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Article

Exogenous Spermidine Alleviated Waterlogging Damages in Two Varieties of Camellia oleifera

by
El-Hadji Malick Cisse
1,2,3,†,
Jin-Fu Huang
1,†,
Da-Dong Li
1,2,3,
Ling-Feng Miao
1,2,
Li-Shan Xiang
1,2 and
Fan Yang
1,2,*
1
School of Ecological and Environmental Sciences, Hainan University, Haikou 570228, China
2
Key Laboratory of Agro-Forestry Environmental Processes and Ecological Regulation of Hainan Province, Center for Eco-Environmental Restoration Engineering of Hainan Province, Haikou 570228, China
3
School of Life Sciences, Hainan University, Haikou 570228, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2023, 14(1), 91; https://doi.org/10.3390/f14010091
Submission received: 29 November 2022 / Revised: 20 December 2022 / Accepted: 25 December 2022 / Published: 3 January 2023
(This article belongs to the Special Issue Physiological and Molecular Adaptable Strategies to Abiotic Stress in Forest Trees)
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Abstract

:
The potential management of waterlogging-damaged plants can be through the promotion of aliphatic polyamine accumulation, such as spermidine (SPD), in non-accumulator and accumulator species under stress. Camellia oleifera, commonly called tea oil, is an evergreen shrub confronting waterlogged soils in Hainan forest plantations during the pluvial season. As far as we know, few studies focused on the responses of C. oleifera to abiotic stresses, such as waterlogging (WL), and the involvement of SPD in WL tolerance remains unclear. Therefore, two cultivars of C. oleifera (CoH1 and CoH2) were subjected to WL and exogenous SPD to shed light on the role of SPD on WL tolerance via the morphological and physio-biochemical responses of C. oleifera under stress. The results showed that the two varieties of C. oleifera were sensitive to WL stress, and spraying SPD enhanced WL tolerance via root activities, photosynthesis, redox-homeostasis, antioxidant machinery, and compatible solute components. Thus, exogenous SPD significantly reduced the damages caused by WL in C. oleifera seedlings. Moreover, the alternative oxidase (AOX) protein content was down regulated by WL in both varieties of C. oleifera, whereas exogenous SPD enhanced the AOX protein under stress. The two varieties of C. oleifera generally had similar morphological and physiological responses to WL. However, CoH2 demonstrated better photosynthesis compared to CoH1. The results of the present study provide a significant outlook to improve the accumulation of SPD in trees under abiotic stress, particularly via genome editing techniques.

1. Introduction

Global climate change has generated significant changes in seasonal precipitation around the world. The prevalence of abiotic stresses, such as waterlogging (WL), or flooding, is expected to increase in many regions, including Southeast Asia [1,2]. The degradation of productive capacity in many lands induced by WL is a global obstacle for agriculture, given that it afflicts approximately one billion hectares in at least sixty countries [3]. In plants, WL can significantly increase anaerobic respiration, which can cause a significant accumulation of harmful substances in the soil. Indeed, it changes soil properties, which reshape plant nutrient availability and results in increased solubility of micronutrients, leading to the deterioration of the rhizosphere environment that perilously affects plant growth and development [4]. Under WL stress, plant growth faces physio-biochemical modifications, such as protein and photosynthesis degradation, reduction in antioxidant enzymatic activities, increased lipid peroxidation, and reactive oxygen species over-accumulation [5]. Indeed, WL inhibits plant growth via a generation of oxygen deficiency or a total depletion in the level of oxygen in the soil [1]. WL-induced high plant mortality reduced the leaf area, dry weight, plant height, and total carbohydrates in Zea mays [6]. It was also reported that WL reduced photosynthesis, dry matter accumulation, and induced stomatal closure and chlorosis in Glycine max [7,8]. To counteract the adverse effects of WL in plant photosynthesis, reactive oxygen species (ROS) accumulation, and antioxidant systems, various exogenous substances such as Ca2+, gamma-aminobutyric acid, or melatonin have been used [9].
The application of exogenous substances, such as plant hormones or osmoprotectants, is one of the effective ways to improve WL tolerance in plants [9,10]. The aliphatic polyamines (PAs) such as putrescine, spermidine (SPD), and spermine have been regarded as a new class of growth substances in improving plant stress resistance ability. Moreover, the implication of these PAs in numerous physiological processes, including plant growth and development, and the mitigation of abiotic stress tolerance in various plant species has been proved [11]. Generally, in plants, PAs are present in three forms: tetraamine spermine, diamine putrescine, and triamine spermidine. Several studies have shown the antioxidant system’s crucial role in plants under submerged conditions [12]. It has been reported that PAs are considered cell membranes, nucleic acids, and protein stabilizers. Indeed, PAs such as SPD could play a prominent role in detoxifying activated oxygen species [13]. Further, some studies have used exogenous SPD to alleviate stress damage in plants due to its ability to inhibit lipid peroxidation and electrolyte leakage (EL) and increase antioxidant enzymatic activities and the activities of enzymes involved in PAs metabolism [14]. It was reported that exogenous SPD enhanced photosynthesis and maintained antioxidant activities and water status under WL in the Welsh onion (Allium fistulosum) [15]. Moreover, the net photosynthetic rate (Pn) was strongly enhanced by exogenous SPD in Cucumis sativus under hypoxic stress [16]. Under salt stress, Kentucky bluegrass exposed to SPD showed a decrease in Na+ and electrolyte leakage (EL) and an increase in proline, endogenous SPD, the activities of ornithine decarboxylase, and adenosylmethionine decarboxylase [17]. As ubiquitous biogenic amines, PAs have prominent functions in plant environmental stress tolerance. In addition, it is of great interest to study the relationships between SPD and WL tolerance in tree species.
Camellia oleifera (tea oil) is a unique oil tree belonging to the genus Camellia of the Theaceae family. More than 4.4 million hectares in China are used to produce more than 2.4 million-ton fruit of C. oleifera in 2019 [18]. Hainan Island is highly vulnerable to typhoons and heavy rainfall during the rainy season, and these natural catastrophes can provoke environmental stresses such as flooding or WL that perilously affect plant growth and development in forest plantations. C. oleifera is mainly distributed in Hainan areas and is influenced by several environmental factors. Recently, to promote flooding watershed ecological restoration in China, a total of 57 plant species, including C. oleifera, were used to investigate growth rates and leaf phenotypes under WL stress. That study was set up to select the most resistant species to WL [19]. It revealed five categories following their tolerance to WL: excellent, good, ordinary, poor, and very poor, and C. oleifera was classified as an ordinary species. Furthermore, it was reported that plants with excellent or good WL tolerance promptly developed new leaves under stress, whereas species with ordinary or poor WL tolerance showed old leaves dropping under stress and were more sensitive to WL.
The role of SPD in plant cell homeostasis via ROS scavenging and the inhibition of lipid peroxidation to provide stress tolerance has been well-documented. However, its function in plants under WL remains elusive. We hypothesized that a spray of SPD in C. oleifera might boost the antioxidant defense system and reduce damages caused by WL. Thus, this present work aims to investigate how exogenous SPD could increase WL tolerance in C. oleifera seedlings via an evaluation of physio-biochemical parameters such as water status, growth traits, root activity, photosynthesis, redox homeostasis, and antioxidant machinery. Moreover, there are controversial reports about the effect of SPD on WL tolerance. Thus, the present study could shed light on the biochemical interactions between SPD and eco-physiological systems under submergence conditions. In addition, it has been reported that alternative oxidase (AOX) is likely involved in the WL tolerance of Citrullus lanatus [2]. Thus, the evaluation of the AOX protein in this present work could provide some insights into the involvement of SPD on the AOX pathway in improving WL tolerance.

2. Materials and Methods

2.1. Plant Material and Growth Conditions

In this experiment, two varieties of C. oleifera (2 years old) cultivated by Hainan Shanyou Science and Technology were used (Hailin1 and Hailin2, abbreviation CoH1 and CoH2). The plants of C. oleifera were pruned to an identical height (10 cm) for re-sprouting after they were transferred into pots (21 cm in diameter and 19 cm in height) containing red soil, coconut coir, and river sand (3:2:1, v/v/v) in a greenhouse at Hainan University (20°03′22.80″ N, 110°19′10.20″ E). From June to October, the average temperature from 8: 00 a.m. to 8:00 p.m. was 32.6 °C, and the average moisture was 72.9%. During the night (8:00 p.m. to 8:00 a.m.), the average temperature was 27.1 °C, and the average humidity was 88.2%. The weight of the soil substrate was 5.0 kg per pot, and the number of plants per pot was one seedling. The physical and chemical properties of the soil substrate were: maximal field capacity (52.99%), N (77 g/kg), P (0.64 g/kg), organic matter (58.01 g/kg), organic carbon (33.65 g/kg), and pH (6.23). The pruned seedlings were well-watered for two months (June-August). Then seedlings of C. oleifera with approximately the same height were chosen to screen the suitable concentration of SPD used in this present experiment. The levels of SPD employed (0.1 mM, 0.5 mM, 1 mM) were based on previous research work [14]. The following formula, U (Xi) = (Xi − Xmin)/Xmax − Xmin described in [20], was used to evaluate the suitable concentration of SPD that had the most positive impact on C. oleifera growth under WL (Table 1). U (Xi) represents the value of subordinate function analysis, Xi is the parameter’s value, Xmin is the minimum value of this parameter, and Xmax is the maximum value. Thus, 0.5 mM SPD was applied in the present study according to the morphological changes from Table 1.

2.2. Experimental Setup

After three months of growth (June to September), healthy seedlings (15–20 cm in height) were selected for the experiment. The entire experiment was set up in a complete, randomized design with two factors, including (i) Waterlogging, WL, and (ii) exogenous substances (SPD),and six replicates (4 seedlings each replicate). The seedlings of C. oleifera were half submerged, and the water surface was 5 cm higher than the soil surface. Treatments were designed as follows: (1) CK: well-watered; (2) WL: WL treatment; and (3) WL + SPD: WL treatment and SPD (0.5 mM). The SPD was sprayed at 6:00 p.m. every two days on the front and back of C. oleifera leaves and applied in the waterlogged soil at a range of 10 mL. The experiment ended when the seedlings appeared yellow with fallen leaves (40 d). Mature functional leaves below the top bud were selected to determine various morphological and physio-biochemical parameters.

2.3. Growth Analysis

The leaf number, plant height, and stem diameter were measured at the start and end of treatment, and their increments were calculated. The below-ground and above-ground fresh weights were measured at the end of treatment. Data were collected from at least eight samples in each treatment. The remaining leaves were harvested and kept at −80 °C for biochemical analysis.

2.4. Photosynthesis Analysis

The net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration (Trr), and water use efficiency (Wue) were measured on intact leaves with an LI-COR 6400 portable photosynthesis system (LI-COR Inc., Lincoln, NE, USA). Measurements were made on the youngest fully expanded leaf from 9:00 a.m. to 12:00 a.m., according to [21].
The leaf chlorophyll content (Chlo-a, Chlo-b, Caro, and T-Chlo) of the fully expanded leaves was measured after chlorophyll extraction with 85% acetone by the colorimetric method at absorbances of 663, 646, and 470 nm. The concentrations of photosynthetic pigment in C. oleifera leaf were calculated according to the equations described by [22].

2.5. Water Status

The dew point water potential (DWP) was estimated with a Dewpoint Potential Meter WP4C (Gene Company Ltd., Pullman, WA, USA). The electrolyte leakage (EL) was determined according to the method described in [23] with some modifications. Fully expanded leaves were cleaned with deionized water. Approximately 10 discs of the fresh leaves were incubated in test tubes containing 15 mL of deionized water. The initial conductivity (C1) was recorded after 2 h, and then samples were boiled for 30 min to determine final conductivity (C2). The EL percentage was calculated by the following formula: EL (%) = C1/C2 × 100.

2.6. Root Activity Measurement

Root activity was determined according to the TTC method described in [24]. Approximately 200 mg of roots were cleaned with deionized water and incubated at 37 °C for 3 h under dark conditions in 5 mL of a solution containing 0.4% TTC and the phosphate buffer. The reaction was stopped by adding 1 mL of the sulfuric acid solution. Then the samples were removed and placed in a mortar containing 5 mL of the ethyl acetate solution to extract the resulting red powder (formazan). The homogenate was transferred into a tube and centrifuged at 5000× g for 8 min. The optical density was read at 485 nm, and the root activity was expressed in µg·g−1·FW/h.

2.7. Proteins, Carbohydrates, and Proline Determination

The soluble protein content was determined by the Bradford method [25] using Coomassie Brilliant Blue G-250. About 2 mL of phosphate buffer (pH 7.8) was used as an extract solution, and approximately 100 mg of fresh leaf samples were homogenized with the phosphate buffer. After centrifugation, the supernatant was mixed with 1 mL of the reagent, and absorbance was read at 595 nm.
The carbohydrate content was determined according to the anthrone method described in [26]. Approximately 2 mL of the reagent (anthrone 0.2%) was reacted with 1 mL of the sample solution. The extraction solution was composed of 3% aqueous sulfosalicylic. The absorbance was recorded at 630 nm, and the concentrations of the carbohydrates were determined via a standard curve.
Proline content was determined based on the method described in [27]. About 200 mg of fresh leaf samples were homogenized into a liquid with 5 mL of 3% aqueous sulfosalicylic acid. The acid-acetic ninhydrin solution was used as a reagent. Approximately 1 mL of the homogenate was added to 1 mL of acid-acetic ninhydrin reagent, and then 1 mL of glacial acetic acid was added. After that, the mixture was incubated at 100 °C for 1 h, and the reaction was stopped by cooling the samples in ice water. Toluene was used to extract the chromophore-containing phase, and the absorbance was recorded at 520 nm.

2.8. Hydrogen Peroxide (H2O2), Hydroxyl Radical (•OH), and Malondialdehyde (MDA) Measurements

The concentration of H2O2 was determined according to [28]. Fresh leaf samples (200 mg) were ground in 0.1% trichloroacetic acid and then centrifuged at 12,000× g for 8 min at 4 °C. Approximately 1 mL of the supernatant was mixed with 1 mL of potassium iodide solution, and 1 mL of 10 mM of phosphate buffer solution was added. The absorbance was recorded at 390 nm, and the concentration of H2O2 was calculated using a standard curve.
The •OH content was determined with a colorimetric Hydroxyl Free Radical Scavenging Capacity Assay Kit (Nanjing Jiancheng Bioengineering Institute) based on the Fenton reaction according to [29]. The concentration of MDA was estimated by the method described in [30]. Fresh leaf samples were homogenized in 0.1% trichloroacetic acid (TCA) and centrifuged at 12,000× g for 10 min. About 4 mL of 20% TCA solution containing 0.5% thiobarbituric acid was added to 1 mL of the supernatant and incubated at 100 °C for 30 min. The reaction mixture was cooled in an ice bath and then centrifuged at 10,000× g for 10 min. The absorbance was recorded first at 530 and corrected at 600 nm.

2.9. Antioxidant Activities Analysis

The activities of antioxidant enzymes such as catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) were quantified according to the manufacturer’s instructions. Approximately 100 mg of fresh leaves were ground in liquid nitrogen and homogenized with PBS (10 mg of fresh leaves to 100 μL of PBS). The homogenates were centrifuged at 10,000 rpm for 30 min at 4 °C, and the assay was performed with the aliquot. The remaining samples were stored at −20 °C, and the supernatant was used for CAT, POD, SOD, and GSH-PX analyses with assay kits from Nanjing Jiancheng Bioengineering Institute.
AOX protein was determined by using an ELISA KIT (JL22749 Plant AOX ELISA KIT; 48T/96T) according to the manufacturer’s instructions. The reaction was based on using a purified plant AOX antibody to coat microstrip plate wells to make a solid-phase antibody. The addition of AOX and the AOX antibody labeled with HRP to wells gives an antibody–antigen–antibody–enzyme complex. In this present experiment, it was considered that an increase in the AOX proteins might enhance the AOX enzymes’ activity.

2.10. Principal Component Analysis

Principal component analysis (PCA), a multivariate method that can reduce the dimension of the multi datasets, was performed using a GraphPad prism 9.0.0. The loadings plot, which can visually represent the relationships between variables and components, was performed for the control group, WL group, and WL + SPD group for each variety.

2.11. Data Analysis

The data were submitted to Two-way ANOVA for a factorial experiment with a completely randomized design using Graph Pad prism 9.0.0 software. Statistically significant differences between means were determined at p ≤ 0.05 using Tukey’s honestly significant difference test. The results were expressed as mean ± standard error, and software was used to draw graphs.

3. Results

3.1. Phenotypic Traits

Growth traits were significantly reduced by WL in C. oleifera (p ≤ 0.05) compared to the control group (Table 2). Growth traits such as plant height, basal stem diameter, and the below-ground and above-ground fresh weight in CoH1 under WL were slightly ameliorated (non-significant) by exogenous SPD compared to CoH2. Indeed, SPD better influenced these growth parameters in CoH2, except for the above-ground fresh weight. In CoH1 seedlings, WL reduced the plant height without SPD application by 59.15% and with SPD by 36.96% compared to the control of CoH1. But the WL treatment decreased the plant height of CoH2 by 37.03% without SPD and by 20.68% with SPD, regardless of the control CoH2. CoH2 showed a significant increase in the number of leaves and plant height increments compared to CoH1 under stress. The morphological response of C. oleifera to WL was slightly different between CoH1 and CoH2. The application of exogenous SPD slightly promoted plant growth in CoH2 compared to the WL-stressed seedlings, whereas it exhibited no statistical difference in CoH1 compared to the seedlings subjected to a single WL treatment.

3.2. Photosynthesis

Photosynthetic pigment concentrations in C. oleifera under control and waterlogged conditions are presented in Table 3. Total chlorophyll was reduced significantly (p ≤ 0.05), by 61.17%, in CoH1 by WL treatment compared to the control of CoH1. In contrast, it decreased by 9.59% in seedlings of CoH1 treated with exogenous SPD under WL compared with the control. In CoH2 seedlings, WL treatment reduced the total chlorophyll content by 47.94%compared to the control of the CoH2 group. Meanwhile, it decreased, by 12.43%, in seedlings exposed to SPD under stress. The application of exogenous SPD enhanced the photosynthetic pigment under WL in both varieties. Chlorophyll a and b were significantly higher in CoH2 compared to CoH1 under stress and when exposed to SPD treatment.
The net photosynthetic rate (Pn) and transpiration rate (Trr) were statistically greater (p ≤ 0.05) in CoH2 than in CoH1 under both stressed and non-stressed conditions (Table 3). Under WL conditions, exogenous SPD strongly alleviated the negative influence of WL in both varieties compared to their control groups. WL diminished the stomatal conductance (Gs) by 85.71% in CoH1 compared to the control and by 73.68% in CoH2, regardless of the control group. The water use efficiency (Wue) was significantly increased in CoH1 under stress, whereas it was decreased in CoH2 (Table 3).

3.3. Water Status and Root Activity

Root activities showed a significant decrease (p ≤ 0.05) of 32.66% in CoH1 and 22.95% in CoH2 under WL compared to their control group (Figure 1A). Root activities were strongly enhanced in both varieties of C. oleifera by exogenous SPD. Moreover, exogenous SPD increased root activities in CoH1 by 10.97% compared to WL treatment; the increase of root activities by SPD was higher (22.14%) in CoH2 compared to CoH1.
Moreover, as indicated in the EL results (Figure 1B), WL conditions increased the membrane damage in CoH1 leaves by 27.82% and in CoH2 leaves by 43.71%compared to their respective control group. Exogenous SPD application significantly protected against (p ≤ 0.05) WL-induced damages in both leaves of CoH1 and CoH2. The damages caused by WL in C. oleifera leaves were statistically similar between CoH1 and CoH2. As for EL activities, DWP from the CoH1 and CoH2 seedlings was significantly ameliorated by exogenous SPD application under WL (Figure 2).

3.4. ROS Accumulation and Lipid Peroxidation

The ROS, such as •OH and H2O2, were drastically enhanced by WL conditions and subsequently increased the lipid peroxidation in both varieties of C. oleifera (Figure 3). Under WL conditions, H2O2 contents showed a significant decrease (36.42%) in CoH2 treated by exogenous SPD compared to its WL control group. Meanwhile, the H2O2 content in CoH1 was not significantly decreased by exogenous SPD, despite a decrease of 33.82%. As was the case for the contents of H2O2 and •OH in CoH2, the MDA contents showed similar behavior in both varieties of C. oleifera under WL stress and exposure to exogenous SPD. Indeed, lipid peroxidation was strongly inhibited in CoH1 and CoH2 seedlings under WL by SPD treatment compared to the seedlings exposed to a single WL treatment (Figure 3C). Moreover, WL significantly increased the MDA contents in both varieties compared to their respective control.

3.5. Osmoprotectants Accumulation and Soluble Proteins

Non-structural carbohydrates and proline contents were strongly increased by WL conditions in CoH1 and CoH2 seedlings compared to their respective control group (Figure 4A,B). Under stress, exogenous SPD enhanced the carbohydrate contents by 29.57% in CoH1 and by 33.87% in CoH2 compared to a single WL treatment. Meanwhile, the proline contents were statistically higher in CoH1 and CoH2 seedlings exposed to SPD compared to WL-stressed plants. Protein degradation caused by WL conditions was strongly alleviated by exogenous SPD in both varieties of C. oleifera (Figure 4C).

3.6. Antioxidant Enzymatic Activities

WL significantly reduced POD and SOD activities in both varieties of C. oleifera seedlings, while the addition of exogenous SPD strongly enhanced the activity of POD and SOD compared to the seedlings exposed to a single WL treatment, particularly in CoH2 seedlings (Figure 5A,B). CAT activity was statistically higher in C. oleifera seedlings under WL compared to the control (Figure 5C). Under WL, there was no significant difference between CoH2 and CoH1 regarding POD and SOD activities when exposed to SPD. Exogenous SPD and WL treatments strongly affected CAT activity in C. oleifera.
Waterlogged conditions significantly decreased (p ≤ 0.05) the AOX protein content in CoH1by 28.58% and in CoH2 by 30.36% compared to their respective control groups (Figure 6A). The application of exogenous SPD alleviated the deterioration of AOX proteins by enhancing AOX in both varieties of C. oleifera under stress conditions. In CoH1, the AOX protein content was statistically similar between the control group and the SPD-WL-treated seedlings. There was no significant difference between CoH1 and CoH2 apropos of the AOX protein under WL stress and exposed to SPD. Furthermore, the GSH-PX activity was significantly increased by waterlogged conditions in C. oleifera seedlings. The application of exogenous SPD did not significantly affect GSH-PX activity in either variety under stress compared to a single WL treatment (Figure 6B).

3.7. PCA Results

The PCAs from loadings were used to draw loadings plots that showed the correlation between photosynthesis activities, osmolytes, antioxidants, and the accumulation of toxic molecules (H2O2, •OH, and MDA) under control, WL, and WL + SPD conditions (Figure 7 and Figure 8). The total cumulative proportion in the variance of PC1 and PC2 that showed the most significant contributions to the explanation of the data correlation was 78.46% under control, 84.89% under WL, and 87.46% under WL + SPD in CoH1. Meanwhile, it was 91.83% under control, 88.22% under WL, and 76.31% under WL + SPD in CoH2.
The correlation profile of the data was not similar based on the type of variety and conditions. Indeed, in CoH1 under control, •OH and MDA were positively correlated, and H2O2 was negatively correlated based on the fact that the vectors for •OH and MDA compared to the vector of H2O2 at nearly a 180° angle (Figure 7A). As shown in Figure 7B, POD, SOD, CAT, and GSH-PX were clustered closely together, indicating that these variables are positively correlated in CoH2 under control. In CoH1 under WL conditions, the PCA showed a strong association among SOD, proteins, and •OH between MDA, proline, and H2O2 (Figure 8A). Meanwhile, in CoH2, H2O2 was positively correlated with proteins, and •OH was positively correlated with MDA, carbohydrates, and proline (Figure 8C). Under waterlogged conditions, SPD application showed a strong correlation between H2O2 and MDA, •OH and POD, and AOX and CAT in CoH1 (Figure 8B). The correlation was positive between H2O2, •OH, and Proteins, SOD, GSH-PX and SOD, and CAT and AOX in CoH2 seedlings under stress and exposed to SPD (Figure 8D).

4. Discussion

Several substances, such as melatonin, glycine betaine, calcium, or gamma-aminobutyric, have been used exogenously to promote plant stress tolerance. Spermidine appears to possess the same function as these substances in improving plant system defense. Indeed, a recent report showed the efficiency of melatonin under water stress by improving the antioxidant system and reducing ROS accumulation in Populus cathayana [31]. Moreover, it has been reported that γ-aminobutyric acid improved antioxidant capacity and growth traits in maize seedlings under waterlogging [32]. The application of exogenous calcium in Handeliodendron bodinieri promoted photosynthesis, water status, and the antioxidant system against water stress [33]. The results of the present study demonstrate that exogenous SPD may have the same role as those substances in plant stress tolerance.

4.1. Spermidine Affected Root Activity and Morphological Traits of C. oleifera

On the one hand, the present study showed that both varieties were affected by waterlogging stress (WL) in terms of phenotypic traits. The supply of spermidine increased significantly in various morphological indexes, such as the below-ground biomass or plant height in CoH2. The enhancement of morphological traits in CoH1 by SPD was not significant. It has been reported that exogenous SPD significantly enhanced the number of leaves and aboveground biomass in rice seedlings under WL [34]. Moreover, this report also showed that SPD application shielded the decrease of fresh leaf weight due to heat stress in rice seedlings. Indeed, spermidine, as one of the main PAs in plants, is involved in the regulation of diverse physiological processes such as organogenesis or senescence [35]. The results suggested that under WL, SPD is able to positively influence plant morphology in one variety. However, as reported in [31], the role of PAs in the regulation of plant growth is still not fully understood.
On the other hand, plants under WL stress face a disturbance between water absorption by roots and transpiration from the leaves. Given that water is vital for plant growth, development, and survival, a saturation of the soil’s water-holding capacity can lead to the inhibition of root respiration and the accumulation of toxic substances, which can provoke severe damage in plant cells or lead to cell death [36]. The results showed that WL decreased the root activities in C. oleifera, which negatively affected the leaves’ water status and the above- and below-ground biomass accumulation in C. oleifera seedlings under WL. Moreover, the mitigation of the roots’ damage in C. oleifera by exogenous SPD under WL might positively affect photosynthesis. In fact, during photosynthesis, the water used by land plants is essentially absorbed from the soil by roots. It has been well-established that water stress can significantly reduce root activity, which negatively affects water absorption [10].

4.2. Photosynthesis in Both Varieties of C. oleifera Was Significantly Increased by SPD under WL

Waterlogged soils are anaerobic and characterized by a reduction in the availability of dissolved oxygen. One of the symptoms of WL in plants is stomatal closure that perilously affects gas exchange, shoot growth, and plant height [37]. WL stress can severely inhibit photosynthesis and respiration in plants by reducing the Pn and chlorophyll activities. In the present study, the loss of photosynthetic pigments and the decrease of Pn were stopped by the application of exogenous SPD in both varieties of C. oleifera under WL. Moreover, the reduction of transpiration in each variety compared to their respective control was prevented by exogenous SPD, which significantly reduced of leaf loss in C. oleifera and ameliorated phenotypic traits. Indeed, it has been well-established that a reduction of transpiration in plants under stress can provoke leaf wilting and early senescence leading to foliar abscission [38]. It was reported for the Welsh onion that WL induced leaf wilting, chlorosis (yellow leaves), and progressive senescence [15]. However, exogenous SPD can prevent the loss of chlorophyll and stabilize the molecular composition of the thylakoid membranes via the inhibition of lipid peroxidation, as demonstrated in Avena sativa [39].
Furthermore, our results showed that WL stress in C. oleifera was accompanied by oxidative stress due to the over-accumulation of ROS, such as H2O2 and •OH, that participated in increasing lipid peroxidation and disturbed homeostasis via an increase of the DWP and EL. Indeed, the over-accumulation of H2O2 and •OH can easily provoke disruption of cellular and chloroplastic membrane function and homeostasis that leads to a decrease in photosynthesis activity. Moreover, previous studies showed that PAs, especially thylakoid-bound PAs, participate in the regulation of the structure and function of the photosynthetic apparatus under abiotic stresses [15]. The application of exogenous SPD can protect the structure and function of the photosynthetic system by enhancing the initial electron transfer in PSII [40].

4.3. SPD Application Alleviated Oxidative Stress in C. oleifera, Particularly in CoH2

Our results showed that waterlogged soil significantly enhanced free radical accumulation and lipid peroxidation in C. oleifera leaves. Meanwhile, exogenous SPD prevented the overproduction of these molecules in CoH2. Plants can accumulate various osmolytes, like non-structural carbohydrates and proline, to prevent ROS accumulation and lipid peroxidation under abiotic stresses. Proline is a compatible solute that can significantly reduce stress-induced cellular acidification and can directly scavenge the ROS in plant cells under stress [41]. SPD application resulted in the alleviation of the salinity-induced damages with an increase in PAs and proline contents in C. sativus [42]. The ability of exogenous SPD to maintain cell homeostasis in plants was attributed to its contribution to osmotic adjustment by increasing the internal proline contents [15]. Furthermore, the role of SPD has been reported in the increase of non-structural carbohydrates in Vitis vinifera. The results suggested that the increase of proline and carbohydrates by exogenous SPD played a crucial role in maintaining cellular redox homeostasis and ROS scavenging.
The role of the enzymes such as CAT, POD, and SOD in ROS scavenging in plants under stress is crucial to maintain cell homeostasis. Although the increase of POD and SOD activities in various plants under WL stress [43] has been well-establish, WL significantly decreased POD and SOD activities in C. oleifera leaves in the present study, whereas exogenous SPD strongly enhanced their activities, particularly in CoH2. The decrease of POD and SOD in the present study might be due to the dysfunctioning cellular respiration caused by long-term WL, as suggested in a previous review article [44]. The increase of CAT by exogenous SPD in C. oleifera leaves under WL contributed significantly to higher H2O2 scavenging. Indeed, it has been reported that deficiencies in CAT activity in plants caused, at least to a part, an over-accumulation of hydrogen peroxide [15]. Moreover, GSH-PX played a crucial role against oxidative stress in C. oleifera under WL treatment. Indeed, their activities were significantly higher in seedlings under WL stress, but a supply of exogenous SPD did not affect the profile of GSH-PX in either variety of C. oleifera under stress. Thus, the results suggest that the role of the SPD in C. oleifera under WL stress is to maintain the antioxidant activity that WL significantly decreases.
It has been demonstrated that short-term WL stress (9 days) increased the AOX activity in watermelon [2]. Exo-substances, such as glycine betaine and melatonin, influenced the AOX protein in D. odorifera to provide stress tolerance [29]. The results of the present study showed that the exogenous SPD application maintained AOX protein under WL, which might participate significantly in scavenging the over-accumulation of reactive oxygen in the mitochondria. Indeed, the AOX pathway is involved in the response to abiotic stresses, such as chilling, nutrient deficiency, or hypoxia [45]. As far as we know, there are no reports that focus on the interactions between SPD and AOX proteins that take part in the AOX pathway in stress tolerance. AOX is a non-energy conserving terminal oxidase in the plant mitochondrial electron transport chain that can reduce dioxygen to water without transferring a proton through the mitochondrial proton pumping complex [46]. The present study suggested that exogenous SPD maintains the AOX protein level to reduce the damages caused by hypoxia in the mitochondrial respiratory processes in C. oleifera leaves. Overall, as shown in Figure 9, exogenous SPD boosted different biochemical defense components to promote significant ROS scavenging and cell homeostasis.

5. Conclusions

The present work presented different physiological data that support the alleviation of WL by exogenous SPD in C. oleifera. Exogenous SPD provided WL tolerance in both varieties by increasing the antioxidant system, growth traits, and photosynthesis. The detoxification of ROS was similar in both varieties under WL by exogenous SPD. However, CoH2 presented a higher root activity, Pn, and Gs compared to CoH1. In conclusion, this study provided several results at morphological and physio-biochemical levels to develop WL resilience in C. oleifera via the use of SPD to increase C. oleifera productivity, particularly in areas with poor soil drainage properties and those constantly affected by heavy rainfall and typhoons. For future studies, the use of genome editing techniques could be an appropriate pathway to promote the accumulation of SPD in C. oleifera. Indeed, recent research clearly showed that climate change is resulting in more extreme weather events, such as soil WL, that negatively affect trees and crop production. It has been well-established that modern sequencing and genome assembly technologies, as well as big data analyses, enable rational crop improvement under both non-stressed and stressed conditions. Thus, the results suggest that manipulating SPD accumulation in C. oleifera diminishes the loss of productivity of C. oleifera in Southeast Asia due to natural catastrophes like typhoons or heavy rain.

Author Contributions

E.-H.M.C. wrote the draft manuscript; J.-F.H. performed the majority of the experiments; E.-H.M.C., J.-F.H., and D.-D.L. collected the data, drew the graphs, and analyzed and interpreted the data; L.-F.M. managed the experiments and provided scientific guidance; L.-S.X. and F.Y. designed the experiments, revised the manuscript, and provided funding. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Hainan Provincial Natural Science Foundation of China (No. 421RC1033, 320RC507, and 421QN192), the National Science Foundation of China (No. 32060240 and 31660165), and the Hainan Province Science and Technology Special Fund (ZDYF2022SHFZ054).

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to the policy of the institute.

Conflicts of Interest

All authors approved the manuscript, and authors have declared no conflict of interest.

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Figure 1. Variations in Root activity (A) and EL (B) in C. oleifera seedlings under WL exposed to SPD. The bars on the top show SE (n = 5), and different lowercases and uppercases indicate significant differences among CK, WL, and WL + SPD treatments within each species according to Tukey’s multiple comparison test, respectively (p < 0.05). The signs such as * and ns in the figures concern the significance difference between the same treatment for CoH1 and CoH2 (*, p < 0.05; ns, Not significant). CK: control; WL: waterlogging; SPD: spermidine.
Figure 1. Variations in Root activity (A) and EL (B) in C. oleifera seedlings under WL exposed to SPD. The bars on the top show SE (n = 5), and different lowercases and uppercases indicate significant differences among CK, WL, and WL + SPD treatments within each species according to Tukey’s multiple comparison test, respectively (p < 0.05). The signs such as * and ns in the figures concern the significance difference between the same treatment for CoH1 and CoH2 (*, p < 0.05; ns, Not significant). CK: control; WL: waterlogging; SPD: spermidine.
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Figure 2. Dew point water potential variations in C. oleifera seedlings under WL exposed to SPD. The bars on the top show SE (n = 5), and different lowercases and uppercases indicate significant differences among CK, WL, and WL + SPD treatments within each species according to Tukey’s multiple comparison test, respectively (p < 0.05). The signs such as * and ns in the figures concern the significance difference between the same treatment for CoH1 and CoH2 (ns, Not significant). CK: control; WL: waterlogging; SPD: spermidine.
Figure 2. Dew point water potential variations in C. oleifera seedlings under WL exposed to SPD. The bars on the top show SE (n = 5), and different lowercases and uppercases indicate significant differences among CK, WL, and WL + SPD treatments within each species according to Tukey’s multiple comparison test, respectively (p < 0.05). The signs such as * and ns in the figures concern the significance difference between the same treatment for CoH1 and CoH2 (ns, Not significant). CK: control; WL: waterlogging; SPD: spermidine.
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Figure 3. H2O2 (A), •OH (B), and MDA (C) levels in C. oleifera under WL and exposed to SPD treatment. The bars on the top show SE (n = 5), and different lowercases and uppercases indicate significant differences among CK, WL, and WL + SPD treatments within each species according to Tukey’s multiple comparison test, respectively (p < 0.05). The signs such as *** and ns in the figures concern the significance difference between the same treatment for CoH1 and CoH2 (***, p < 0.0005; ns, Not significant). CK: control; WL: waterlogging; SPD: spermidine.
Figure 3. H2O2 (A), •OH (B), and MDA (C) levels in C. oleifera under WL and exposed to SPD treatment. The bars on the top show SE (n = 5), and different lowercases and uppercases indicate significant differences among CK, WL, and WL + SPD treatments within each species according to Tukey’s multiple comparison test, respectively (p < 0.05). The signs such as *** and ns in the figures concern the significance difference between the same treatment for CoH1 and CoH2 (***, p < 0.0005; ns, Not significant). CK: control; WL: waterlogging; SPD: spermidine.
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Figure 4. Carbohydrates (A), proline (B), and soluble proteins (C) contents in C. oleifera under WL stress exposed to SPD. The bars on the top show SE (n = 5), and different lowercases and uppercases indicate significant differences among CK, WL, and WL + SPD treatments within each species according to Tukey’s multiple comparison test, respectively (p < 0.05). The signs such as ****, ** and ns in the figures concern the significance difference between the same treatment for CoH1 and CoH2 (****, p < 0.0001; **, p < 0.005; ns, Not significant). CK: control; WL: waterlogging; SPD: spermidine.
Figure 4. Carbohydrates (A), proline (B), and soluble proteins (C) contents in C. oleifera under WL stress exposed to SPD. The bars on the top show SE (n = 5), and different lowercases and uppercases indicate significant differences among CK, WL, and WL + SPD treatments within each species according to Tukey’s multiple comparison test, respectively (p < 0.05). The signs such as ****, ** and ns in the figures concern the significance difference between the same treatment for CoH1 and CoH2 (****, p < 0.0001; **, p < 0.005; ns, Not significant). CK: control; WL: waterlogging; SPD: spermidine.
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Figure 5. POD (A), SOD (B), and CAT (C) activities in C. oleifera under WL and exposed to SPD. The bars on the top show SE (n = 5), and different lowercases and uppercases indicate significant differences among CK, WL, and WL + SPD treatments within each species according to Tukey’s multiple comparison test, respectively (p < 0.05). The signs such as ns in the figures concern the significance difference between the same treatment for CoH1 and CoH2 (ns, Not significant). CK: control; WL: waterlogging; SPD: spermidine.
Figure 5. POD (A), SOD (B), and CAT (C) activities in C. oleifera under WL and exposed to SPD. The bars on the top show SE (n = 5), and different lowercases and uppercases indicate significant differences among CK, WL, and WL + SPD treatments within each species according to Tukey’s multiple comparison test, respectively (p < 0.05). The signs such as ns in the figures concern the significance difference between the same treatment for CoH1 and CoH2 (ns, Not significant). CK: control; WL: waterlogging; SPD: spermidine.
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Figure 6. AOX (A) and GSH-PX (B) activities in C. oleifera under WL and exposed to SPD treatment. The bars on the top show SE (n = 5), and different lowercases and uppercases indicate significant differences among CK, WL, and WL + SPD treatments within each species according to Tukey’s multiple comparison test, respectively (p < 0.05). The signs such as NS in the figures concern the significance difference between the same treatment for CoH1 and CoH2 (***, p < 0.0005; ****, p < 0.0001; ns, Not significant). CK: control; WL: waterlogging; SPD: spermidine.
Figure 6. AOX (A) and GSH-PX (B) activities in C. oleifera under WL and exposed to SPD treatment. The bars on the top show SE (n = 5), and different lowercases and uppercases indicate significant differences among CK, WL, and WL + SPD treatments within each species according to Tukey’s multiple comparison test, respectively (p < 0.05). The signs such as NS in the figures concern the significance difference between the same treatment for CoH1 and CoH2 (***, p < 0.0005; ****, p < 0.0001; ns, Not significant). CK: control; WL: waterlogging; SPD: spermidine.
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Figure 7. Loadings plot of the PCAs showing correlation between antioxidant enzymes, osmolytes, ROS, MDA, and proteins in control group CoH1 (A) and CoH2 (B).
Figure 7. Loadings plot of the PCAs showing correlation between antioxidant enzymes, osmolytes, ROS, MDA, and proteins in control group CoH1 (A) and CoH2 (B).
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Figure 8. Loadings plot of the PCAs showing correlation between antioxidant enzymes, osmolytes, ROS, MDA, and proteins in CoH1 ((A) WL; (B), WL + SPD) and in CoH2 ((C) WL; (D) WL + SPD).
Figure 8. Loadings plot of the PCAs showing correlation between antioxidant enzymes, osmolytes, ROS, MDA, and proteins in CoH1 ((A) WL; (B), WL + SPD) and in CoH2 ((C) WL; (D) WL + SPD).
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Figure 9. A model design showing how SPD influenced the physio-biochemical systems in C. oleifera seedlings under WL.
Figure 9. A model design showing how SPD influenced the physio-biochemical systems in C. oleifera seedlings under WL.
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Table
Table 1. Subordinate function analysis of exogenous SPD at different concentrations in C. oleifera seedlings under WL.
Table 1. Subordinate function analysis of exogenous SPD at different concentrations in C. oleifera seedlings under WL.
ControlWL + 0.1 mM SPDWL + 0.5 mM SPDWL + 1 mM SPD
Basal diameter of stem100.4030.06
Plant height00.7110.79
Number of leaves10.550.820
Mean0.670.420.740.28
Table
Table 2. Variations of the phenotypic traits in C. oleifera seedlings under WL and treated with exogenous SPD.
Table 2. Variations of the phenotypic traits in C. oleifera seedlings under WL and treated with exogenous SPD.
VarietyTreatmentsNumber of
Leaves
Increment		Plant Height Increment (cm)Basal Diameter of Stem Increment (mm)Below-Ground Fresh Weight (g)Above-Ground Fresh Weight (g)
C. oleifera
(CoH1)		CK11.50 ± 1.29 a11.00 ± 1.77 a3.07 ± 0.55 ab13.48 ± 3.26 ab5.60 ± 1.02 ab
WL6.25 ± 1.25 c4.82 ± 0.85 c2.63 ± 0.36 ab9.48 ± 2.17 b4.60 ± 1.50 b
WL + SPD7.75 ± 1.05 bc5.42 ± 1.97 c2.84 ± 0.84 ab9.65 ± 1.44 b4.41 ± 1.32 b
C. oleifera
(CoH2)		CK12.00 ± 1.41 a7.87 ± 1.65 b3.48 ± 0.76 a17.01 ± 2.77 a7.05 ± 1.75 a
WL8.25 ± 1.70 bc7.05 ± 1.05 b2.37 ± 0.44 b8.85 ± 1.06 b4.81 ± 0.75 ab
WL + SPD9.75 ± 1.70 ab9.00 ± 1.47 ab2.84 ± 0.33 ab10.65 ± 1.76 ab5.00 ± 0.70 ab
The different lowercases indicate significant differences among CK, WL, and WL + SPD treatments within each species according to Tukey’s multiple comparison test, respectively (p < 0.05).Values are expressed as mean ± SE (n = 5). CK: control; WL: waterlogging; SPD: spermidine.
Table
Table 3. Variations of the photosynthetic parameters in C. oleifera leaves under WL and exposed to SPD.
Table 3. Variations of the photosynthetic parameters in C. oleifera leaves under WL and exposed to SPD.
VarietyTreatmentsT-Chl (µg/g.Fw)Chl-a (µg/g.Fw)Chl-b (µg/g.Fw)Car (µg/g.Fw)Pn (μmol·m−2·s−1)Gs (mol·m−2·s−1)Ci (μmol·mol−1)Trr (mmol·m−2·s−1)Wue (µmol·mmol−1)
C. oleifera
(CoH1)		CK813.56 ± 13.1 b606.7 ± 8.23 b206.8 ± 16.30 c202.50 ± 11.40 b5.77 ± 0.99 b0.10 ± 0.020 b396.00 ± 31.17 d3.64 ± 0.84 b1.58 ± 0.43 c
WL432.42 ± 17.6 e317.1 ± 5.03 f115.3 ± 4.06 f132.90 ± 8.50 e2.76 ± 0.90 e0.04 ± 0.009 d 403.50 ± 40.21 c1.74 ± 0.42 f1.75 ± 0.95 a
WL + SPD739.06 ± 22.3 c546.7 ± 12.60 d192.2 ± 7.31 d187.03 ± 13.01 c3.76 ± 0.61 d0.05 ± 0.010 cd389.00 ± 16.98 e2.24 ± 0.54 e1.67 ± 0.28 b
C. oleifera
(CoH2)		CK900.83 ± 26.4 a654.2 ± 20.01 a246.6 ± 9.13 a231.70 ± 8.80 a7.35 ± 1.64 a0.13 ± 0.014 a354.66 ± 41.13 f4.1 ± 1.09 a1.78 ± 0.98 a
WL552.45 ± 17.4 d393.3 ± 7.36 e159.1 ± 14.11 e132.15 ± 4.21 e3.53 ± 0.58 d0.06 ± 0.010 c436.57 ± 23.56 b2.37 ± 0.76 d1.64 ± 0.65 bc
WL + SPD795.37 ± 10.3 c559.6 ± 5.12 c235.6 ± 8.03 b162.82 ± 3.83 d4.06 ± 0.40 c0.09 ± 0.012 b445.80 ± 38.92 a3.41 ± 1.29 c1.34 ± 0.59 d
The different lowercases indicate significant differences among CK, WL, and WL + SPD treatments within each species according to Tukey’s multiple comparison test, respectively (p < 0.05).Values are expressed as mean ± SE (n = 5). CK: control; WL: waterlogging; SPD: spermidine; T-Chl: total of chlorophyll; Chl-a: chlorophyll a; Chl-b: Chlorophyll b; Car: carotenoids; Pn: net photosynthetic rate; Gs: stomatal conductance; Ci: intercellular CO2 concentration; Trr: transpiration rates; Wue: water use efficiency.
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MDPI and ACS Style

Cisse, E.-H.M.; Huang, J.-F.; Li, D.-D.; Miao, L.-F.; Xiang, L.-S.; Yang, F. Exogenous Spermidine Alleviated Waterlogging Damages in Two Varieties of Camellia oleifera. Forests 2023, 14, 91. https://doi.org/10.3390/f14010091

AMA Style

Cisse E-HM, Huang J-F, Li D-D, Miao L-F, Xiang L-S, Yang F. Exogenous Spermidine Alleviated Waterlogging Damages in Two Varieties of Camellia oleifera. Forests. 2023; 14(1):91. https://doi.org/10.3390/f14010091

Chicago/Turabian Style

Cisse, El-Hadji Malick, Jin-Fu Huang, Da-Dong Li, Ling-Feng Miao, Li-Shan Xiang, and Fan Yang. 2023. "Exogenous Spermidine Alleviated Waterlogging Damages in Two Varieties of Camellia oleifera" Forests 14, no. 1: 91. https://doi.org/10.3390/f14010091

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