The Effect of Low Temperature and Low Illumination Intensity on the Photosynthetic Characteristics and Antioxidant Enzyme Activity in the Strawberry

Abstract

Low temperature (LT) and low illumination (LI) are common meteorological factors posing a great risk to plants. This study aimed to clarify and quantify the effects of LT, LI, and their combined stress (LTLI) on the photosynthetic physiological processes of strawberry plants during the flowering stage. The results indicated that LI stress increased Chla and b levels in strawberry plants while lowering the chlorophyll a/b ratio. In contrast, LT and LTLI stress reduced chlorophyll content. All stress conditions (LT, LI, and LTLI) decreased net photosynthetic rate, stomatal conductance, transpiration rate, the maximum photochemical efficiency of photosystem II, photosynthetic electron transport rate, and actual photochemical quantum efficiency. These stresses also raised intercellular carbon dioxide concentration, non-photochemical quenching coefficient, and levels of malondialdehyde, proline, hydrogen peroxide, and peroxide ion content. Moreover, LI stress treatment boosted the activity of superoxide dismutase, peroxidase, and catalase, while LT and LTLI stress initially raised the activity of these enzymes before it eventually declined. Importantly, the previously mentioned photosynthetic physiological parameters showed notable changes under the combined stress conditions. Ultimately, the TOPSIS model was used to quantitatively evaluate the impact levels of different stressors and treatment durations on the photosynthetic system of strawberry plants. In conclusion, the synergistic impact of LT and LI results in a reduction in photosynthetic rate and photosystem II activity, a disruption in the equilibrium of the antioxidant system, and an intensification of photoinhibition, ultimately leading to diminished photosynthetic efficiency in plants.

1. Introduction

Temperature and light are critical meteorological factors influencing the flowering, fruiting, pollination, fertilization, fruit yield, and quality development of strawberries [1]. Global climate change has exhibited abnormal patterns, with extreme weather events occurring more frequently [2]. The flowering phase signifies the transition from vegetative to reproductive growth in strawberries and constitutes a crucial stage that influences both yield and fruit quality [3], which is especially susceptible to biotic and abiotic stresses. In recent years, the flowering period of strawberries in the middle and lower reaches of the Yangtze River has frequently coincided with prolonged rainy conditions and low light levels, accompanied by low temperatures. This combination of low temperature and weak light intensity has constrained the growth and development of strawberries in the region.

Light and temperature are crucial meteorological factors influencing plant photosynthesis [4,5]. Temperature and light significantly impact the photosynthetic enzymes, stomatal regulation, metabolite accumulation, and cytochrome composition in plants [6,7]. Extreme temperatures and light intensities typically exert detrimental effects on plant photosynthesis, resulting in a marked reduction in plant yield [8]. Furthermore, light and temperature do not act independently in affecting photosynthesis and growth characteristics of crops; rather, they exhibit a complementary and mutually enhancing relationship [9,10]. The effects of low temperature on photosynthesis mainly focus on the structure and activity of photosynthetic organs, chloroplast function, enzyme activity involved in photosynthesis, photosynthetic electron transfer rate, and carbon assimilation process [11,12]. In addition, the carbon assimilation process of photosynthesis involves multiple complex enzymatic reactions, and low-temperature stress mainly inhibits the carbon assimilation process of photosynthesis by suppressing the activity of various biological enzymes [13]. Low temperatures disrupt the balance of reducing agents in photosynthesis, impairing thylakoid electron transport [14]. This leads to reduced CO₂ availability for carbon cycling and stomatal regulation, causing photoinhibition and photooxidative damage to photosystems II and I, which in turn inhibits normal plant growth and development [15]. Additionally, suboptimal light conditions can also impact the photosynthetic process. Sun et al. [16] demonstrated that shading treatment led to an increase in the levels of chlorophyll a, chlorophyll b, and carotenoids in summer maize leaves, while concurrently reducing the photosynthetic rate. Additionally, under conditions of low light, sweet pepper leaves demonstrated a reduction in chlorophyll a content and stomatal conductance efficiency, accompanied by an increase in intercellular CO₂ concentration and malondialdehyde levels, collectively leading to a reduction in photosynthetic activity [17]. The combined stress of low temperature and weak light further intensifies the inhibition of photosynthesis in greenhouse-grown tomatoes [18], peppers [19], and yellow light leaves. Furthermore, exposure to low temperatures increases reactive oxygen species (ROS) in plants, causing cellular damage and affecting antioxidant enzymes and photosynthesis [20]. Plants counteract this stress with antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX), as well as non-enzymatic antioxidants [21]. Chlorophyll fluorescence parameters are key tools for studying the link between plant photosynthesis and environmental conditions [22]. Notably, the maximum photochemical efficiency (F_v/F_m) is a crucial indicator of the maximum photon efficiency of photosystem II (PSII), providing insights into the plant’s photosynthetic capacity and the extent to which environmental stress impacts photosynthetic processes [23].

There have been relevant research reports on effects of individual treatments involving either temperature or light on strawberry production [24,25]. We hypothesized that extended exposure to low-temperature and low-light stress during the flowering phase will surpass the plant’s adaptive capacity, resulting in an imbalance in the antioxidant system, an increased peroxidation of cell membranes, and a consequent reduction in photosynthetic efficiency. Additionally, we hypothesized that the combined stress of low temperature and low illumination exerts a more detrimental effect on photosynthetic physiological processes than the individual stressors of low temperature or low illumination alone. This study employed controlled environment experiments to simulate the impacts of low temperature and reduced light intensity on the flowering period of strawberries. The objectives were twofold: (1) to assess the effects of varying low temperature and light intensity, as well as their duration during the flowering period, on gas exchange parameters, fluorescence parameters, reactive oxygen species, and antioxidant enzyme activity in strawberries, and (2) to quantify the relative contributions of low temperature, light intensity, and their duration to the photosynthetic physiological processes in strawberries. The findings will provide a theoretical foundation and technical support for stress-resistant cultivation and high-quality and high-yield production of strawberries.

2. Materials and Methods

2.1. Plant Material and Treatments

The control experiments were conducted in the artificial climate chamber from September 2023 to January 2024 at the Jiangxi Academy of Agricultural Sciences, located in Nanchang, Jiangxi Province, China (39°96′ N, 116°33′ E). The strawberry variety utilized in the experiment was Fragaria × ananassa Duch. ‘Benihoppe’, sourced from Jiayi Flower and Horticultural Nursery Co., Ltd., Jiangsu, China. The strawberry plants were cultivated in nutrient pots with the following specifications: a bottom diameter of 25 cm, a top diameter of 30 cm, and a height of 30 cm. The potting medium consisted of a 1:1 ratio of vermiculite to perlite, with each component weighing 2.0 kg. A single strawberry plant was planted per pot. During the flowering phase, strawberry plants exhibiting robust health and uniform growth were selected for environmental control experiments, to investigate the effects of varying low temperature and low light intensity and duration.

The experiment was structured into four distinct groups, each subjected to specific low-temperature and low-light treatments as outlined in

Table 1. Low-temperature and weak-light treatment of strawberry seedlings in a phytotron.

Treatment	Temperature (°C)	Illumination Intensity (μmol m−2 s−1)
NT	20	800
LT	6	800
LI	20	100
LTLI	6	100

Note: NT—normal temperature and light illumination conditions, LT—low-temperature stress treatment, LI—low-illumination stress treatment, and LTLI—combined low-temperature and low-illumination stress treatment.

: (1) group with normal temperature and normal light conditions (NT, 25 °C + 800 μmol m⁻² s⁻¹), (2) group ex-posed to low-temperature conditions (LT, 6 °C + 800 μmol m⁻² s⁻¹), (3) group exposed to low illumination conditions (LI, 25 °C + 100 μmol m⁻² s⁻¹), and (4) group exposed to both low-temperature and low-illumination conditions (LTLI, 6 °C + 100 μmol m⁻² s⁻¹). Throughout the experimental period, the relative humidity within the climate chamber was consistently maintained at approximately 60%. Additionally, the strawberry plants were irrigated and fertilized daily using a half-strength Hoagland nutrient solution. Samples were collected on the 1st, 3rd, 5th, and 7th days of the experimental process to assess pertinent photosynthetic and physiological biochemical parameters. Each experiment was conducted in triplicate, utilizing approximately 10 strawberry seedlings per iteration.

2.2. Leaf Pigment Levels

Introduce 20 mg of strawberry leaf sample, ensuring the removal of primary veins, into a test tube containing 10 milliliters of 95% ethanol. Seal the tube and incubate it in darkness for 24 h to facilitate complete chlorophyll extraction. Subsequently, measure the absorbance of the resulting solution at wavelengths of 649 nm, 665 nm, and 470 nm using a spectrophotometer (DU 730, Beckman Coulter, Inc., Brea, CA, USA). The concentrations of chlorophyll a, chlorophyll b, and carotenoids can then be determined by applying Equations (1)–(3) as referenced in [26]. The units of Chla and Chlb are mg g⁻¹.

$$Chlorophylla=13.95A_{665}-6.88A_{649}$$

(1)

$$Chlorophyllb=13.95A_{665}-6.88A_{649}$$

(2)

$$Carotenoid=(1000A_{470}-2.05\times chlorophylla-114.8\times chlorophyllb)/245$$

(3)

2.3. Measurement of Photosynthetic Properties

The LI 6400 XT portable photosynthetic system (Li-Cor Inc., Lincoln, NE, USA) was employed to assess leaf gas exchange parameters on fully developed leaves between 9:00 a.m. and 11:00 a.m. following treatment durations of 1, 3, 5, and 7 days. In the course of the measurement process, the parameters of the portable photosynthesis analyzer’s leaf chamber were configured to a CO₂ concentration of 380 μmol mol⁻¹, a temperature of 25 °C, a photosynthetic photon flux density (PPFD) of 1000 μmol m⁻² s⁻¹, and a relative air humidity of 80%. This instrument autonomously analyzes and documents the net photosynthetic rate (P_n), stomatal conductance (g_s), intercellular carbon dioxide concentration (C_i), and transpiration rate (T_r) in the strawberry leaves [27]. The units of P_n, g_s, C_i, and T_r are μmol m⁻² s⁻¹, mol m⁻² s⁻¹, μmol mol⁻¹, and mmol mol⁻¹.

2.4. Determination of Chlorophyll Fluorescence

The selection and repetition times for measuring leaves are consistent with the determination of gas exchange indicators. A PAM-2500 pulse-modulated fluorometer (Walz, Effeltrich, Germany) was employed to measure chlorophyll fluorescence kinetic parameters in vivo, ensuring that the midrib of the leaves was avoided. Prior to measurement, the leaves were dark-adapted in a leaf clamp for 20 min. Subsequently, the chlorophyll fluorescence parameters of strawberry leaves were assessed, including the maximum photochemical efficiency of PSII (F_v/F_m), the photosynthetic electron transport rate (ETR), and non-photochemical quenching coefficient (NPQ), and actual photochemical quantum efficiency (φPSII) [28].

2.5. Quantification of Malondialdehyde and Proline

The procedure for quantifying malondialdehyde (MDA) entails the precise measurement of 2 g of fresh strawberry leaves, with the primary vein excised. These leaves are then ground in an ice bath using 5 milliliters of phosphate buffer solution (pH 7.8) and a minimal quantity of quartz sand. Following this, the mixture undergoes homogenization through centrifugation at 12,000 rpm at 4 °C for a duration of 10 min. The supernatant obtained post-centrifugation serves as the MDA extraction solution. Subsequently, 1 mL of MDA extraction solution combined with 3 mL of 27% trichloroacetic acid and 1 mL of 2% thiobarbituric acid (TBA) was then incubated at 95 °C for 30 min, followed by rapid cooling in an ice bath and centrifugation for 10 min. Optical density (OD) values of the mixed solution were measured at wavelengths of 532 nm and 600 nm, with the detailed calculation process described in the study by Guo et al. [29]. The unit of MDA is μmol g⁻¹.

To determine the proline (Pro) content, precisely weigh 0.20 g of fresh strawberry leaves, ensuring the removal of the main vein, and finely chop the leaves before transferring them into a test tube. Introduce 5 mL of a 3% sulfosalicylic acid solution to the test tube and immerse it in a boiling water bath for 10 min. Allow the mixture to cool to room temperature, then carefully extract 2 mL of the supernatant. To this, add 2 mL of glacial acetic acid and 3 mL of a 2.5% acidic indole-3-acetic acid colorimetric solution. Subject the mixture to a boiling water bath for 40 min to facilitate color development, then cool the solution. Then, extract the mixture with 5 mL of toluene, measure the OD values of the extraction solution at 520 nm, and the comprehensive calculation methodology was elaborated upon the method of Guo et al. [29]. The unit of Pro is mg g⁻¹.

2.6. Content of Reactive Oxygen Species

Precisely weigh 1 g of healthy strawberry leaves to assess the content of reactive oxygen species. The production rate of superoxide anion (O²⁻) was determined following the methodology outlined by Lee et al. [30], with minor modifications. The production of H₂O₂ was quantified using a sodium nitrite standard curve and expressed in units of nmol min⁻¹ g⁻¹. The quantification of hydrogen peroxide content was based on the approach described by Xu et al. [10], with slight adjustments, and was reported in units of μmol g⁻¹.

2.7. Antioxidant Enzyme Activity Assay

Accurately weigh 0.3 g of de-veined strawberry leaves and then place them into a pre-cooled mortar. Add 8 mL of 50 mmol L⁻¹ phosphate buffer (pH 7.8) and grind the mixture into a homogenate. Transfer the homogenate to a centrifuge tube and centrifuge at 12,000 rpm for 20 min. The resulting supernatant was used for the determination of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities. SOD activity was assessed using the nitroblue tetrazolium (NBT) method, POD activity was measured using the guaiacol method, and CAT activity was determined using the dynamic UV absorption method [31]. The units of SOD, POD, and CAT are U g⁻¹ min⁻¹.

2.8. Determination of Comprehensive Evaluation Model for Low Temperature and Low Illumination Level

2.8.1. Construction of Standardized Matrix

The standardized matrix for evaluating disaster levels is shown in Equation (4).

$$R=\left[\begin{array}{cccc}{r}_{11}& r_{12}& \cdot \cdot \cdot & r_{1n}\\ r_{21}& r_{22}& \cdot \cdot \cdot & r_{2n}\\ \cdot \cdot \cdot & \cdot \cdot \cdot & \cdot \cdot \cdot & \cdot \cdot \cdot \\ r_{m1}& r_{m2}& \cdot \cdot \cdot & r_{mn}\end{array}\right]$$

(4)

where R is the standardized matrix for disaster levels, m is the number of evaluation indicators, and n is the number of influencing factors.

2.8.2. Determination of Evaluation Index Weights

The entropy weight method is employed to compute the weights of each indicator, as demonstrated in Equations (5)–(7).

$$w_i={\displaystyle \frac{1-H_i}{m-{\displaystyle \sum _{i=1}^m{H}_i}}}$$

(5)

$$H_i=-{\displaystyle \frac{1}{\ln n}}{\displaystyle \sum _{j=1}^n{f}_{ij}\ln }{f}_{ij}$$

(6)

$$f_{ij}={\displaystyle \frac{r_{ij}}{{\displaystyle \sum _{j=1}^n{r}_{ij}}}}$$

(7)

where w_i represents the weight value, H_i denotes information entropy, and f_ij is the characteristic weight of the indicator.

2.8.3. Construction of Evaluation Model Based on Entropy Weight TOPSIS

A weighted normalized evaluation matrix is constructed using the entropy weight (w_i), and the calculations are performed as shown in Equation (8). In this study, the degree of closeness (T_j) is used to indicate the intensity of the disaster, with values ranging from 0 to 1. The value approaching 1 indicates minimal disaster damage. The relevant calculation formulas are provided in Equations (9)–(13) [32].

$$Y=\left[\begin{array}{cccc}{v}_{11}{w}_1& v_{12}{w}_1& \cdot \cdot \cdot & v_{1n}{w}_1\\ v_{21}{w}_2& v_{22}{w}_2& \cdot \cdot \cdot & v_{2n}{w}_2\\ \cdot \cdot \cdot & \cdot \cdot \cdot & \cdot \cdot \cdot & \cdot \cdot \cdot \\ v_{m1}{w}_m& v_{m2}{w}_m& \cdot \cdot \cdot & v_{mn}{w}_m\end{array}\right]$$

(8)

$$T_j={\displaystyle \frac{{D_j}^{-}}{{D_j}^{+}+{D_j}^{-}}}$$

(9)

$${D_j}^{+}=\sqrt{{\displaystyle \sum _{i=1}^m{({y_i}^{+}-y_{ij})}^2}}$$

(10)

$${D_j}^{-}=\sqrt{{\displaystyle \sum _{i=1}^m{({y_i}^{-}-y_{ij})}^2}}$$

(11)

$$Y^{+}=\left\{\underset{1\le i\le m}{\max }{y}_{ij} | i=1,2,\cdot \cdot \cdot ,m\right\}=\left\{{y_i}^{+},{y_i}^{+},\cdot \cdot \cdot ,{y_m}^{+}\right\}$$

(12)

$$Y^{-}=\left\{\underset{1\le i\le m}{\min }{y}_{ij} | i=1,2,\cdot \cdot \cdot ,m\right\}=\left\{{y_i}^{-},{y_i}^{-},\cdot \cdot \cdot ,{y_m}^{-}\right\}$$

(13)

where Y⁺ and Y⁻ denote the positive and negative ideal solutions, respectively. Similarly, D_j⁺ and D_j⁻ represent the distances between the i-th indicator and y_i⁺ and y_i⁻, respectively.

2.9. Statistical Analysis

The experimental data were organized using Excel 2013 software, while OriginPro 9.0 Software (Origin Lab, Northampton, MA, USA) was employed for data visualization. Duncan’s multiple comparison method was utilized to assess the significance levels among multiple samples. A significance level of p < 0.05 was established. The data results presented in the text are expressed as the mean ± standard deviation (SD) of three biological replications, with three samples for each replication.

3. Results

3.1. Effects of Low-Temperature and Low-Illumination Stress on Photosynthetic Pigments of Strawberry Leaves During the Flowering Stage

Table 2. Changes in photosynthetic pigments in strawberry leaves under low-temperature and low-illumination stress.

Treatments	Chla (mg g−1)				Chlb (mg g−1)				Chla/b
	1d	3d	5d	7d	1d	3d	5d	7d	1d	3d	5d	7d
NT	3.21 ± 0.01 b	3.20 ± 0.01 b	3.22 ± 0.01 b	3.22 ± 0.02 b	1.14 ± 0.03 a	1.13 ± 0.01 a	1.13 ± 0.02 b	1.14 ± 0.01 b	2.82 ± 0.02 c	2.83 ± 0.01 c	2.85 ± 0.01 d	2.84 ± 0.01 c
LT	3.14 ± 0.02 c	3.12 ± 0.03 c	3.11 ± 0.02 c	3.08 ± 0.01 c	1.11 ± 0.01 a	1.03 ± 0.01 b	0.98 ± 0.01 c	0.82 ± 0.02 c	2.83 ± 0.01 a	3.03 ± 0.02 b	3.17 ± 0.02 b	3.15 ± 0.03 b
LI	3.29 ± 0.02 a	3.29 ± 0.01 a	3.47 ± 0.02 a	3.49 ± 0.03 a	1.16 ± 0.01 a	1.17 ± 0.01 a	1.22 ± 0.01 a	1.31 ± 0.01 a	2.84 ± 0.01 c	2.81 ± 0.01 c	2.97 ± 0.02 c	2.66 ± 0.01 d
LTLI	3.12 ± 0.01 c	3.08 ± 0.02 c	3.01 ± 0.01 d	2.96 ± 0.02 d	1.05 ± 0.01 b	0.99 ± 0.01 a	0.84 ± 0.02 d	0.78 ± 0.02 c	2.95 ± 0.02 b	3.11 ± 0.02 a	3.58 ± 0.03 a	3.79 ± 0.02 a

Note: Chl a—chlorophyll a, Chl b—chlorophyll b, Chla/b—the ratio of chlorophyll a to chlorophyll b, NT—normal temperature and light illumination conditions. The data in the table are the average values of three replicated samples. Different letters indicate significant differences under the same column at p < 0.05.

illustrates that, in comparison to the control group, the LTLI stress resulted in a reduction of Chla and Chlb content in strawberry leaves, while concurrently increasing the Chla/b ratio. As the duration of the treatment was extended, both Chla and Chlb contents progressively declined, whereas the Chla/b ratio exhibited an upward trend. Specifically, following LT stress treatment on days 1, 3, 5, and 7, Chla content decreased by 2.19%, 2.51%, 3.42%, and 4.34%, respectively, relative to the control. Similarly, Chlb content decreased by 2.63%, 8.85%, 13.27%, and 28.07%, respectively, and the Chla/b ratio increased by 0.36%, 7.07%, 11.23%, and 10.92%, respectively. The observed trends in Chla, Chlb, and Chla/b under LT treatment were largely consistent with those observed under LTLI treatment. Conversely, the trends observed under LI stress were entirely opposite to those under LTLI treatment. LI stress treatment led to an increase in Chla and Chlb contents in strawberry leaves, accompanied by a reduction in the Chla/b ratio. Over time, Chla and Chlb content continued to rise, while the Chla/b ratio declined. Following a 7d treatment with LI stress, the Chla and Chlb contents exhibited increases of 9.39% and 14.91%, respectively, relative to the control group. Conversely, the Chla/b ratio experienced a reduction of 6.34%. During the entire treatment duration, the Chla/b ratio consistently ranged from 2.66 to 2.84.

3.2. Effects of Low-Temperature and Low- Illumination Stress on Photosynthetic Gas Exchange Parameters of Strawberry Leaves During the Flowering Stage

Under conditions of LT, LI, and LTLI stresses, significant alterations were observed in the P_n, g_s, T_r, and C_i of strawberry leaves, as illustrated in Figure 1. Both LT and LI stresses led to a reduction in the P_n of strawberry leaves; however, the patterns of decrease exhibited distinct characteristics. Under LT stress, the P_n of strawberry leaves progressively declined with the duration of stress exposure, showing reductions of 16.77%, 41.62%, and 66.10% compared to the control at 3, 5, and 7 days of stress, respectively. In contrast, the decline in P_n under LI stress was relatively moderate. Under the combined stress of low temperature and low light, the P_n of strawberry leaves demonstrated a linear decrease, reaching 3.25 μmol m⁻² s⁻¹ by the end of the 7 d treatment, which constituted only 17.41% of the control group’s Pn. The g_s of strawberry leaves exhibited sensitivity to the treatments of LT, LI, and LTLI stresses, with a notable reduction observed by the third day of treatment. By the fifth day, g_s decreased by 63.52%, 48.31%, and 72.73% under LT, LI, and LTLI treatments, respectively, compared to the control. On the seventh day, g_s under LT and LI conditions showed a slight decline relative to the fifth day, whereas under LTLI stress, the stomata were nearly closed. The impact of LT, LI, and LTLI on the T_r mirrored the changes in g_s. As the experiment progressed, T_r exhibited a decreasing trend, with the extent of reduction following the order LTLI > LT > LI. LT, LI, and LTLI stresses exerted differential impacts on the C_i in strawberry leaves. Both LT and LI stresses significantly elevated the C_i in strawberry leaves, with the treatment group exhibiting higher levels than the control group throughout the experimental period. Under the LTLI stress, the C_i in the treatment group of strawberry leaves progressively decreased as the duration of the treatment extended.

3.3. Effects of Low-Temperature and Low-Illumination Stress on Chlorophyll Fluorescence Parameters of Strawberry Leaves During the Flowering Stage

Figure 2 illustrated the impact of LT, LI, and LTLI stress treatments on chlorophyll fluorescence parameters in the leaves of strawberry plants. In the control group, the F_v/F_m values of strawberry leaves remained between 0.80 and 0.82. In contrast, F_v/F_m values in strawberry leaves subjected to LT, LI, and LTLI stress treatments exhibited a reduction, with a continuous decline observed as the treatment duration increased. Notably, the variations in F_v/F_m values under LT, LI, and LTLI stress conditions were minimal throughout the experimental period.

Under LT stress, the NPQ of strawberry leaves showed a progressive increase, with increments of 29.73%, 50.22%, and 55.26% on days 3, 5, and 7, respectively, compared to the control. The NPQ trend under LTLI stress mirrored that of LT stress but demonstrated a more pronounced increase, reaching 63.16% above the control by the 7th day. Conversely, under LI stress, the NPQ increase in strawberry leaves was relatively gradual, remaining comparable to the control for the initial 5 days and rising by 3.51% on the 7th day of treatment. The pattern of variation in φPSII was entirely contrary to that observed in NPQ.

The ETR in strawberry leaves exhibited a declining trend as the treatment progressed. Under LT stress, the ETR of strawberry leaves significantly diminished starting from the fifth day of treatment, ultimately reaching a value of 1.98 by the conclusion of the experiment. In contrast, under LI stress, the reduction in ETR was comparatively moderate, with a decrease to 2.25 by the seventh day of treatment. Under LTLI stress, the ETR trend in strawberry leaves mirrored that observed under LT stress. By the seventh day of treatment, the ETR had decreased to 1.74, representing a 29.84% reduction compared to the control.

3.4. Effects of Low-Temperature and Low-Illumination Stress on Malondialdehyde and Proline Contents of Strawberry Leaves During the Flowering Stage

Figure 3 illustrated the impact of LT, LI, and LTLI stress treatments on MDA and Pro contents in strawberry leaves. Both LT and LTLI stress treatments resulted in a significant elevation of MDA levels in the leaves. Throughout the experimental period, the treatment groups consistently exhibited higher MDA levels compared to the control group at corresponding time points. However, no significant difference was observed between the LT and LTLI treatments at any given time. Under LI stress treatment, MDA content showed a slight increase during the initial 5 days, which was not statistically significant compared to the control. Nevertheless, a significant increase was observed after 7 days of LI stress. Throughout the treatment period, MDA levels under LI stress remained significantly lower than those under LT and LTLI stress treatments at corresponding time points. The alterations in Pro content under LT, LI, and LTLI stress treatments mirrored the patterns observed in MDA content. Specifically, Pro content under LT and LTLI stress treatments was significantly elevated compared to the control from day 3 onwards, whereas a significant increase in Pro content under LI stress treatment was not observed until day 7.

3.5. Effects of Low-Temperature and Low-Illumination Stress on Reactive Oxygen Species of Strawberry Leaves During the Flowering Stage

The impact of LT, LI, and LTLI stress treatments on the levels of H₂O₂ and O₂⁻ in strawberry leaves was illustrated in Figure 4. Under the stress conditions of LT, LI, and LTLI, the H₂O₂ content in strawberry leaves exhibited an increase, which further escalated with the extension of the treatment duration. On the initial day of stress exposure, no significant difference was observed in the H₂O₂ content of strawberry leaves subjected to LT, LI, and LTLI stress treatments compared to the control group. However, by the third and fifth days of stress, the H₂O₂ content in strawberry leaves was markedly higher under LT and LTLI stress treatments compared to the LI treatment group and the control. No significant difference in H₂O₂ content was detected between the LT and LTLI stress treatments, nor between the LI treatment group and the control group. By the seventh day of stress, the effects of LT, LI, and LTLI stress treatments on H₂O₂ levels in strawberry leaves were significantly elevated compared to the control group, with increases of 16.78%, 5.10%, and 23.49%, respectively. The pattern of O₂⁻ production paralleled the changes observed in H₂O₂ content.

3.6. Effects of Low-Temperature and Low-Illumination Stress on Antioxidant Enzyme Activity of Strawberry Leaves During the Flowering Stage

Under the stress conditions of LT, LI, and LTLI, significant alterations were observed in the levels of SOD, CAT, and POD in strawberry leaves (

Table 3. Changes in antioxidant enzyme activity in strawberry leaves under low-temperature and low-illumination stress.

Treatments	SOD (U g−1 min−1)				CAT (U g−1 min−1)				POD (U g−1 min−1)
	1d	3d	5d	7d	1d	3d	5d	7d	1d	3d	5d	7d
NT	61.31 ± 0.02 b	61.28 ± 0.03 c	60.90 ± 0.02 d	61.89 ± 0.03 b	12.32 ± 0.02 c	12.32 ± 0.01 d	12.32 ± 0.01 d	12.41 ± 0.02 b	0.18 ± 0.01 c	0.18 ± 0.01 c	2.01 ± 0.02 d	0.19 ± 0.01 d
LT	62.41 ± 0.02 a	69.23 ± 0.03b	74.31 ± 0.02 b	61.22 ± 0.03 c	13.12 ± 0.01 b	14.32 ± 0.02 b	15.22 ± 0.02 b	12.11 ± 0.02 c	2.03 ± 0.01 a	2.94 ± 0.02 b	3.45 ± 0.02 b	2.12 ± 0.01 c
LI	61.22 ± 0.03 b	64.77 ± 0.01 c	65.31 ± 0.01 c	66.89 ± 0.01 a	12.62 ± 0.02 c	12.74 ± 0.02 c	13.12 ± 0.02 c	13.12 ± 0.02 a	0.19 ± 0.01 c	2.21 ± 0.01 c	2.37 ± 0.01 c	2.46 ± 0.02 b
LTLI	62.44 ± 0.01 a	77.89 ± 0.02 a	83.87 ± 0.01 a	61.23 ± 0.02 c	13.52 ± 0.01 a	15.24 ± 0.01 a	16.43 ± 0.02 a	11.08 ± 0.02 c	2.55 ± 0.01 b	3.18 ± 0.01 a	4.65 ± 0.02 a	2.99 ± 0.01 a

Note: SOD—superoxide dismutase, POD—peroxidase, CAT—catalase. The data in the table are the average values of three replicated samples. Different letters indicate significant differences under the same column at p < 0.05.

). Both LT and LI stress treatments resulted in elevated SOD levels; however, the patterns of increase differed between the two groups. Specifically, under LT stress, SOD levels in strawberry leaves progressively rose with the duration of stress exposure, consistently remaining above the control levels throughout the treatment period. In contrast, the increase in SOD levels under LI stress was more gradual. Under LTLI stress, SOD levels exhibited a linear increase, surpassing the control by 18.43%, 27.11%, 37.72%, and 52.25% at 1, 3, 5, and 7 days of treatment, respectively. The trends observed for CAT and POD were analogous to those of SOD, with increases noted as stress duration was extended under LT, LI, and LTLI treatments. Notably, the increases in CAT and POD under LT and LTLI stress were substantially greater than those observed under LI stress.

3.7. A Quantitative Assessment of the Physiological Impact on Strawberry Plants Subjected to Low-Temperature and Low-Illumination Conditions

Table 4. Comprehensive assessment of stress levels across various treatments and durations.

Treatments	Comprehensive Evaluation Value of Stress Levels
	1d	3d	5d	7d
LT	0.87	0.82	0.79	0.63
LI	0.96	0.95	0.93	0.92
LTLI	0.87	0.80	0.79	0.60

provided a detailed account of the stress level scores across various treatments and durations, with values ranging from zero to one. The score approaching one indicates minimal stress-induced damage. The data in Table 4 clearly demonstrate that, under identical stress treatments, the damage to the photosynthetic organs of strawberry plants escalated as the duration of stress exposure increased. When considering treatments of equal duration, the LTLI treatment results in the most significant damage, followed by the LT treatment, with the LI treatment causing the least damage.

4. Discussion

Chlorophyll is a critical component of photosynthesis in higher plants and serves as one of the most active light quantum receptors in nature [33]. The present study demonstrated that LI stress treatment led to an increase in Chla and Chlb contents in strawberry leaves, accompanied by a reduction in the Chla/b ratio. Furthermore, with prolonged treatment duration, the content of Chla and Chlb continued to rise, while the Chla/b ratio exhibited a decreasing trend (Table 2). The findings of this study align with those of Longo et al. [34] and Singh at al. [35], potentially due to the differential absorption properties of Chla and Chlb, as more Chlb helps utilize blue-violet light, boosting light-harvesting efficiency, enhancing chloroplasts’ ability to reduce 2,6-dichloroindophenol, and increasing photosynthetic activity. Additionally, the study observed that under LT conditions, the Chla and Chlb contents in strawberry leaves decreased (Table 2). The findings of various scholars regarding the causes of chlorophyll reduction at low temperatures are inconsistent. It is widely accepted that the decline in chlorophyll content under LT conditions is not attributed to the inhibition of the pigment synthesis system, but rather to the degradation of existing chlorophyll [13].

Photosynthesis is vital for plant growth and yield, and changes in its activity under adverse stress can indicate plant stress resistance [36,37]. In this study, stresses such as LT, LI, and LTLI were found to reduce the P_n in strawberry leaves (Figure 1a). The reduction in P_n was relatively moderate under LI stress, whereas LT stress exerted a more pronounced impact on strawberries compared to LI stress. However, under LTLI stress, the P_n of strawberry leaves exhibited a linear decline, approaching zero by the seventh day of treatment. This observation suggested that LTLI stress severely impairs the carbon assimilation process in strawberry photosynthesis. These findings are consistent with those reported by Xu et al. [10]. The decrease in P_n due to stress is primarily attributable to two factors [38]. Firstly, the reduction in stomatal opening and conductance limits the influx of external CO₂ into the leaves, thereby affecting the intensity of photosynthesis, which is referred to as a stomatal factors limitation. Secondly, the diminished photosynthetic activity of mesophyll cells impedes the utilization of CO₂, resulting in decreased photosynthesis, which is referred to as a non-stomatal factors limitation. This study demonstrated that under LT and LTLI stress conditions, an increase in stress intensity resulted in a significant decline in P_n, accompanied by a gradual reduction in g_s and T_r, as well as a gradual increase in C_i(Figure 1b–d). This suggests that the decrease in P_n is primarily attributable to non-stomatal factors. Conversely, under LI stress, the significant reduction in P_n was accompanied by a gradual decrease in g_s and T_r and a gradual increase in C_i, indicating that the decline in P_n is mainly due to stomatal factors. Xu et al. [27] conducted a study on citrus, revealing that under severe LT stress, P_n was initially restricted by stomatal factors and subsequently by non-stomatal factors in the later stages of stress. The reduction in P_n was the result of the combined effects of both factors, which contrasts with the findings of this study. This discrepancy may be associated with the intensity or duration of the low-temperature stress.

Photoinhibition is a prevalent phenomenon in the photosynthetic process of higher plants [39]. During photoinhibition, excessive light energy not only reduces the F_v/F_m but also inflicts varying degrees of damage to the photosynthetic apparatus in leaves [40,41]. Research conducted by Yang et al. [42] demonstrated that under LI conditions, the F_v/F_m of cucumber leaves exhibited minimal change, suggesting that appropriate shading can enhance the photochemical efficiency of PSII and mitigate photoinhibition. The findings of this study align with the results of our experiment. The application of LTLI stress led to a marked reduction in F_v/F_m in strawberry plants, whereas other treatments, particularly those involving LI stress conditions, exerted a comparatively minor effect on F_v/F_m (Figure 2a). This suggested that LTLI stress induces more pronounced photoinhibition. The minor drop in F_v/F_m observed with other treatments might serve as a photoprotective mechanism, with the decline in photosynthetic activity primarily resulting from diminished dark reaction processes. NPQ reflects the dissipation of light energy absorbed by PSII pigments as heat energy [43]. Under LT conditions, plant defense mechanisms against photo damage, including the lutein cycle, high-energy state quenching, and enhanced non-radiative energy dissipation in thylakoid membranes, contribute to an increase in the NPQ [44]. Dong et al. [45] observed an increase in NPQ in tomato, while Ashrostaghi et al. [46] reported a similar increase in sweet potatoes under LT conditions. This study revealed that LTLI stress contributes to a continuous increase in NPQ in strawberries (Figure 2b). This indicated that strawberry leaves absorb excess energy and dissipate it as heat, protecting against photosynthetic damage from excessive light energy. Additionally, the present study revealed a significant reduction in both φPSII and ETR under LTLI stress treatment (Figure 2c,d), suggesting that LTLI stress reduces the reoxygenation ability of the primary electron receptor QA within PSII, weakening electron transfer activity. The impediment in transferring electrons from the reduced state QA to subsequent electron transporters disrupts the overall photosynthetic electron transfer process, thereby inhibiting electron transfer in strawberry leaves. Notably, similar findings have been corroborated in grapevines [47].

ROS are harmful byproducts of oxygen metabolism that damage cells and also regulate signaling pathways during stress [48]. Typically, the production and scavenging of ROS in plants are maintained in a state of dynamic equilibrium [49,50]. In this study, LT and LTLI treatments significantly elevated the rate of O₂⁻ production and the content of H₂O₂ in strawberry leaves (Figure 4), resulting in the accumulation of MDA (Figure 3a). These findings align with previous research conducted by Xu et al. [10]. Malondialdehyde (MDA) is a key byproduct of plant lipid peroxidation that affects intracellular proteins and cellular membrane integrity, making it an important biomarker for evaluating oxidative stress in plants [51,52]. Pro is a critical organic osmoregulatory compound in plants [53,54]. Studies have demonstrated that proline plays a role in regulating the stability of plant cell membranes, scavenging reactive oxygen species, and stabilizing cell structures [55,56]. Under LT stress, elevated Pro levels enhance cellular or tissue water retention capacity and function as a protective agent for enzymes and cell structures, in addition to serving as a carbohydrate source [57]. Research by Araz et al. [58] indicates that under low-temperature conditions, peppers alleviate cellular dehydration by enhancing proline production, thereby mitigating cellular damage induced by LT stress. Furthermore, plants synthesize substantial quantities of ROS scavengers, including SOD, POD, and CAT, to mitigate ROS-induced damage to cell membranes and preserve cellular stability [59]. This study observed that under LT and LTLI treatments, the activities of SOD, POD, and CAT initially increased and subsequently decreased (Table 3). This phenomenon may be attributed to the initial upregulation of ROS scavenging enzymes in strawberry leaf cells as an adaptive response to counteract various stress conditions. However, with prolonged exposure to LT or LTLI stress, the accumulation of ROS in strawberry leaf cells surpasses the antioxidant capacity of enzymes such as SOD. This imbalance leads to membrane lipid peroxidation and damage to the membrane system or other cellular organelles, ultimately compromising the protective mechanisms of strawberries and diminishing their ability to withstand adverse conditions.

5. Conclusions

This study examined the impact of low temperature and low illumination intensity on gas exchange parameters, chlorophyll fluorescence parameters, reactive oxygen species, and antioxidant enzyme activity in strawberry plants during the flowering period. The findings indicated that LTLI stress significantly decreased the net photosynthetic rate, stomatal conductance, transpiration rate, maximum photochemical efficiency of photosystem II, photosynthetic electron transport rate, and actual photochemical quantum efficiency, but increased intercellular carbon dioxide concentration, non-photochemical quenching coefficient, and the levels of malondialdehyde, proline, hydrogen peroxide, and peroxide ions. Additionally, LTLI stress initially enhanced the activity of superoxide dismutase, peroxidase, and catalase, but subsequently led to a decline in their activity. The combined stress of LT and LI exerts a more pronounced effect on the photosynthetic physiological processes of strawberry leaves during flowering than either stressor alone. These findings provide theoretical support for preventing and managing low-temperature, low-light disasters and assessing their impact on strawberries grown in controlled environments.