, whereas male plants adapted a more conservative strategy [46]. In this study, females exhibited a more effective protective mechanism than males under natural drought conditions. This is likely due to the elevated ABA and JA levels, which induce stomatal closure to maintain water status. Additionally, antioxidant and osmotic adjustment mechanisms mitigate the downstream consequences of oxidative damage by suppressing ROS accumulation and pigment degradation. Notably, all plant material in our research was sampled in August, coinciding with the peak period of essential oil accumulation in female plants. However, a specific phenological stage may influence both primary and secondary metabolism. Sampling at multiple developmental stages should be carried out in the future, which will help reveal the interaction between genotypes and phenology. In the future, we will focus on the physiological and transcriptomic differences between drought stress and normal conditions to further clarify the sexual differences in the response strategies and mechanisms under drought stress in L. cubeba.

5. Conclusions

This study demonstrated that natural drought stress significantly influences the physiological and biochemical differences between female and male L. cubeba. It revealed that although female L. cubeba plants exhibited higher membrane lipid peroxidation than male plants, they possessed more efficient antioxidant systems, enhanced osmotic adjustment capacities, superior phytohormone contents, and higher levels of total chlorophyll and carotenoids under drought stress. This suggests that female plants may endure lower levels of drought stress compared to male plants due to a flexible balancing strategy involving osmotic adjustment, the antioxidant system, and phytohormonal regulation. Our research enhances our comprehension of sexually dimorphic responses to environmental challenges such as drought. Due to the sexual dimorphism of L. cubeba under drought, more severe shifts in the sex ratio of L. cubeba forests may occur under future climate change scenarios with aggravated aridity. In future research, we will further explore the different strategies and molecular mechanisms of male and female L. cubeba plants in response to drought stress.

Author Contributions

Conceptualization, Y.W. and M.G.; software, M.G.; validation, M.G., Y.C. and Y.Z.; formal analysis, M.G.; investigation, M.G., Y.C. and Y.Z.; resources, M.G.; data curation, Y.W. and M.G.; writing—original draft preparation, Y.W. and M.G.; writing—review and editing, M.G., Y.C. and Y.W.; visualization, M.G.; funding acquisition, M.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Fundamental Research Funds of CAF [Grant No. CAFYBB2020QA002] and Zhejiang Science and Technology Major Program on Agricultural New Variety Breeding [Grant No. 2021C02070-3].

Data Availability Statement

The data are contained within this article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The rainfall and temperature data for 2021 to 2022 of the experimental site: (a) the rainfall of the experimental site for 2021 to 2022; (b) the mean high temperature of the experimental site for 2021 to 2022; (c) the extreme high temperature of the experimental site for 2021 to 2022.
Figure 1. The rainfall and temperature data for 2021 to 2022 of the experimental site: (a) the rainfall of the experimental site for 2021 to 2022; (b) the mean high temperature of the experimental site for 2021 to 2022; (c) the extreme high temperature of the experimental site for 2021 to 2022.
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Figure 2. The MDA content and DHA, CAT, POD, and SOD activities of different sexes of four L. cubeba varieties under drought stress: (a) the MDA content of different sexes of four L. cubeba varieties under drought stress; (b) the H2O2 content of different sexes of four L. cubeba varieties under drought stress; (c) the DHA activities of different sexes of four L. cubeba varieties under drought stress; (d) the CAT activities of different sexes of four L. cubeba varieties under drought stress; (e) the POD activities of different sexes of four L. cubeba varieties under drought stress; (f) the SOD activities of different sexes of four L. cubeba varieties under drought stress. The lowercase letters “a” and “b” indicate significant differences between sexes, and the lowercase letters “x” and “y” indicate significant differences between organs (p < 0.05). Identical letters indicate the absence of significant differences. Error bars show standard errors.
Figure 2. The MDA content and DHA, CAT, POD, and SOD activities of different sexes of four L. cubeba varieties under drought stress: (a) the MDA content of different sexes of four L. cubeba varieties under drought stress; (b) the H2O2 content of different sexes of four L. cubeba varieties under drought stress; (c) the DHA activities of different sexes of four L. cubeba varieties under drought stress; (d) the CAT activities of different sexes of four L. cubeba varieties under drought stress; (e) the POD activities of different sexes of four L. cubeba varieties under drought stress; (f) the SOD activities of different sexes of four L. cubeba varieties under drought stress. The lowercase letters “a” and “b” indicate significant differences between sexes, and the lowercase letters “x” and “y” indicate significant differences between organs (p < 0.05). Identical letters indicate the absence of significant differences. Error bars show standard errors.
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Figure 3. Pro and soluble sugar contents of four L. cubeba varieties with varying sexes under drought stress: (a) pro contents of four L. cubeba varieties with varying sexes under drought stress; (b) soluble sugar contents of four L. cubeba varieties with varying sexes under drought stress. Statistical analyses are illustrated in Figure 2.
Figure 3. Pro and soluble sugar contents of four L. cubeba varieties with varying sexes under drought stress: (a) pro contents of four L. cubeba varieties with varying sexes under drought stress; (b) soluble sugar contents of four L. cubeba varieties with varying sexes under drought stress. Statistical analyses are illustrated in Figure 2.
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Figure 4. JA and ABA contents of four L. cubeba varieties with varying sexes under drought stress: (a) JA contents of four L. cubeba varieties with varying sexes under drought stress; (b) ABA contents of four L. cubeba varieties with varying sexes under drought stress. Statistical analyses are illustrated in Figure 2.
Figure 4. JA and ABA contents of four L. cubeba varieties with varying sexes under drought stress: (a) JA contents of four L. cubeba varieties with varying sexes under drought stress; (b) ABA contents of four L. cubeba varieties with varying sexes under drought stress. Statistical analyses are illustrated in Figure 2.
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Figure 5. The principal component analysis (PCA) was conducted on all analyzed traits in the total L. cubeba population exposed to drought stress. Colored symbols represent females, and uncolored symbols denote males. The rhombus, square, triangle, and circle indicate varieties 6, 10, 21, and 24, respectively. MDA, malondialdehyde; H2O2, hydrogen peroxide; CAT, catalase; SOD, superoxide dismutase; POD, peroxidase; Pro, proline; SS, soluble sugar; Chl, chlorophyll; Car, carotenoids; DHA, dehydrogenase; ABA, abscisic acid; JA, jasmonic acid.
Figure 5. The principal component analysis (PCA) was conducted on all analyzed traits in the total L. cubeba population exposed to drought stress. Colored symbols represent females, and uncolored symbols denote males. The rhombus, square, triangle, and circle indicate varieties 6, 10, 21, and 24, respectively. MDA, malondialdehyde; H2O2, hydrogen peroxide; CAT, catalase; SOD, superoxide dismutase; POD, peroxidase; Pro, proline; SS, soluble sugar; Chl, chlorophyll; Car, carotenoids; DHA, dehydrogenase; ABA, abscisic acid; JA, jasmonic acid.
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Figure 6. The principal component analysis (PCA) was conducted on all analyzed traits of female L. cubeba exposed to drought stress. MDA, malondialdehyde; H2O2, hydrogen peroxide; CAT, catalase; SOD, superoxide dismutase; POD, peroxidase; Pro, proline; SS, soluble sugar; Chl, chlorophyll; Car, carotenoids; DHA, dehydrogenase; ABA, abscisic acid; JA, jasmonic acid.
Figure 6. The principal component analysis (PCA) was conducted on all analyzed traits of female L. cubeba exposed to drought stress. MDA, malondialdehyde; H2O2, hydrogen peroxide; CAT, catalase; SOD, superoxide dismutase; POD, peroxidase; Pro, proline; SS, soluble sugar; Chl, chlorophyll; Car, carotenoids; DHA, dehydrogenase; ABA, abscisic acid; JA, jasmonic acid.
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Figure 7. Principal component analysis (PCA) was conducted on all analyzed traits of male L. cubeba exposed to drought stress. MDA, malondialdehyde; H2O2, hydrogen peroxide; CAT, catalase; SOD, superoxide dismutase; POD, peroxidase; Pro, proline; SS, soluble sugar; Chl, chlorophyll; Car, carotenoids; DHA, dehydrogenase; ABA, abscisic acid; JA, jasmonic acid.
Figure 7. Principal component analysis (PCA) was conducted on all analyzed traits of male L. cubeba exposed to drought stress. MDA, malondialdehyde; H2O2, hydrogen peroxide; CAT, catalase; SOD, superoxide dismutase; POD, peroxidase; Pro, proline; SS, soluble sugar; Chl, chlorophyll; Car, carotenoids; DHA, dehydrogenase; ABA, abscisic acid; JA, jasmonic acid.
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Table 1. Source of four cultivars.
Table 1. Source of four cultivars.
Litsea cubeba Selections Used in This StudyOrigin
6Bijie county, Guizhou Province, China
10Lingyun county, Guangxi Province, China
21Yuexi county, Anhui Province, China
24Jianou county, Fujian Province, China
Table 2. Soil moisture content data.
Table 2. Soil moisture content data.
Plant SexSoil Layer Depth (cm)SWC (%)
Under Drought Stress
(16 August 2022)		Non-Stressed Levels (20 August 2021)
Female>0~1013.4 ± 0.220.4 ± 0.3
>10~3015.2 ± 0.222.2 ± 0.4
>30~6017.3 ± 0.324.5 ± 0.2
Male>0~1013.2 ± 0.121.1 ± 0.2
>10~3015.6 ± 0.322.6 ± 0.1
>30~6017.4 ± 0.424.8 ± 0.3
Mean 15.35 ± 0.222.6 ± 0.2
Table 3. F values of three-way ANOVA for parameters of L. cubeba under drought stress.
Table 3. F values of three-way ANOVA for parameters of L. cubeba under drought stress.
VariableSex
(S)		Variety (V)Organ (O)S × V × OS × VV × OS × O
MDA518.680 ***17.012 ***15711.770 ***36.475 ***19.945 ***28.511 ***264.042 ***
H2O22391.170 ***325.167 ***4240.718 ***333.294 ***736.063 ***175.849 ***789.521 ***
SOD77.755 ***136.095 ***152.126 ***8.986 ***2.422 ***130.586 ***24.882 ***
CAT513.311 ***39.791 ***2344.433 ***43.532 ***21.162104.328 ***139.786 ***
POD377.157 ***690.778 ***3043.903 ***11.217 ***4.204 *233.945 ***7.389 *
JA6606.920 ***712.558 ***1114.566 ***1030.721 ***371.936 ***542.481 ***346.581 ***
ABA1244.458 ***910.037 ***10909.375 ***214.865 ***360.426 ***941.893 ***1304.941 ***
SS6.727 ***18.884 ***3573.972 ***43.561 ***35.228 ***53.506 ***12.840 **
Pro518.735 ***35.815 ***3.142 ***45.615 ***23.108 ***254.150 ***5.417 *
Note: ***, **, and * indicate significant differences at the p < 0.001, p < 0.01, and p < 0.05 level, respectively.
Table 4. F values of two-way ANOVA for parameters of L. cubeba under drought stress.
Table 4. F values of two-way ANOVA for parameters of L. cubeba under drought stress.
VariableSex (S)Variety (V)S × V
DHA3784.008 ***1712.571 ***144.415 ***
Total chlorophyll4548.496 ***1041.385 ***528.176 ***
Carotenoids5509.921 ***1370.861 ***523.607 ***
Note: *** indicates significant differences at the p < 0.001 level.
Table 5. Total chlorophyll levels and carotenoid levels in four L. cubeba varieties with varying sexes subjected to drought stress.
Table 5. Total chlorophyll levels and carotenoid levels in four L. cubeba varieties with varying sexes subjected to drought stress.
VariablesTotal Chl (mg g−1 FW)Carotenoid (µg g−1 FW)
FemaleMaleFemaleMale
62.97 ± 0.02 a2.54 ± 0.02 b342.68 ± 2.68 a202.40 ± 1.25 b
102.90 ± 0.01 a1.81 ± 0.01 b376.71 ± 1.19 a214.67 ± 1.46 b
213.46 ± 0.02 a2.56 ± 0.02 b417.94 ± 3.11 a348.39 ± 1.47 b
242.49 ± 0.01 a2.35 ± 0.01 b301.37 ± 0.45 a272.45 ± 2.20 b
Note: The lowercase letters “a” and “b” indicates significant differences between sexes (p < 0.05).
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Gao, M.; Zhao, Y.; Chen, Y.; Wang, Y. Drought Stress Drives Sex-Specific Physiological and Biochemical Differences in Female and Male Litsea cubeba. Horticulturae 2025, 11, 594. https://doi.org/10.3390/horticulturae11060594

AMA Style

Gao M, Zhao Y, Chen Y, Wang Y. Drought Stress Drives Sex-Specific Physiological and Biochemical Differences in Female and Male Litsea cubeba. Horticulturae. 2025; 11(6):594. https://doi.org/10.3390/horticulturae11060594

Chicago/Turabian Style

Gao, Ming, Yunxiao Zhao, Yicun Chen, and Yangdong Wang. 2025. "Drought Stress Drives Sex-Specific Physiological and Biochemical Differences in Female and Male Litsea cubeba" Horticulturae 11, no. 6: 594. https://doi.org/10.3390/horticulturae11060594

APA Style

Gao, M., Zhao, Y., Chen, Y., & Wang, Y. (2025). Drought Stress Drives Sex-Specific Physiological and Biochemical Differences in Female and Male Litsea cubeba. Horticulturae, 11(6), 594. https://doi.org/10.3390/horticulturae11060594

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