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Exogenous diethyl aminoethyl hexanoate alleviates the damage caused by low-temperature stress in Phaseolus vulgaris L. seedlings through photosynthetic and antioxidant systems

BMC Plant Biology volume 25, Article number: 75 (2025) Cite this article

Abstract

Background

Phaseolus vulgaris is a warm-season crop sensitive to low temperatures, which can adversely affect its growth, yield, and market value. Exogenous growth regulators, such as diethyl aminoethyl hexanoate (DA-6), have shown potential in alleviating stress caused by adverse environmental conditions. However, the effects that DA-6 has on P. vulgaris plants subjected to low-temperature stress are not well understood. This study aimed to investigate the impact DA-6 has on the growth, photosynthesis, antioxidant system, and gene expression in cold-tolerant (YJ009763) and cold-sensitive (Baibulao) P. vulgaris seedlings under low-temperature stress.

Results

To simulate low-temperature stress, P. vulgaris seedlings were exposed to 5 °C, and 25 mg/L DA-6 solution applied to their leaves. This study revealed that DA-6 significantly enhanced the growth and photosynthetic performance of P. vulgaris seedlings under low-temperature stress. Specifically, DA-6 increased chlorophyll content and photosynthetic rates, reducing stomatal limitation and enhancing carbon assimilation. It also improved the photosynthetic efficiency by boosting electron transport in the reaction center. The antioxidant enzyme activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were markedly increased following DA-6 treatment. After 24 h of low-temperature stress, the cold-tolerant seedlings showed a 68.95% increase in POD activity, whereas the cold-sensitive seedlings displayed a 160.63% increase in SOD activity and an 85.56% increase in CAT activity. In addition, DA-6 significantly reduced the production rate of superoxide anion radical generation, with a 25.24% reduction in cold-tolerant seedlings and a 49.38% reduction in cold-sensitive seedlings. Under low-temperature stress, exogenous DA-6 could upregulate the relative expression of antioxidant enzyme-related genes, such as PvSOD and PvAPX. DA-6 also promoted the expression of key antioxidant genes, including PvMDHAR and PvDHAR2, which accelerated the ascorbate-glutathione cycle and mitigated oxidative stress.

Conclusion

Exogenous application of DA-6 effectively alleviates low-temperature stress in P. vulgaris by enhancing photosynthetic capacity and regulating the antioxidant defense system. Cold-tolerant varieties exhibited a stronger response to DA-6, demonstrating a greater ability to withstand cold stress. These findings suggest that DA-6 treatment could serve as a promising approach for improving the resilience of P. vulgaris to low temperatures.

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Introduction

Low-temperature stress can be classified into two main categories: chilling injury (0–15 ℃) and freezing injury (< 0 °C) [1, 2]. Crops, fruit trees, and even vegetables can be damaged by low temperatures. Low temperatures can cause various adverse effects on plants, including leaf wilting, necrosis, reduction in chlorophyll content, damage to the photosynthetic apparatus, and a decline in photosynthetic capacity. This can lead to an increase in the formation of reactive oxygen species (ROS) [3,4,5,6]. Under cold stress, plants induce physiochemical processes that help them grow and develop, promoting stress tolerance, i.e., increased antioxidant activity, which scavenges ROS and protects cells from oxidative damage [7]. Plants can mitigate the detrimental effects of ROS through a variety of physiological adaptations. Non-enzymatic components of the antioxidant defense system include l-ascorbic acid (AsA) and glutathione (GSH), whereas enzymatic antioxidant components include superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate POD (APX), and GSH reductase (GR) [8, 9]. The activities of SOD, APX, and CAT in maize seedlings were found to be significantly enhanced after exposure to low-temperature stress at 2 ℃ for different periods, which promoted resistance to the damage caused by ROS [10]. The content of hydrogen peroxide (H₂O₂) in Phaseolus vulgaris seedlings increases under low-temperature stress, and its accumulation aggravates membrane lipid peroxidation and causes damage to P. vulgaris [11]. The AsA-GSH cycle represents a non-enzymatic scavenging system that enables plants to scavenge endogenously produced ROS. Low temperatures have been demonstrated to impair efficiency of the AsA-GSH cycle in P. vulgaris seedlings, leading to adverse effects on plant health and development [12]. In response to cold stress conditions, plants upregulate the expression of genes involved in membrane protection and stress responses, such as those encoding antioxidant enzymes and heat shock proteins [13].

A simple and effective method to improve plant stress resistance (such as resistance to cold, drought, salts, etc.) is by using exogenous growth regulators. Exogenous application of chitosan oligosaccharide has been demonstrated to enhance the photosynthetic rate (Pn) and antioxidant activity of wheat leaves under low-temperature stress conditions [14]. Exogenous spraying of AsA has been demonstrated to enhance photosynthesis and antioxidant capacity, improving the response of tolerant beans to low-temperature stress [15]. Diethyl aminoethyl hexanoate (DA-6) is a novel plant growth regulator [16] characterized by a high efficiency, low cost, and low residue levels [17, 18]. Previous studies have shown that exogenous DA-6 activates chlorophyll synthesis and the expression of photosystem-related genes, resulting in higher photosynthetic activity and chlorophyll yield. DA-6 acts on tomatoes to regulate the synthesis and expression of endogenous cytokinins in tomato leaves under low night temperature stress, stabilize chloroplast structure, reduce oxidative damage, and maintain leaf photochemical activity [19]. DA-6 acts on maize and soybean to accelerate chloroplast biosynthesis and promote seedling growth by altering photosynthesis [20]. The application of DA-6 on Chinese cabbage can improve its yield and quality [21]. At present, comprehensive knowledge of the effects DA-6 has on P. vulgaris is limited; therefore, this study aimed to explore the cold resistance mechanism of DA-6 on two extreme P. vulgaris varieties. Spraying a low concentration of DA-6 at the two-leaf stage of wild barley was found to significantly accelerate seedling growth, and spraying a high concentration of DA-6 at the four-leaf stage significantly increased seedling biomass and improved photosynthesis [22].

The P. vulgaris plant is adapted to warmer climates and is not tolerant to cold conditions; its seedlings are susceptible to chilling injury at 8 °C, which results in alterations to cell membrane permeability, diminished chlorophyll content, and an imbalance in active oxygen metabolism systems. Low-temperature stress exerts a significant constraint on the quality and yield of P. vulgaris [23, 24]. The objective of this study was to elucidate the physiological mechanism through which exogenous DA-6 enhances the cold tolerance of bean seedlings and to investigate whether DA-6 regulates cold tolerance through photosynthetic and antioxidant pathways. These findings would provide a theoretical foundation for further research on the high-yield and high-quality cultivation of vegetables under low-temperature stress.

Materials and methods

Test materials

In the pre-experimental treatment, cold-tolerant ‘YJ009763’ and cold-sensitive ‘Baibulao’ P. vulgaris beans were screened from 31 beans. All P. vulgaris seeds and materials were sourced from the Sichuan Agricultural University College of Horticulture Vegetable Research Laboratory (Chengdu, China).

Test method

Only cold-tolerant ‘YJ009763’ and cold-sensitive ‘Baibulao’ P. vulgaris full seeds that exhibited a consistent size were selected. Seeds were soaked at 25 °C for 2 h, whereafter they were germinated in a 25 °C artificial incubator. After germination, the P. vulgaris seeds were sown. The matrix formula used comprised peat: vermiculite: perlite (3:1:1), and routine management was performed during seedling raising. When the seedlings grew to the two-leaf stage (7-days-old), P. vulgaris plants of the same growth stage were selected and randomly divided into two groups (control and treatment groups). In the early stage, two varieties were sprayed with different concentrations of DA-6 throughout the experiments. When the spraying concentration was 25 mg/L, the soluble sugar, protein, and proline contents of the two varieties reached peak levels compared with when other DA-6 concentrations were sprayed. Therefore, the treatment group was sprayed with 25 mg/L DA-6, and leaves of the control group sprayed with deionized water. Plants were sprayed once every night for three consecutive days; thereafter placed in an artificial climate chamber with a temperature of 25/18°C, light cycle of 12 h/12 h (day/night), light intensity of 300 µmol·m− 2 s− 1, and relative humidity of 80%; and allowed to adapt under these conditions for 24 h before low-temperature treatment. The low-temperature conditions included a climate chamber temperature of 5/5°C and light cycle of 12 h/12 h (day/night), where the light exposure remained unchanged during this period.

The specific treatments were as follows. The control group comprised deionized water and cold-tolerant P. vulgaris varieties (Nck), whereas the treatment group comprised DA-6 and cold-tolerant bean varieties (Nt). Cold-sensitive bean varieties (Sck) were used in the control group, and cold-sensitive bean varieties (St) used in the treatment group.

Each of the above treatments were repeated thrice, with 40 plants per repetition, for a total of 120 plants per treatment. Therefore, the total number of plants in the four treatments was 480. At 0, 6, 12, 24, and 48 h of low-temperature treatment, the plant height, stem diameter, aboveground dry weight, and fresh weight of P. vulgaris plants were measured. Fresh leaves were obtained for the determination of photosynthetic pigments and relative conductivity, as well as to measure the photosynthetic gas exchange and chlorophyll fluorescence parameters of P. vulgaris leaves. Young P. vulgaris leaves were rapidly frozen in liquid nitrogen and stored in an ultralow temperature refrigerator at − 80 °C for the determination of other physiological indicators.

The leaves of P. vulgaris seedlings at 0 and 24 h were rapidly frozen in liquid nitrogen and stored in an ultralow temperature refrigerator at − 80 °C for RT-qPCR analysis to determine the relative expression of AsA-GSH cycle-related genes because gene-expression responses to stress are typically observed within the first 24 h, at which time the necessary metabolic reactions also take place.

Determination of test indexes

Determination of the vegetative growth index of bean seedlings

Plant height was determined via direct measurement with a ruler, and stem diameter assessed using a Vernier caliper. The plants were subjected to a cleansing process involving the use of distilled water, after which they were separated into two portions: aboveground and underground portions. Fresh weight was subsequently determined, after which the plants were subjected to heat treatment at 105 °C for 15 min and subsequently dried to a constant weight at 75 °C. The dry weight was then determined.

Determination of related indexes of the photosynthetic system in leaves of Phaseolus vulgaris seedlings

Measurement of gas exchange parameters: The photosynthetic gas exchange parameters of bean seedlings were determined using a portable photosynthetic instrument (LI-6400XT; LI-COR, St. Lincoln, NE, USA). The PAR and actinic light intensity value in the photosynthetic apparatus was set to 1000 µmol·m− 2·s− 1. The measured parameters included Pn, stomatal conductance (Gs), transpiration rate (Tr), and the intercellular CO2 concentration (Ci).

Chlorophyll fluorescence parameters were measured using a PAM2500 high-performance chlorophyll fluorometer (Walz, Germany) with the saturated pulse analysis method. Following a 30-min period of dark adaptation, the leaves were irradiated to detect light, and the slow kinetic curve subsequently measured. The chlorophyll fluorescence kinetic parameters included the maximum fluorescence yield (Fm), maximum photochemical efficiency of PSII (Fv/Fm), photosynthetic electron transport rate (ETR), photochemical quenching coefficient (qP), non-photochemical quenching (NPQ), and effective photochemical efficiency of PSII under light (Y(II)). The determination time for these parameters was 10 min [25].

Determination of photosynthetic pigment content in leaves: The total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid contents were determined using the ethanol/acetone volumetric mixing method [26]. Fresh leaf samples (0.2 g) were collected and divided into three parts, with the requisite reagents added to grind the tissue until it turned white. The absorbance at A663, A645, and A470 was measured using a spectrophotometer, and the colors compared.

Total chlorophyll concentration: CT (mg/L) = Ca + Cb = 20.29 A645 + 8.05 A663.

Chlorophyll a concentration: Ca (mg/L) = 12.72 A663 − 2.59 A645.

Chlorophyll b concentration: Cb (mg/L) = 202.88 A645 − 4.67 A663.

Carotenoid concentration: Cc (mg/L) = 4.367 A470 − 0.014 × Ca − 0.454 × Cb.

Chlorophyll content (mg/g) = (C × V × N)/(m × 1000).

In the formulae above, A470, A663, and A645 represent the absorbance of the extraction solution at wavelengths of 470, 663, and 645 nm, resepectively; C the pigment content (mg/L); V the volume of the extraction solution (mL); N the dilution factor; M the sample mass (g); and 1000 represents 1 L = 1000 mL.

Determination of the related indicators of ROS byproducts in leaves of Phaseolus vulgaris seedlings

The production rate of O2− was determined via spectrophotometry [27], and the H2O2 content through fluorescence spectrometry [28]; 2 g plant tissue was required for these measurements.

Determination of antioxidant enzyme activity in leaves of Phaseolus vulgaris seedlings

The activity of SOD was determined via the nitroblue tetrazolium photochemical method [29], POD activity via the guaiacol method [30], and CAT activity using the ultraviolet absorption method [31]. Plant tissue (1 g) was required for these measurements.

Determination of AsA-GSH cycle-related indicators in bean seedling leaves

The contents of AsA, GSH, dehydroascorbic acid (DHA), and oxidized GSH (GSSG) were determined using a kit (Solarbio, Beijing, China); the contents of AsA + DHA and GSH/GSSG were calculated [32]; and the activities of APX, GSH peroxidase (GPX), GSH S-transferase (GST), monodehydroascorbate reductase, dehydroascorbate reductase (DHAR), and GR were measured using a kit (Solarbio) and calculated [33]. Plant tissue (0.1 g) was required for these measurements.

Validation of antioxidant and AsA-GSH system-related genes in leaves of Phaseolus vulgaris seedlings via RT-qPCR

Genes related to low-temperature stress and antioxidants were selected for RT-qPCR. Primers were designed on NCBI, and the specific primer sequences are shown in Table 1. RT-qPCR was performed using the Bimake kit. The RT-qPCR amplification and sequencing conditions were as follows: initial denaturation at 95 °C for 3 min, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s.

Table 1 Specific primers used for RT-qPCR
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Statistical analysis

All data were organized and analyzed using Excel 2019 software, assisted by the SPSS 22.0 program for variance and correlation analysis. Duncan’s multiple range method was used for multiple comparisons, with a significance level set at P < 0.05. Data are presented as mean ± standard deviation (n = 3).

Results

Effects of exogenous DA-6 on growth and biomass of Phaseolus vulgaris seedlings under low-temperature stress

As illustrated in Table 2, exogenous DA-6 treatment markedly enhanced the shoot dry weight of cold-tolerant bean seedlings by 30.43% and the root dry weight by 10.64% under low-temperature conditions at 0 h. However, no notable impact was observed on the plant height, stem diameter, or fresh weight of both the shoot and root portions. Following a 48-h period of exposure to low-temperature stress, the shoot fresh weight of cold-tolerant seedlings significantly decreased by 27.61% compared with that of the control group maintained under low-temperature conditions for 0 h. Following the exogenous application of DA-6, the plant height of the leaves of cold-tolerant P. vulgaris seedlings remained largely unchanged compared with that of the control group at the corresponding time point. Following a 12-h period of stress, the stem diameter exhibited a significant increase of 15.05% compared with that of the low-temperature control group at the same time point. At 48 h of stress, the shoot dry weight significantly increased by 54.34% compared with that of the low-temperature control group at the same time point. In addition, the root fresh weight significantly increased by 39.80%, and the root dry weight exhibited a significant increase of 92.11%.

Table 2 Effects of DA-6 on growth and biomass of cold-tolerant common bean seedlings under low-temperature stress
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As illustrated in Table 3, exogenous DA-6 markedly enhanced the shoot fresh weight of cold- sensitive bean seedlings by 52.52% and the root dry weight by 88.36% under low-temperature conditions at 0 h. However, this treatment did not have a notable effect on plant height, stem diameter, or underground dry and fresh weights. Following a 12-h period of exposure to low-temperature stress, a significant reduction in the root fresh weight was observed, with a 13.37% decrease compared with that of the control group maintained under low-temperature conditions for 0 h. Following a 48-h period of exposure to low-temperature stress, the root dry weight exhibited a significant reduction of 54.76% compared with that of the control group maintained under low-temperature conditions for 0 h. The application of low-temperature stress did not result in significant changes in plant height, stem diameter, or shoot fresh weight. In the context of low-temperature stress, the exogenous application of DA-6 did not result in a significant change in plant height or stem diameter in cold-sensitive bean seedlings when compared with those of the low-temperature control group at the same time point. At the 6-h mark, the fresh weight of the aboveground portion exhibited a notable increase of 62.77% compared with that of the low-temperature control group, whereas the dry weight of the same region demonstrated a significant increase of 63.36%. Following a 24-h period of stress exposure, the root fresh and dry weights exhibited significant increases of 48.04% and 100.00%, respectively, compared with those of the low-temperature control group at the same time point.

Table 3 Effects of DA-6 on growth and biomass of cold-sensitive common bean seedlings under low-temperature stress
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Effects of exogenous DA-6 on photosynthetic pigments in leaves of Phaseolus vulgaris seedlings under low-temperature stress

As illustrated in Fig. 1, the application of 25 mg/L DA-6 at standard temperatures did not elicit a notable effect on chlorophyll a, chlorophyll b, chlorophyll a + b, and carotenoid concentrations in cold-tolerant bean leaves when compared with those of the control group (Fig. 1A, C, E, and G). Following exposure to low temperatures, the concentrations of chlorophyll a, chlorophyll b, and chlorophyll a + b in the leaves of cold-tolerant beans initially declined and then exhibited a gradual increase with prolongation of the stress duration. In the context of low-temperature stress, the exogenous application of DA-6 enhanced the levels of chlorophyll a, chlorophyll b, and chlorophyll a + b in the leaves of cold-tolerant bean seedlings while concurrently reducing the concentration of carotenoids. Following a 12-h period of stress exposure, the concentration of chlorophyll a in the leaves of cold-tolerant beans exhibited a notable increase of 3.87% compared with that in the low-temperature control group at the corresponding time point (Fig. 1A). At 24 h of stress exposure, the chlorophyll b content significantly increased by 54.91% compared with that in the low-temperature control group (Fig. 1C). Similarly, chlorophyll a + b content showed a notable increase of 19.40% (Fig. 1E). Conversely, the carotenoid content exhibited a pronounced decline of 61.84% (Fig. 1G).

As illustrated in Fig. 1, under standard temperatures, the application of 25 mg/L DA-6 resulted in a notable increase in the chlorophyll a + b content in the leaves of cold-sensitive bean seedlings, with a 13.48% enhancement (Fig. 1F), whereas the carotenoid content exhibited a pronounced reduction of 66.48% (Fig. 1H). The chlorophyll a and chlorophyll b content was not significantly altered (Fig. 1B and D). Following exposure to low temperatures, the levels of chlorophyll a, chlorophyll b, and chlorophyll a + b in leaves of the cold-sensitive bean seedlings declined. However, this decline was not consistent, but showed a biphasic pattern, decreasing and then increasing with the prolongation of stress time. This trend was similar to that observed for the cold-tolerant beans. Exogenous application of DA-6 enhanced the levels of chlorophyll a, chlorophyll b, and chlorophyll a + b in the leaves of cold-sensitive P. vulgaris seedlings, concomitant with a reduction in carotenoid content. Following a 6-h period of stress exposure, the concentration of chlorophyll b exhibited a marked increase of 114.29% compared with that in the low-temperature control group (Fig. 1D). At the 24-h mark, the content of chlorophyll a + b demonstrated a notable increase of 43.37% in relation to that in the low-temperature control group (Fig. 1F), whereas the carotenoid concentration exhibited a pronounced decline of 35.96% (Fig. 1H).

Fig. 1
figure 1

Effects of DA-6 on photosynthetic pigments in bean seedlings under low-temperature stress. a–b, chlorophyll a content of (a) cold-tolerant and (b) cold-sensitive beans; c–d, chlorophyll b content of (c) cold-tolerant and (d) cold-sensitive beans; e–f, chlorophyll a + b content of (e) cold-tolerant and (f) cold-sensitive beans; g–h, carotenoid content of (g) cold-tolerant and (h) cold-sensitive beans. Nck, cold-tolerant variety sprayed with equal amounts of clean water, low-temperature control; Nt, cold-tolerant variety sprayed with DA-6; Sck, sensitive variety sprayed with an equal amount of clean water, low-temperature control; St, sensitive variety sprayed with DA-6. The abscissa represents the exposure time to low-temperature stress, and the ordinate the value of each parameter. Data are presented as the means ± SD of three different experiments with three replicated measurements; different letters within rows indicate significant differences (P < 0.05) of variation

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Effects of exogenous DA-6 on photosynthetic (gas exchange) parameters of Phaseolus vulgaris seedling leaves under low-temperature stress

As illustrated in Fig. 2, at low temperatures for 0 h, the application of 25 mg/L DA-6 resulted in a notable increase in the Gs of cold-tolerant bean seedling leaves, reaching 144.45% that of the control group (Fig. 2C). Similarly, a significant 50.18% increase in Tr was observed (Fig. 2G), with no discernible impact on Pn and Ci (Fig. 2A and E). Following exposure to low-temperature stress, the Pn, Gs, and Tr of cold-tolerant bean seedlings exhibited a decline when compared with those of the control group maintained under low temperatures for 0 h. Conversely, Ci initially decreased and then increased. Under low-temperature stress, exogenous spraying of DA-6 increased the Pn, Gs, and Tr in leaves of cold-tolerant bean seedlings, whereas the Ci was reduced. Following a 24-h period of low-temperature stress, the Pn of these leaves significantly increased by 427.63% (Fig. 2A), Gs increased by 584.32% (Fig. 2C), and Tr increased by 275.32% (Fig. 2G). After 48 h of stress exposure, the concentration of CO₂ in the leaves of cold-tolerant bean seedlings was significantly reduced by 42.29% (Fig. 2E).

As illustrated in Fig. 2, under low temperatures for 0 h, the application of 25 mg/L DA-6 resulted in a notable enhancement in the photosynthetic performance of cold-sensitive bean seedlings. This was evidenced by a 35.3% increase in leaf Pn (Fig. 2B), a 90.1% surge in Gs (Fig. 2D), a 98.2% increase in Tr (Fig. 2H), and a relatively minimal change in Ci (Fig. 2F). As the duration of low-temperature stress was extended, the Pn and Tr declined, followed by an increase and a subsequent decline. The Ci increased, decreased, and subsequently increased, whereas the Gs gradually declined. The exogenous application of DA-6 showed a significant enhancement in the photosynthetic efficiency of cold-sensitive bean seedlings under low-temperature stress. This was evidenced by a notable increase in the maximum photochemical efficiency of Pn, accompanied by a substantial reduction in the Ci and Tr. At 12 h of stress exposure, the Pn of cold-sensitive bean seedlings exhibited a marked increase of 256.3% compared with that of the low-temperature control group (Fig. 2B). Concurrently, the Ci concentration declined significantly by 30.7% (Fig. 2F), and the Tr decreased significantly at 24 h and 48 h.

Fig. 2
figure 2

Effects of DA-6 on photosynthetic (gas exchange) parameters of common bean seedlings under low-temperature stress. a–b, Pn values of (a) cold-tolerant and (b) cold-sensitive beans; c–d, Gs values of (c) cold-tolerant and (d) cold-sensitive beans; e–f, Ci values of (e) cold-tolerant and (f) cold-sensitive beans; g–h, Tr values of (g) cold-tolerant and (h) cold-sensitive beans. Nck, cold-tolerant variety sprayed with equal amounts of clean water, low-temperature control; Nt, cold-tolerant variety sprayed with DA-6; Sck, sensitive variety sprayed with an equal amount of clean water, low-temperature control; St, sensitive variety sprayed with DA-6. The abscissa represents the exposure time of low-temperature stress, and the ordinate the value of each parameter. Data are presented as the means ± SD of three different experiments with three replicated measurements; different letters within rows indicate significant differences (P < 0.05) of variation

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Effects of exogenous DA-6 on chlorophyll fluorescence parameters of Phaseolus vulgaris seedling leaves under low-temperature stress

As illustrated in Fig. 3, at low temperatures for 0 h, exogenous DA-6 did not affect the Fm, Fv/Fm, NPQ, qP, ETR, or Y(II) in the leaves of cold-tolerant bean seedlings compared with those of the control group. Following exposure to low temperatures, the Fm trend of cold-tolerant bean seedling leaves exhibited minimal changes. The variables, Fv/Fm, qP, ETR, and Y(II), exhibited a biphasic pattern, decreasing and subsequently increasing, whereafter they then decreased. In contrast, the trend of NPQ exhibited an inverse pattern: increasing, decreasing, and then subsequently increasing. In the context of low-temperature stress, exogenous spraying of DA-6 reduced the Fm and NPQ of cold-tolerant P. vulgaris seedling leaves and increased the Fv/Fm, qP, ETR, and Y(II) compared with those of the low-temperature control group measured at the same period. Following a 6-h period of stress exposure, a significant 22.89% reduction in Fm was observed compared with that in the low-temperature control group at the same time point (Fig. 3A). Conversely, a significant increase (13.91%) in Fv/Fm was observed (Fig. 3C). After 12 h of stress exposure, the NPQ was significantly reduced by 74.51% compared with that in the low-temperature control group (Fig. 3E); qP significantly increased by 40.43% (Fig. 3G), ETR significantly increased by 53.54% (Fig. 3I), and Y(II) significantly increased by 57.05% (Fig. 3K).

As illustrated in Fig. 3, under low temperatures for 0 h, exogenous DA-6 did not significantly affect the Fm, NPQ, qP, ETR, and Y(II) in the leaves of cold-sensitive P. vulgaris seedlings, with Fv/Fm exhibiting a 3.93% reduction (Fig. 3D). Following the imposition of low-temperature stress, a decline was observed for the Fm, Fv/Fm, qP, ETR, and Y(II) measured in the leaves of cold-sensitive bean seedlings as the duration of stress was extended. Conversely, the trend observed for the NPQ was reversed. In the context of low-temperature stress, the exogenous application of DA-6 was observed to enhance the qP, ETR, and Y(II) while concurrently reducing the NPQ in cold-sensitive P. vulgaris seedlings compared with those in the low-temperature control group at the same period. Conversely, the initial increase in Fv/Fm observed was followed by its subsequent decline. The Fv/Fm ratio exhibited an increase at the 12-h mark, followed by a decline at subsequent time points. At 24 h of stress exposure, the NPQ of cold-sensitive bean seedling leaves exhibited a notable decline of 38.22% compared with that of the low-temperature control group measured at the same time point (Fig. 3F). Conversely, a marked increase in the qP (58.12%) (Fig. 3H), ETR (50.78%) (Fig. 3J), and Y(II) (51.90%) (Fig. 3L) was observed. After 48 h of stress exposure, significant increases in qP of 112.43% (Fig. 3H) and ETR of 68.90% (Fig. 3J) were measured.

Fig. 3
figure 3

Effects of DA-6 on chlorophyll fluorescence parameters of common bean seedlings under low-temperature stress. a–b, Fm values of (a) cold-tolerant and (b) cold-sensitive beans; c–d, Fv/Fm values of (c) cold-tolerant and (d) cold-sensitive beans; e–f, NPQ values of (e) cold-tolerant and (f) cold-sensitive beans; g–h, qP values of (g) cold-tolerant and (h) cold-sensitive beans; i–j, ETR values of (i) cold-tolerant and (j) cold-sensitive beans; k–l, Y(II) values of (k) cold-tolerant and (l) cold-sensitive beans. Nck, cold-tolerant variety sprayed with equal amounts of clean water, low-temperature control; Nt, cold-tolerant variety sprayed with DA-6; Sck, sensitive variety sprayed with an equal amount of clean water, low-temperature control; St, sensitive variety sprayed with DA-6. The abscissa represents the exposure time of low-temperature stress, and the ordinate the value of each parameter. Data are presented as the means ± SD of three different experiments with three replicated measurements; different letters within rows indicate significant differences (P < 0.05) of variation

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Effects of exogenous DA-6 on ROS byproducts in leaves of Phaseolus vulgaris seedlings under low-temperature stress

As illustrated in Fig. 4, under low temperatures for 0 h, exogenous DA-6 caused a notable enhancement in the O2− production rate of cold-tolerant bean seedling leaves, exhibiting a 24.15% increase (Fig. 4A). However, this treatment did not have a discernible impact on the H2O2 content. As the duration of low-temperature stress exposure increased, the O2− production rate and H2O2 content of cold-tolerant beans increased correspondingly. Exogenous application of DA-6 was demonstrated to reduce the O2− production rate and H2O2 content of cold-tolerant beans under low-temperature stress. Following a 24-h period of stress exposure, the O2− production rate of cold-tolerant beans was significantly reduced by 25.24% compared with that of the low-temperature control group (Fig. 4A). In addition, the H2O2 content was significantly reduced by 31.41% (Fig. 4C).

As illustrated in Fig. 4, under low temperatures for 0 h, exogenous DA-6 caused a notable reduction in the O2− production rate of cold-sensitive beans, showing a 14.66% decrease (Fig. 4B). However, this treatment did not have a discernible impact on the H2O2 content. As the duration of low-temperature stress exposure increased, the O2− production rate and H2O2 content of cold-sensitive beans exhibited corresponding increases. Exogenous spraying of DA-6 was demonstrated to reduce the O2− production rate and H2O2 content of cold-sensitive P. vulgaris under low-temperature stress. After 6 h of stress exposure, the H₂O₂ content of cold-sensitive P. vulgaris was significantly reduced by 26.35% compared with that of the low-temperature control group measured at the same time point (Fig. 4D). After 24 h of stress exposure, the O2− production rate was significantly reduced by 49.38% (Fig. 4B).

Fig. 4
figure 4

Effects of exogenous DA-6 on ROS byproducts in Phaseolus vulgaris seedling leaves under low-temperature stress. a–b, O2− production rate of (a) cold-tolerant and (b) cold-sensitive beans; c–d, H2O2 content of (c) cold-tolerant and (d) cold-sensitive beans. Nck, cold-tolerant variety sprayed with equal amounts of clean water, low-temperature control; Nt, cold-tolerant variety sprayed with DA-6; Sck, sensitive variety sprayed with an equal amount of clean water, low-temperature control; St, sensitive variety sprayed with DA-6. The abscissa represents the exposure time of low-temperature stress, and the ordinate the value of each parameter. Data are presented as the means ± SD of three different experiments with three replicated measurements; different letters within rows indicate significant differences (P < 0.05) of variation

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Effects of exogenous DA-6 on antioxidant enzyme activities in leaves of Phaseolus vulgaris seedlings under low-temperature stress

As illustrated in Fig. 5, under low temperatures for 0 h, exogenous DA-6 markedly elevated POD activity in the leaves of cold-tolerant bean seedlings by 30.65% (Fig. 5C), whereas APX activity was significantly diminished by 58.53% (Fig. 5G). However, no notable impact was observed on SOD and CAT activities. Following exposure to low temperatures, the activities of SOD, POD, CAT, and APX in leaves of the cold-tolerant P. vulgaris seedlings gradually increased. Application of DA-6 via spraying in the presence of low-temperature stress was observed to enhance the activities of SOD, POD, CAT, and APX in the leaves of cold-tolerant bean seedlings compared with those of the low-temperature control group measured at the same period. At the 6-h mark, APX activity in the leaves of cold-tolerant bean seedlings exhibited a notable increase of 165.44% compared with that in the low-temperature control group (Fig. 5G). At 24 h of stress exposure, POD activity reached its highest level, exhibiting a significant increase of 68.95% compared with that of the low-temperature treatment group (Fig. 5B). At 48 h of stress exposure, SOD and CAT activities reached their highest levels, with SOD and CAT activities showing significant increases of 71.06% (Fig. 5A) and 123.49% (Fig. 5C), respectively, compared with those in the low-temperature treatment group at the same period.

Figure 5 illustrates that under normal temperatures, exogenous DA-6 significantly increased POD activity in the leaves of cold-sensitive P. vulgaris seedlings by 91.27% (Fig. 5D) and CAT activity by 189.05% (Fig. 5F), but did not significantly affect SOD and APX activity. Following the imposition of low-temperature stress, the activities of SOD, POD, and CAT in the leaves of cold-sensitive bean seedlings initially increased and subsequently declined with prolonged stress exposure time. In the presence of low-temperature stress, the exogenous application of DA-6 led to an increase in the activities of SOD, POD, CAT, and APX in the leaves of cold-sensitive P. vulgaris seedlings compared with those in the low-temperature control group measured at the same period. At the 24-h mark, the activities of SOD and CAT peaked. Compared with the low-temperature treatment group at the same time point, SOD activity significantly increased by 160.63% (Fig. 5B), whereas CAT activity significantly increased by 85.56% (Fig. 5F). At 48 h of stress exposure, the highest level of POD activity was recorded, which was significantly increased by 318.34% compared with that in the low-temperature treatment group measured at the same time point (Fig. 5D).

Fig. 5
figure 5

Effects of DA-6 on antioxidant enzyme activity in common bean seedlings under low-temperature stress. a–b, SOD content of (a) cold-tolerant and (b) cold-sensitive beans; c–d, POD content of (c) cold-tolerant and (d) cold-sensitive beans; e–f, CAT content of (e) cold-tolerant and (f) cold-sensitive beans; g–h, APX content of (g) cold-tolerant and (h) cold-sensitive beans. Nck, cold-tolerant variety sprayed with equal amounts of clean water, low-temperature control; Nt, cold-tolerant variety sprayed with DA-6; Sck, sensitive variety sprayed with an equal amount of clean water, low-temperature control; St, sensitive variety sprayed with DA-6. The abscissa represents the exposure time of low-temperature stress, and the ordinate the value of each parameter. Data are presented as the means ± SD of three different experiments with three replicated measurements; different letters within rows indicate significant differences (P < 0.05) of variation

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Effects of exogenous DA-6 on the AsA-GSH cycle in leaves of Phaseolus vulgaris seedlings under low-temperature stress

As illustrated in Fig. 6, under typical temperatures, 25 mg/L exogenous DA-6 caused no notable differences in AsA, DHA, AsA + DHA, GSH, GSSG, and GSH/GSSG contents in the leaves of cold-tolerant bean seedlings compared with those in the control group. Following exposure to low-temperature stress, the contents of AsA, DHA, AsA + DHA, and GSH in the leaves of cold-tolerant bean seedlings increased by varying degrees. In the context of low-temperature stress, exogenous application of DA-6 enhanced the levels of AsA, GSH, GSSG, and GSH/GSSG in the leaves of cold-tolerant bean seedlings. However, the increase in DHA levels was only statistically significant after 24 h, exhibiting a 9.19-fold increase (Fig. 6C). Similarly, the increase in AsA + DHA levels was significant only after 24 h, with an increase of 154.02% (Fig. 6E). At the 24-h mark, the AsA content exhibited a notable increase of 74.81% compared with that of the low-temperature control group (Fig. 6A). Similarly, GSH content significantly increased by 36.97% (Fig. 6G). The content of GSSG increased significantly by 32.19% at the 6-h mark (Fig. 6I), whereas the ratio of GSH to GSSG demonstrated a notable increase of 53.46% at the 12-h interval (Fig. 6K).

As illustrated in Fig. 6, under normal temperatures, 25 mg/L exogenous DA-6 did not significantly affect the AsA, DHA, AsA + DHA, GSH, GSSG, and GSH/GSSG contents in the leaves of cold-sensitive bean seedlings when compared with those in the control group. As the duration of low-temperature stress increased, AsA content in the leaves of cold-sensitive bean seedlings remained relatively stable. Conversely, DHA, AsA + DHA, and GSH contents exhibited gradual increases. In the context of low-temperature stress, exogenous application of DA-6 enhanced the levels of DHA, AsA + DHA, and GSH/GSSG while concurrently reducing the concentration of GSSG. At the 6-h mark, the GSSG content was the lowest, exhibiting a significant reduction of 33.82% compared with that of the low-temperature control group (Fig. 6G). After 24 h of stress exposure, DHA content significantly increased by 55.93% (Fig. 6D), and AsA + DHA content significantly increased by 83.00% (Fig. 6F). After 48 h of stress exposure, the GSH/GSSG ratio significantly increased by 62.00% (Fig. 6L).

Thus, the application of 25 mg/L DA-6 under low-temperature stress conditions has the potential to enhance the levels of AsA, DHA, GSH, and GSH/GSSG in the P. vulgaris leaves while simultaneously reducing the GSSG levels. This may mitigate the adverse effects that low-temperature stress has on P. vulgaris.

Fig. 6
figure 6

Effects of DA-6 on AsA-GSH cycle-related substances in common bean seedlings under low-temperature stress. a–b, AsA content of (a) cold-tolerant and (b) cold-sensitive beans; c–d, DHA content of (c) cold-tolerant and (d) cold-sensitive beans; e–f, AsA + DHA content in (e) cold-tolerant and (f) cold-sensitive beans; g–h, GSH content of (g) cold-tolerant and (h) cold-sensitive beans; i–j, GSSG content of (i) cold-tolerant and (j) cold-sensitive beans; k–l, GSH/GSSG content of (k) cold-tolerant and (l) cold-sensitive beans. Nck, cold-tolerant variety sprayed with equal amounts of clean water, low-temperature control; Nt, cold-tolerant variety sprayed with DA-6; Sck, sensitive variety sprayed with an equal amount of clean water, low-temperature control; St, sensitive variety sprayed with DA-6. The abscissa represents the exposure time of low-temperature stress, and the ordinate the value of each parameter. Data are presented as the means ± SD of three different experiments with three replicated measurements; different letters within rows indicate significant differences (P < 0.05) of variation

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Effects of exogenous DA-6 on the activities of AsA-GSH cycle-related enzymes in leaves of Phaseolus vulgaris seedlings under low-temperature stress

As illustrated in Fig. 7, under standard temperatures, exogenous DA-6 markedly elevated the MDHAR activity of cold-tolerant bean seedling leaves by 222.19% (Fig. 7C) compared with that of the control group. Conversely, DHAR activity was significantly diminished by 24.45% (Fig. 7A), whereas the effects on GPX, GR, and GST activities were not statistically significant. As the duration of low-temperature stress exposure increased, the activity of MDHAR, GPX, GR, and GST in the leaves of cold-tolerant bean seedlings exhibited a general upward trend. Exogenous application of DA-6 was demonstrated to enhance the activities of DHAR, MDHAR, GPX, GR, and GST in the leaves of cold-tolerant P. vulgaris seedlings subjected to low-temperature stress. Compared with the low-temperature control group at the same time point, the DHAR activity of cold-tolerant P. vulgaris exhibited a notable increase of 498.71% after 48 h of low-temperature stress exposure (Fig. 7A), whereas MDHAR activity significantly increased by 58.90% after 12 h of stress exposure (Fig. 7C). Following a 6-h period of stress exposure, the GR activity of cold-tolerant beans exhibited a notable increase of 97.13% (Fig. 7E), whereas GPX activity showed a significant increase of 37.70% (Fig. 7G) compared with that of the low-temperature control group measured at the corresponding time point. A significant increase in GST activity was observed, reaching 26.51% at the 48-h mark (Fig. 7I).

As illustrated in Fig. 7, under standard temperatures, exogenous DA-6 administration resulted in a notable reduction in MDHAR activity in cold-sensitive bean varieties, with a 74.08% decrease compared with that in the control group (Fig. 7D). Conversely, no significant alterations in the activities of DHAR, GPX, GR, and GST were observed. As the duration of low-temperature stress exposure increased, the activity of DHAR and GST in cold-sensitive beans also increased. Following the exogenous application of DA-6, DHAR activity in the leaves of cold-sensitive bean seedlings significantly increased (133.61%) compared with that in the low-temperature control group measured at the same time point (24 h) (Fig. 7B). MDHAR activity exhibited a significant increase of 190.14% at the 12-h mark (Fig. 7D). After 24 h of stress exposure, the GR activity of cold-sensitive beans significantly increased by 218.67% compared with that of the low-temperature control group (Fig. 7F). Similarly, GPX activity showed a notable increase of 133.50% (Fig. 7H), whereas GST activity significantly increased by 39.27% (Fig. 7G).

Fig. 7
figure 7

Effects of DA-6 on AsA-GSH cycle-related enzyme activity in bean seedlings under low-temperature stress. a–b, DHAR activity of (a) cold-tolerant and (b) cold-sensitive beans; c–d, MDHAR activity of (c) cold-tolerant and (d) cold-sensitive beans; e–f, GR activity of (e) cold-tolerant and (f) cold-sensitive beans; g–h, GPX activity of (g) cold-tolerant (h) cold-sensitive beans; i–j, GST activity of (i) cold-tolerant (j) cold-sensitive beans. Nck, cold-tolerant variety sprayed with equal amounts of clean water, low-temperature control; Nt, cold-tolerant variety sprayed with DA-6; Sck, sensitive variety sprayed with an equal amount of clean water, low-temperature control; St, sensitive variety sprayed with DA-6. The abscissa represents the exposure time of low-temperature stress, and the ordinate the value of each parameter. Data are presented as the means ± SD of three different experiments with three replicated measurements; different letters within rows indicate significant differences (P < 0.05) of variation

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Effects of exogenous DA-6 on antioxidant and AsA-GSH system-related gene expression in leaves of Phaseolus vulgaris seedlings under low-temperature stress

The previous experiments indicate that the most favorable effect was achieved at 24 h of stress exposure, mainly due to the significant changes that occurred in photosynthetic pigments, photosynthetic fluorescence parameters, ROS byproducts, and other indicators caused by DA-6-spraying at 24 h of stress exposure compared with those at other times; gene expression responses to stress are typically observed within the first 24 h, at which time the necessary metabolic reactions also take place. The relative expression levels of antioxidant enzyme-related genes in leaves of the two bean seedlings at 0 and 24 h were analyzed via RT-qPCR. Figure 8 illustrates that under standard temperatures, exogenous DA-6 had no discernible impact on the expression of PvSOD and PvAPX genes of cold-tolerant and cold-sensitive beans when compared with that of the control group. The relative expression levels of PvSOD and PvAPX in the leaves of cold-tolerant bean seedlings were upregulated following exogenous spraying with DA-6 under low-temperature stress. Compared with the low-temperature control group at the same time point, the relative expression of PvSOD was significantly upregulated by 27.76% (Fig. 8A), and the relative expression of PvAPX was significantly upregulated by 52.17% (Fig. 8C). Following the application of DA-6 to cold-sensitive P. vulgaris, the relative expression of PvSOD was observed to increase significantly by 775.87% (Fig. 8C), and the relative expression of PvAPX increased by 200.34% (Fig. 8D) at the 24-h mark. These findings suggest that exogenous DA-6 may upregulate the expression of PvSOD and PvAPX.

The relative expression levels of AsA-GSH-related genes in the leaves of two bean seedlings at 0 and 24 h were analyzed via RT-qPCR. As illustrated in Fig. 8, under standard temperatures, exogenous DA-6 did not significantly affect the relative expression levels of PvMDHAR, PvDHAR2, and PvGR in the leaves of cold-tolerant bean seedlings when compared with those of the control group. Following the application of DA-6, the relative expression levels of PvMDHAR, PvDHAR2, and PvGR in the leaves of cold-tolerant P. vulgaris seedlings exhibited a notable increase after 24 h of low-temperature stress exposure. Compared with the low-temperature control group at the same time point, PvDHAR2 exhibited a significant 38.31% increase (Fig. 8E); PvMDHAR showed a significant increase of 48.13% (Fig. 8G); and PvGR displayed a significant increase of 66.83% (Fig. 8H). Following application of DA-6 under low temperatures for 0 h, the relative expression of PvDHAR2 significantly increased by 82.59% (Fig. 8F), and the relative expression of PvGR exhibited a notable increase of 71.13% (Fig. 8J). Conversely, the relative expression of PvMDHAR significantly decreased by 29.75% (Fig. 8H). After spraying DA-6 onto the leaves of cold sensitive P. vulgaris seedlings, no significant difference in PvMDHAR and PvGR levels were observed compared with those measured for the low-temperature control at the same period after 24 h of low-temperature stress exposure. However, both showed an upward trend, and PvDHAR2 was significantly upregulated. Therefore, exogenous DA-6 can upregulate the relative expression levels of PvDHAR2, PvMDHAR, and PvGR in P. vulgaris under low-temperature stress.

Fig. 8
figure 8

AsA-GSH-related gene expression changes induced by exogenous DA-6 in common bean seedlings at low temperatures. a–b, Relative expression of PvSOD in (a) cold-tolerant and (b) cold-sensitive beans; c–d, relative expression of PvAPX in (c) cold-tolerant and (d) cold-sensitive beans; e–f, relative expression of PvDHAR2 in (e) cold-tolerant and (f) cold-tolerant beans; g–h, relative expression of PvMDHAR in (g) cold-tolerant and (h) cold-sensitive beans; i–j, relative expression of PvGR in (i) cold-tolerant and (j) cold-sensitive beans. Nck, cold-tolerant variety sprayed with equal amounts of clean water, low-temperature control; Nt, cold-tolerant variety sprayed with DA-6; Sck, sensitive variety sprayed with an equal amount of clean water, low-temperature control; St, sensitive variety sprayed with DA-6. The abscissa represents the exposure time of low-temperature stress, and the ordinate the value of each parameter. Data are presented as the means ± SD of three different experiments with three replicated measurements; different letters within rows indicate significant differences (P < 0.05) of variation

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Discussion

The typical growth and development of plants are contingent upon the presence of suitable environmental conditions, with temperature being a pivotal factor. Low temperatures can impede plant growth and development, potentially leading to mortality [34]. The current study demonstrated that exogenous DA-6 significantly enhances fresh and dry weights of the roots and shoots of two P. vulgaris seedlings subjected to low-temperature stress. At 6 h of stress exposure, DA-6 significantly increased the aboveground fresh weight of cold-sensitive beans by 62.77% compared with that of the low-temperature control group measured at the same time point. At 24 h of stress exposure, the dry weight of the underground parts was significantly increased by 100.00% compared with that of the low temperature control group measured at the same time point. At 12 h of stress exposure, DA-6 significantly increased the stem diameter of cold-tolerant beans by 15.05% compared with that of the low temperature control group measured at the same time point. These findings indicate that exogenous DA-6 mitigated the adverse effects that low-temperature stress exerts on P. vulgaris seedlings, sustained the water content of these seedlings, and enhanced their biomass. Similar results were observed in a previous study conducted on tomatoes [19]. The optimal stress duration was 24 h. This outcome may be attributable to the robust cold stress observed during the initial stages of low-temperature exposure [35].

Photosynthesis is important for plants because it serves as the basis for plant growth and survival, and low-temperature stress has been shown to inhibit photosynthesis in plants. In the present study, the application of exogenous DA-6 resulted in a notable increase in the contents of chlorophyll a, chlorophyll b, and total chlorophyll under low-temperature stress conditions. Conversely, carotenoid content was significantly reduced. This may be due to the effect that low temperatures have on chlorophyll synthesis in P. vulgaris, which prevents damage to the plants by absorbing excessive light energy at low temperatures [36]. Exogenous pre-spraying with DA-6 was observed to significantly increase the Pn, Gs, and Ci of the two P. vulgaris seedlings tested. These results demonstrate that exogenous DA-6 can enhance the photosynthetic capacity of P. vulgaris seedlings. Exogenous DA-6 was previously demonstrated to enhance various chlorophyll fluorescence indices of Camellia oleifera, including the Pn, Gs, Tr, Ci, Fm, Fv/Fm, Y(II), qP, and ETR [37]. In the current study, exogenous pre-spraying of DA-6 under low-temperature stress increased the Fv/Fm, ETR, Y(II), and qP under dark adaptation in P. vulgaris seedlings, indicating that exogenous DA-6 can enhance the photosynthetic capacity and electron transfer efficiency of chloroplasts. DA-6 increases the ETR of P. vulgaris, thereby increasing the electron transfer rate and preventing photoinhibition. NPQ is a self-protection mechanism of the photosynthetic system, where excess light energy absorbed by antenna pigments cannot be used for photosynthetic electron transfer and is dissipated as heat energy [38]. The higher the NPQ value, the stronger the resistance to damage from external harmful light systems [39]. In this study, NPQ did not increase significantly after DA-6-spraying, but rather decreased under certain stress periods. This may be due to the fact that low-temperature stress caused the bean seedlings to mainly improve their own photosynthetic capacity and photochemical dissipation, resulting in a decrease in the amount of light energy dissipated from thermal energy. DA-6 is postulated to protect the photosynthetic apparatus from low-temperature stress by stimulating activity of the antioxidant defense system and reducing ROS accumulation [40].

Following exposure to low-temperature stress, plants generate a substantial quantity of ROS [41]. Plants use two pathways to scavenge free radicals: enzymatic and non-enzymatic pathways [42]. In plants, ROS acts as a double-edged sword; they exert various beneficial effects at low concentrations, whereas at high concentrations, ROS and related redox-active compounds cause cell damage through oxidative stress [43]. In this study, both types of P. vulgaris materials tested accumulated a large amount of ROS byproducts (such as O2− and H2O2) after being subjected to low-temperature stress, leading to membrane lipid peroxidation. The activities of SOD, POD, and CAT were significantly increased. The cold-tolerant material showed a continuous increase in activity with prolonged stress exposure time, whereas the cold-sensitive P. vulgaris showed an increase followed by a decrease with prolonged stress exposure time. This indicates that cold-tolerant P. vulgaris can respond to low-temperature stress for a longer period than the cold-sensitive P. vulgaris can. In this study, the exogenous application of DA-6 resulted in a notable reduction in O2− and H2O2 levels in the two types of bean seedlings tested, a finding corroborated by the results of a similar study conducted on maize [44].

The AsA-GSH cycle is a non-enzymatic system used for scavenging free radicals and plays a significant role in the context of plant stress. This study demonstrated that DA-6-spraying significantly increased the AsA and reduced the GSH contents of the two bean materials tested, with a notable increase in the GSH/GSSG ratio observed. The AsA and GSH levels of cold-tolerant bean seedlings exhibited a notable increase at the 24-h mark, with the duration of stress displaying discernible variations. After cold-sensitive P. vulgaris were exposed to low-temperature stress, the GSH content after DA-6-spraying did not increase significantly; however, the GSH/GSSG ratio increased significantly. Previous studies have demonstrated that the ratio of GSH to GSSG is a more reliable indicator of the efficiency of the AsA-GSH cycle than the GSH content is, which is relevant in the context of plant stress [45]. The results indicated a significant increase in the activity of GST and GPX. In addition, the antioxidant defense capacity of transgenic tobacco was augmented after a notable elevation in the activities of GST and GPX. APX is a pivotal enzyme within chloroplasts and can scavenge H₂O₂, and the activities of APX and GR are typically regarded as indicators of plant tolerance to adversity [46]. In this study, the activities of APX and GR significantly increased following the application of exogenous DA-6, indicating that DA-6 can effectively enhance the activities of APX and GR. DHAR and MDHAR are enzymes that facilitate the regeneration of AsA [47]. In this study, the activities of DHAR and MDHAR in cold-tolerant bean seedlings exhibited a notable increase following exposure to low-temperature stress. The sharp decrease in DHAR activity in the cold-tolerant bean control group observed after 48 h of low-temperature stress exposure may be due to internal damage caused by the prolonged low-temperature exposure time. Under low-temperature stress, exogenous pre-spraying of DA-6 significantly increased the DHAR activity of both types of P. vulgaris tested, whereas the MDHAR activity of cold-tolerant P. vulgaris seedlings increased. The MDHAR activity of cold-sensitive P. vulgaris only increased after 12 h of stress exposure, but decreased overall. This indicates that the regeneration of AsA in cold-tolerant P. vulgaris seedlings is mainly achieved through the combined action of MDHAR and DHAR, which maintain high MDHAR and DHAR activity even after cold stress, whereas cold-sensitive P. vulgaris mainly rely on DHAR rather than on MDHAR. These findings may be due to the inherent differences between the varieties. This study indicates that indices of the AsA-GSH cycle in cold-tolerant P. vulgaris seedlings were significantly higher than those in cold-sensitive varieties, a finding corroborated by the previous chilling injury index results. DA-6 can scavenge free radicals by enhancing the activity of pivotal enzymes and accumulation of non-enzymatic substances within the AsA-GSH cycle system. This mitigates the adverse effects of low-temperature stress and exhibits differential response efficiencies across diverse varieties.

The enhancement of antioxidant enzyme activity plays an important role in the regeneration and cycling of AsA and GSH. APX maintains the metabolic balance of intracellular free radicals by reducing H2O2 to H2O [48]. In this study, DA-6 alleviated membrane lipid peroxidation damage and improved the cold resistance of P. vulgaris by promoting the activity of antioxidant enzymes in the AsA-GSH cycle and upregulating the expression levels of related genes. The upregulation of PvSOD plays a crucial role in alleviating oxidative damage in plant stress. In this study, two types of bean seedlings showed significant upregulation of PvSOD and PvAPX genes after exogenous spraying of DA-6, which is consistent with the trend of changes in PvSOD and PvAPX enzyme activities observed. This indicates that exogenous DA-6 can reduce ROS produced during stress by upregulating PvSOD and PvAPX expression, alleviate membrane damage, and improve the cold resistance of the two extreme bean materials tested. Sang Hoon Lee et al. [49] found that in their study on tall fescue, overexpression of CuZnSOD and APX can clear ROS and improve tolerance to abiotic stress. In the current study, exogenous pre-spraying of DA-6 upregulated PvMDHAR and PvDHAR2 expression to promote enzyme activity, thereby accelerating the AsA-GSH cycling rate to alleviate the accumulation of ROS and low-temperature damage of P. vulgaris. However, expression of PvMDHAR in cold-sensitive P. vulgaris seedlings was significantly downregulated not only after low-temperature stress exposure, but also after exogenous pre-spraying of DA-6. Combined with the changes in MDHAR and DHAR activities and the gene changes mentioned earlier, the differences in PvMDHAR expression observed between cold-tolerant and cold-sensitive P. vulgaris may be due to differences between varieties. Cold-sensitive P. vulgaris mainly promotes AsA accumulation through the DHAR enzyme pathway [50].

In summary, exogenous DA-6 showed varying degrees of stress-alleviation effects in photosynthesis, the antioxidant system, and AsA-GSH cycle of the two extreme types of P. vulgaris tested under low-temperature stress conditions. Its stress-alleviating effect on seedling growth, biomass indicators, photosynthetic pigments, and other parameters was stronger in cold-sensitive P. vulgaris than in cold-tolerant P. vulgaris. Therefore, the application of DA-6 to both types of P. vulgaris could be beneficial. Even though cold-tolerant P. vulgaris have stronger cold resistance than other varieties do, the addition of DA-6 could alleviate the damage that low temperatures cause to P. vulgaris.

Conclusion

In the early stage of this study, cold-tolerant (YJ009763) and cold-sensitive (Baibulao) bean materials were screened out, and the optimal concentration of DA-6 found to be 25 mg/L. Exogenous DA-6 mitigated the damage caused by low-temperature stress in two varieties of P. vulgaris seedlings with disparate cold tolerances. The most efficacious treatment was observed at 24 h with a stress period of 0–48 h. DA-6 increased the fresh weight of both the shoots and roots of P. vulgaris seedlings, although no effect was noted on plant height or stem diameter within the short timeframe investigated.

After DA-6-spraying under low-temperature stress, the chlorophyll a, chlorophyll b, and chlorophyll a + b contents, as well as the Pn, Gs, Tr, Fv/Fm, Y(II), qP, and ETR, of P. vulgaris seedling leaves increased, whereas the NPQ, carotenoid content, and Ci decreased. Simultaneously, DA-6 exposure increased the content of AsA, GSH, and AsA + DHA in the AsA GSH cycle; increased the GSH/GSSG ratio; enhanced the activity of antioxidant enzymes, such as SOD, POD, CAT, APX, MDHAR, GR, DHAR, GPX, and GST; enhanced the photosynthetic capacity and electron transfer efficiency; and alleviated PSII photoinhibition in P. vulgaris seedlings. Further, DA-6-spraying reduced the content of O2− and H2O2; increased the AsA-GSH cycling efficiency; and reduced ROS accumulation, which could eliminate the accumulation of ROS, alleviate membrane damage, and improve the cold tolerance of P. vulgaris seedlings. After RT-qPCR analysis, DA-6-spraying upregulated PvSOD and PvAPX expression levels to activate the cold response of P. vulgaris, promote the enhancement of antioxidant enzyme activity, and eliminate ROS produced by adversity; upregulation of PvGR, PvMDHAR, and PvDHAR2 in the AsA-GSH cycle accelerates the AsA-GSH cycling rate. The improvement of cold tolerance in P. vulgaris seedlings by DA-6 is attributable to the result of the combined action of antioxidant and photosynthetic systems.

In agricultural production, after 24 h of low-temperature stress exposure, timely spraying of 25 mg/L DA-6 could alleviate the damage caused by low temperatures to P. vulgaris and greatly reduce the adverse effects that external environmental factors have on the P. vulgaris industry. Overall, the application of DA-6 in bean agriculture can reduce the damage caused by low temperatures to beans and increase their yield, providing a theoretical basis for further exploration of the high-yield and high-quality cultivation of vegetables under low-temperature stress.

Data availability

All data generated or analysed during this study are included in this manuscript.

Abbreviations

APX:

Ascorbate Peroxidase

AsA:

l-Ascorbic Acid

CAT:

Catalase

Chl a:

Chlorophyll a

Chl a + b:

Chlorophyll a + b

Chl b:

Chlorophyll b

Ci:

Intercellular CO2 Concentration

DHA:

Dehydroascorbic Acid

DHAR:

Dehydroascorbate Reductase

ETR:

Electron Transport Rate

Fm:

Maximum Fluorescence Yield

Fv/Fm:

Maximum photochemical efficiency of PS II

GPX:

Glutathione Peroxidase

GR:

Glutathione Reductase

Gs:

Stomatal Conductance

GSH:

Reduced Glutathione

GSSG:

Oxidized Glutathione

GST:

Glutathione S-transferase

MDHAR:

Monodehydroascorbate Reductase

NPQ:

Non-Photochemical Quenching

Pn:

Photosynthetic Rate

POD:

Peroxidase

qP:

Photochemical Quenching Coefficient

SOD:

Superoxide Dismutase

Tr:

Transpiration rate

Y(II):

effective photochemical efficiency of PSII under light

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Acknowledgements

We would like to thank Editage (www.editage.cn) for English language editing.

Funding

This research was supported by the Sichuan Innovation Team Project of the National Modern Agricultural Industry Technology System (SCCXTD-2024-5).

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Author notes

  1. Yu Bai and Qiya Dai contributed equally to this work.

Authors and Affiliations

  1. College of Horticulture, Sichuan Agricultural University, Chengdu, 611130, China

    Yu Bai, Qiya Dai, Yanheng He, Li Yan, Jianpo Niu, Xuan Wang, Xuena Yu, Wen Tang, Huanxiu Li, Zhi Huang, Bo Sun, Guochao Sun, Xun Wang & Yi Tang

  2. Institute of Agro-products Processing and Storage, Chengdu Academy of Agriculture and Forestry Sciences, Chengdu, 611130, China

    Yongdong Xie

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Contributions

YT and HXL: Conceived the study and designed the experiments. ZH, BS, GCS and XW: Experimental technique adviser. YB, QYD, YHH, LY, JPN, XW, YDX, XNY and WT: Preparation of plant materials, indicator determination and data analysis. YB: Manuscript editing. All authors contributed to the article and approved the submitted version.

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Correspondence to Yi Tang.

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Bai, Y., Dai, Q., He, Y. et al. Exogenous diethyl aminoethyl hexanoate alleviates the damage caused by low-temperature stress in Phaseolus vulgaris L. seedlings through photosynthetic and antioxidant systems. BMC Plant Biol 25, 75 (2025). https://doi.org/10.1186/s12870-025-06083-y

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BMC Plant Biology

ISSN: 1471-2229

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