The study was conducted from March to October 2022–2024 at the Horticulture Experimental Center of Shanxi Agricultural University. The grape branches for the test were the Eurasian species of table grapes ‘Lihongbao’. The grapes were harvested from the National Grape Germplasm Resource Nursery of Shanxi Agricultural University, Taigu, Shanxi, China (112°32′ E, 37°23′ N, mean annual temperature 9.8°C). The cuttings of the winter sand-stored ‘Lihongbao’ grape branches were rooted on 25 March 2022 and planted in moist fine sand for rooting. After 2 months, the plants were transplanted into pots containing raw soil, perlite, and humus (2:1:1, v/v/v). The seedlings were grown in a greenhouse for 9 weeks and irrigated once a week with tap water and 1/2 Hoagland nutrient solution. After the grapes had grown to 8–10 functional leaves, 60 uniformly sized, uniformly growing grape seedlings were selected for subsequent trials. A total of 60 grape seedlings were grouped into four according to a completely randomized design. There were 15 seedlings within each group, with five representing one biological replicate for each of the five low-temperature stress durations, replicated three times in total.

Grape seedlings were used for four treatments with deionized water only (as control, CK), 0.2 μM EBR (EBR), 10 μM BRZ (BRZ), and 0.2 μM EBR and 10 μM BRZ (EBR + BRZ). In particular, the above reagents in four treatments were dissolved with 98% ethanol at a content of 0.1% (v/v) and then diluted with deionized water, and it was also used as an unfolding agent with Tween-20 at a content of 0.1% (v/v). Three of the four groups were sprayed with reagents on the front and back of the leaves until they dripped. The seedlings were transferred to a light incubator (16°C/4°C day/night, 55% relative humidity, 10,000 1× light intensity, 14/10 h light/dark photoperiods) for low-temperature stress following 6 days of continuous treatment. The seedling morphology was observed at five time points (0 h, 12 h, 24 h, 48 h, and 96 h) from each treatment group. Seedlings were selected for photosynthetic fluorescence parameters by counting the third to fifth fully expanded functional leaves from bottom to top. Samples were immediately transferred to liquid nitrogen and stored at −80°C for further analysis.

Plant growth was recorded by capturing photographs with a digital Canon camera at five time points of low-temperature stress from three seedlings randomly selected from the treatment groups.

In this experiment, small square slices of 1 × 1 cm in the center of leaves, adjacent to the main vein for each treatment, were selected and rapidly placed in formalin–acetic acid–alcohol (FAA) fixative for fixation. This was followed by paraffin sectioning, ethanol gradient dehydration, xylene transparency, paraffin embedding, toluidine blue staining, and sealing with Canada gum. The sections were then placed under an ordinary light microscope to select the sections for observation and photographed using an Olympus DP71 (Japan) microimaging system. Simultaneously, the system’s tool was used to measure upper epidermal thickness (UET), lower epidermal thickness (LET), fence tissue thickness (PT), sponge tissue thickness (ST), and leaf thickness (LT) for five indices. The blade organization and structure parameters such as palisade-to-sea ratio (P/S), blade structural compactness (CTR), and leaf tissue structure sparseness (SR) were calculated according to the following equations:

The photosynthetic pigment contents of the leaves were measured using direct ethanol extraction (Anwar et al., 2018). 1 g of samples was placed in a 25 mL stoppered test tube, and 10 mL of 95% ethanol was added for thorough mixing. The tubes were left in the dark for 48 h to ensure that leaves lost their green color and whitened, then shaken well every 12 h. The filtrate was used as the extract, and the absorbance values were determined at 470 nm, 665 nm, and 649 nm.

Photosynthetic gas exchange parameters of leaves at the same position in seedlings were measured using a LI-6800 Portable Photosynthesis System (LI-COR, Nebraska, USA) from 9:00 to 11:00 am. The main measured parameters were transpiration rate (Tr), photosynthetic rate (Pn), intercellular CO₂ concentration (Ci), and stomatal conductance (Gs). The specific parameters of the photosynthesizer for the index measurements were set to the leaf chamber light intensity of 500 μmol·m⁻²·s⁻¹, the airway flow rate of 300 μmol·s⁻¹, and the leaf chamber mixing fan speed of 5,000 r·min⁻¹.

A portable fluorometer (FluorPen FP110, Czech Republic) was used to determine the functional leaves of each fully expanded treatment at the same leaf position at night, and the plants were adequately dark-adapted for at least 2 h prior to measurement. The maximum photochemical efficiency of Photosystem II (Fv/Fm), the actual photochemical efficiency of Photosystem II (φPSII), the non-photosynthetic burst coefficient (NPQ), and the photochemical burst coefficient (qP) were determined.

(1) O₂·⁻ content

O₂·⁻ content was determined using the method described by Zhao et al. (2016). 1 g of samples was weighed and added to 50 mM phosphate buffer (PBS) in batches and was fully ground, homogenized, and centrifuged for 15 min. Then, 2 mL of the supernatant of the extract and 2 mL of p-aminobenzenesulfonic acid (17 mM) was added sequentially with 2 mL of α-naphthylamine (7 mM). Next, the tubes were incubated in a 30°C water bath for 30 min and cooled to 20°C, and then the absorbance values at 530 nm were determined.

(2) H₂O₂ content

The H₂O₂ content was measured with a kit (Beijing Solepol Technology Company, Ltd.). 0.1 g of samples was added to the extract in batches to be ground and homogenized in an ice bath. They were added to 1 mL of precooled acetone for overnight maceration. This was followed by centrifugation at freezing temperature for 20 min, and the supernatant was aspirated to determine the H₂O₂ content.

(1) AsA content

AsA content was determined using the method described by Liang et al. (2018). 1 g of samples and 6 mL of 5% trichloroacetic acid (TCA) solution were ground into a homogenate in an ice bath, added into tubes. The test tubes were subjected to freeze centrifugation for 15 min, and the supernatant was collected. Next, 1 mL of the supernatant was added to a test tube, followed by 1 mL of 5% TCA solution, 0.5 mL of 0.4% phosphoric acid–ethanol solution, 1 mL of anhydrous ethanol, 0.5 mL 0.03% FeCl₃–ethanol solution, and BP–ethanol solution (1 mL). The mixture was shaken well and then placed in a water bath at 30°C for 60 min. The absorbance was measured at 525 nm.

(2) GSH content

GSH content was determined using the method described by Liang et al. (2018). 1 g of samples and 10 mL of precooled 5% TCA (containing 5 mM Na₂EDTA) solution were ground into a homogenate in an ice bath, added into tubes. The tubes were then cryocentrifuged for 15 min, and the supernatant was collected. Next, 1 mL of supernatant, 1 mL of 0.1 mM PBS solution, and 0.5 mL of 4 mM DTNB solution were added with sufficient shaking. The absorbance value was measured at 412 nm.

0.6 g of samples and 6 mL of PBS (50 mM, pH 7.8) were ground into a homogenate in an ice bath, added into tubes. This was followed by centrifugation at freezing for 30 min, and the supernatant was collected for enzyme activity assays (Ozlem, 2022).

(1) SOD activity

The SOD activity was determined using the nitrogen blue tetrazolium method (Zeng et al., 2022). 0.1 mL of supernatant and 3 mL of reaction solution were added. The reaction solution was prepared with 130 mM methionine, 20 μM riboflavin, 750 μM nitro blue tetrazolium (NBT), and 100 μM EDTA with 50 mM PBS (pH 7.8). The reaction solution was placed under 10,000 1× culture light for 10 min, and the absorbance was measured at 560 nm under light-avoidance conditions.

(2) POD activity

The POD activity was determined using the guaiacol method (Zeng et al., 2022). A mixture of 3 mL containing 76 μL of guaiacol, 112 μL of H₂O₂, and 200 mL of PBS (pH 7.0) was mixed with 200 μL of enzyme solution. The CK tube was used with the mixture and 200 μL of phosphate buffer, and the timing was started immediately. Absorbance was measured at 470 nm.

(3) CAT activity

The CAT activity was determined using the hydrogen peroxide decomposition method (Zeng et al., 2022). The prepared enzyme solution 200 μL was placed in a cuvette. 3 mL of reaction solution containing 100 mL PBS (pH 7.0) and 154.6 μL H₂O₂ were added. The absorbance was measured at 240 nm.

(4) APX activity

The APX activity was determined using the hydrogen peroxide decomposition method (Zeng et al., 2022). 100 μL of enzyme solution and 2.8 mL of 50 mM PBS (pH 7.0) containing 0.1 mM EDTA and 0.5 mM AsA were added and then mixed thoroughly. Finally, 100 μL of 20 mM H₂O₂ was added to start the reaction. Absorbance was measured at 290 nm.

MDA content was determined using the colorimetric method of thiobarbituric acid (Zhong et al., 2020). 0.5 g of samples and 0.5% (w/v) TCA were added in the ice bath and thoroughly ground, and then cryocentrifuged for 20 min. The supernatant and 0.5% TCA were placed in a tube, shaken well, boiled in a water bath for 30 min, and finally centrifuged for 10 min for twice. The supernatant was determined at 450 nm, 532 nm, and 600 nm.

(1) Free proline content

Free proline content was determined using the ninhydrin colorimetric method (Wang et al., 2022). 0.2 g of samples and 5 mL of 3% sulfosalicylic acid were placed in tubes and covered with a boiling water bath for 15 min. The samples were centrifuged for 15 min for twice. Subsequently, 2 mL of the second supernatant was added to a centrifuge tube with 2 mL glacial acetic acid and 3 mL acidic ninhydrin, allowed to react for 30 min in a boiling water bath. Next, 5 mL of toluene was added, shaken well, and left to stand for 2–3 h away from light. Absorbance was measured at 520 nm.

(2) Soluble sugar content

Soluble sugar content was determined using the anthrone-ethyl acetate method (Li et al., 2022). 0.05 g of samples and 10 mL of deionized water were added, shaken well, and boiled in a water bath for 30 min. The tubes were then centrifuged for 10 min. Next, 0.5 mL of the supernatant, 1.5 mL of deionized water, and ethyl anthrone acetate (0.5 mL) were added into the tubes. 5 mL of concentrated H₂SO₄ was slowly added, and the reaction was carried out in a boiling water bath for 1 min with rapid shaking. Absorbance was measured at 620 nm.

Statistical analysis of the data was performed using Microsoft Office 2016. SPSS 26.0 was used for correlation analysis (p < 0.05), cluster analysis, and principal component analysis. All the data results were expressed as mean ± SE of three replicates. OriginPro 2021 and RStudio were used to plot the graphs, and Photoshop 2023 was used for drawing.

Leaves of all the grape seedlings exhibited different degrees of damage over time, as shown in Figure 1A . Exogenous EBR treatment minimized low-temperature injury in grape seedlings compared with the CK group, and at 0 h, there were no substantial differences in all treatments. After 12 h of low-temperature stress, the leaves in the CK were slightly water-lost and drooped. The leaf margins were curled. However, no notable changes in leaf morphology were observed in the EBR, BRZ, and EBR + BRZ groups. After 24 h, the leaves in the BRZ lost some water and drooped. The edges of the leaves were slightly curled. The CK group showed significant water loss in the leaves of grape seedlings, with prominent curling of the leaf margins and low-temperature damage. After 48 h, the water loss drooping of leaves with curled edges gradually recovered in the EBR group. In the CK group of grapevine seedlings, water loss and drooping leaves were obvious; the edges of the leaves showed fewer yellowing spots, and the plants were more severely damaged. After 96 h, there were no obvious signs of damage to the leaves in the EBR group. The leaves observed in the EBR + BRZ group showed minimal signs of dehydration, accompanied by a subtle degree of wilting. The leaves in the CK group showed significant water loss and wilting, accompanied by leaf abscission. The leaves showed different degrees of damage with the prolongation of cold stress, and exogenous EBR helped alleviate the damage to the leaves.

Figure 1B shows that the fenestrated and spongy tissues of the leaves were closely arranged under normal growth conditions, and the upper and lower epidermal cells were morphologically intact. There was a different degree of damage to the grape plants in each treatment group following low-temperature treatment. Exogenous EBR mitigated the damage to the morphological structure of leaves after 96 h. Specific manifestations showed that the cellular morphology was more complete, and the fenestrated and spongy tissues were more tightly arranged. The anatomical structures of the leaves in the EBR group could not be distinguished. However, the main manifestations were broken upper and lower epidermal cells of the leaves, spongy tissue, and poorly differentiated fenestrated tissue in the CK, BRZ, and EBR + BRZ groups. Exogenous BRZ mitigated damage to the morphological structure of leaves but was relatively ineffective, and BRZ inhibited the effects of EBR. Exogenous EBR protected the structure of leaves under low-temperature stress. In Figure 1C , the stress caused a decrease in seedling leaf UET, LET, PT, ST, LT, P/S, and CTR, with a significant decrease in PT and CTR. The exogenously sprayed EBR group showed a significant reduction of 19.59% in PT and 11.65% in CTR compared with the CK group after 96 h. Exogenous EBR treatment significantly alleviated the damage to leaf anatomical structures from low temperature.

Photosynthetic pigments are crucial in plant physiology, with their concentration in leaves directly indicating the plant’s photosynthetic activity. The Chl a, Chl b, Chl total, and carotenoid contents under different treatments were determined at five sampling periods. Figure 2A shows that the response of different treatments and different pigment contents was not the same. There was an overall decreasing trend, followed by an increasing trend, and then decreasing Chl a, Chl b, and Chl total contents in the leaves of the CK, BRZ, and EBR + BRZ groups. This was characterized by a rapid decline within 12 h, a steady increase from 12 h to 48 h, and a rapid decline from 48 h to 96 h. The Chl a content significantly increased by 9.27% in the BRZ group at 96 h compared with CK. The contents of Chl a, Chl b, and Chl total significantly increased by 31.39%, 43.96%, and 39.44%, respectively, in the EBR + BRZ group at 96 h compared with CK. However, the trends in Chl a, Chl b, and total Chl content in EBR group were different from those in the CK group. It showed a rapidly increasing trend, reaching a peak after 12 h, followed by a gradual decrease. The Chl a, Chl b, and Chl total contents of the exogenous EBR treatment grapevine seedling leaves were higher than in the CK group. The contents of Chl a, Chl b, and Chl total significantly increased by 30.24%, 48.51%, and 39.75%, respectively, at 96 h compared with CK. The carotenoid content in CK, BRZ, and EBR + BRZ grape seedlings steadily decreased during 96 h. However, the trend in carotenoid content in the EBR group was different from the CK group, with an overall decreasing trend followed by an increasing trend. Exogenous EBR treatment leaves had a higher carotenoid content than the CK during 48 h, but the difference was insignificant. A significant difference was observed at 96 h compared with the CK group, which increased by 34.66%. The photosynthetic pigment contents decreased with prolonged low-temperature stress. Exogenous EBR powerfully mitigated the harm caused by low-temperature stress in grape seedlings and increased the content of photosynthetic pigments, while it enhanced the photosynthetic intensity. However, combined application of EBR and BRZ reduced photosynthesis.

Photosynthesis not only dominates the conversion and accumulation of energy in the plant but also determines whether or not the entire plant can grow and thrive. Tr, Pn, Ci, and Gs were determined for each treatment plant. As shown in Figure 2B , the response to the cold stress was not the same for the different treatments and gas exchange parameters. The trend of Tr in the grape seedlings in the four treatment groups was the same from 0 h to 24 h, with a decreasing and then increasing trend. The changes occurred after 24 h, and Tr in grape seedlings of the CK, BRZ, and EBR + BRZ groups showed a gradual decrease with prolonged stress. However, Tr in the grape seedlings of the EBR group showed an increasing trend. It reached its highest value at 48 h and then gradually decreased to reach a significant level compared with CK. The four treatment groups showed a decreasing trend in Pn. The Pn values of EBR and CK groups did not reach significant differences after 96 h, but the Pn values in the BRZ and EBR + BRZ treatment groups were obviously different from the CK group. The overall trend of Ci in grapevine seedlings in the four treatment groups was upward, and the change in Ci in grapevine seedlings in the CK group stabilized after a significant increase at 12 h. Compared with the CK group, the Ci in the EBR, BRZ, and EBR + BRZ groups first decreased obviously at 12 h, then increased at 24 h, and then showed a relatively stable trend after reaching the highest value. The Gs in the CK, BRZ, and EBR + BRZ groups showed a significant decrease with the prolongation. However, the trend of Gs changes in EBR treatment differed from the CK group. After 12 h, it decreased rapidly and then increased significantly with the extension of the cold time to reach the highest value and then stabilize. The Tr, Pn, and Gs of grape seedling leaves decreased, and Ci increased with prolonged stress. Exogenous EBR alleviated the decrease in Tr and Gs and the increase in Ci but did not significantly alleviate the changes in Pn. Exogenous BRZ helped to alleviate the decrease in Tr and Gs but promoted the decline of Pn and did not significantly alleviate the changes in Ci under low-temperature stress.

The chlorophyll fluorescence parameter could reflect the degree of harm and the level of cold tolerance. The Fv/Fm, φPSII, NPQ, and qP parameters were determined for each treatment plant at different low-temperature times. Figure 2C shows that the different treatments and chlorophyll fluorescence parameters did not respond equally to cold stress. The change trends in Fv/Fm, φPSII, NPQ, and qP parameters in grape seedlings was the same in the four treatment groups. Fv/Fm decreased and then increased with the prolongation of stress. The Fv/Fm in BRZ treated was significantly different from the CK group after 96 h, and BRZ effectively suppressed the increase in Fv/Fm values. The trends of PSII, NPQ, and qP decreased with the prolongation of stress. Among them, the use of BRZ alone did not significantly affect φPSII, NPQ, and qP after 96 h, but coupled with EBR, it significantly promoted the reduction. Meanwhile, the EBR group only significantly promoted NPQ after 96 h and did not form an obvious difference in the changes of Fv/Fm, φPSII, and qP values. Fv/Fm in grape seedling leaves did not change significantly with the extension, and φPSII, NPQ, and qP decreased with the extension of stress time. The exogenous EBR helped to promote the rise in Fv/Fm and the decline of φPSII and NPQ in the leaves and alleviated the decline in qP. Exogenous EBR coupled with BRZ improved the chlorophyll fluorescence parameters of leaves.

ROS are metabolic by-products in plant chloroplasts, mitochondria, and peroxisomes and have a dual function in adversity stress with a dose effect. The contents of O₂·⁻ and H₂O₂ were determined under each treatment plant. Figure 2D shows that the O₂·⁻ content of the CK group had an overall increasing trend except for 48 h. However, the trend of O₂·⁻ content changes in EBR, BRZ, and EBR + BRZ treatment grape seedlings was different from that of the CK, with a significant increase at 12 h. This was followed by a notable decreasing trend with the prolongation of stress. Among them, the O₂·⁻ content in the leaves of EBR-, BRZ-, and EBR + BRZ-treated grape seedlings was significantly reduced by 23.66%, 46.50%, and 38.16%, respectively, at 96 h compared with CK. The H₂O₂ content of the CK group increased steadily before 24 h and then decreased and increased from 24 h to 96 h, with an overall increasing trend. However, the H₂O₂ content in the EBR, BRZ, and EBR + BRZ groups tended to increase and then decrease, reaching the highest value at 24 h, following by a gradual decline. Subsequently, the H₂O₂ content was lower than the CK group at 24 h, 48 h, and 96 h, with significant reductions of 19.13%, 17.65%, and 29.88%, respectively, in the EBR group. The H₂O₂ and O₂·⁻ content in grape seedling leaves increased with the duration of stress. Exogenous EBR in the late stage of low-temperature stress could effectively alleviate the low-temperature damage suffered by grape plants and inhibit the rise in O₂·⁻ and H₂O₂ contents.

Antioxidants, such as AsA and GSH, regulate the redox state of plant cells and balance ROS levels. In this experiment, the AsA and GSH contents of treated plants were determined at different times. Figure 2E shows that the AsA content in the CK group gradually increased, peaked after 24 h, and then gradually decreased. The change in the AsA content of grape seedling leaves in the EBR, BRZ, and EBR + BRZ groups showed a trend that was the same as the CK group. The AsA content of leaves peaked at 24 h, was notably higher than CK, and then showed a decreasing trend with prolonged stress time. AsA content of grape seedling leaves at 24 h significantly increased by 57.63% and 29.07% in the EBR and EBR + BRZ groups, respectively. At 48 h, it significantly increased by 27.08% and 17.48% in the EBR and EBR + BRZ groups, respectively. EBR effectively increased AsA content in the leaves of the plants, and BRZ exhibited some inhibitory effects. The trend in the GSH content in grape plants in the four treatment groups remained the same throughout the experiment. The GSH content decreased in the early stage of stress, followed by a gradual increase and then a decrease in GSH content. GSH peaks appeared in the four treatment groups after 48 h. The GSH content of EBR-treated was substantially greater than in the CK group. At 24 h, 48 h, and 96 h, the GSH content significantly increased by 45.36%, 41.99%, and 33.63%, respectively, in the EBR group compared with CK. The AsA and GSH content of grape plants increased with prolonged stress. Exogenous EBR enhanced the content of AsA and GSH in grape plants and mitigated the low-temperature harm to grape plants.

Antioxidant enzymes are crucial components of the plant antioxidant enzyme system, which work together to scavenge excess ROS in the defense against cold stress. EBR-treated effectively mitigated low-temperature damage in grape plants, increased the contents of antioxidant substances, and enhanced the antioxidant capacities of grape to tolerate cold stress. Therefore, we conducted SOD, POD, CAT, and APX analyses for each treatment group at different cold times. As shown in Figure 2F , SOD activity in CK grape seedlings increased and then stabilized, reaching a peak after 24 h. Meanwhile, the SOD activity in EBR, BRZ, and EBR + BRZ groups showed a trend of increasing and then decreasing, reaching a peak at 48 h. SOD activity was obviously higher in the EBR and EBR + BRZ groups than in the CK group. Compared with CK at 24 h, 48 h, and 96 h, the SOD activity of grapevine seedling leaves significantly increased by 34.80%, 84.84%, and 42.70% in the EBR group; 19.21%, 36.53%, and 15.98% in the BRZ group; and 34.79%, 82.48%, and 28.34% in the EBR + BRZ group, respectively, during the middle and late stages of stress. The trends in POD, CAT, and APX activities in all groups first increased and then decreased. The POD activity of grape seedling leaves peaked at 48 h and increased significantly by 52.97% in EBR compared with CK. The CAT and APX activities peaked after 24 h and were significantly reduced by 23.18% and 45.96% in EBR compared with CK. At the end of the stress period, the activities of SOD, POD, and APX were markedly higher, and the activity of CAT was lower in the EBR group than in the CK group. Meanwhile, BRZ and EBR + BRZ treatments either enhanced or suppressed antioxidant enzyme activities in grape seedlings, but the effects were not as prominent as those of the EBR treatment. The enzymatic antioxidant activity of grapevine seedling leaves increased with stress. Exogenous EBR significantly elevated the activities of SOD, POD, and APX, in grape seedling leaves and inhibited the activity of CAT.

As shown in Figure 2G , the MDA changes in the CK group showed an upward trend, and the MDA content of the EBR, BRZ, and EBR + BRZ groups showed an increasing and then a decreasing trend. MDA content significantly decreased after reaching its maximum value after 24 h. At 48 h and 96 h, EBR treatment had the strongest inhibitory effect on the MDA content, which was reduced by 32.95% and 47.96%, respectively, compared with the CK group. An indicator of oxidative stress of grapevine seedlings, the MDA content gradually increased with the prolongation of cold stress. Exogenous EBR treatment markedly reduced the accumulation of MDA content exposed to low-temperature stress. This intervention significantly suppressed the rise in MDA levels.

Plants can synthesize osmoregulatory substances to regulate osmotic pressure and change water potential to improve plant resistance. The free proline and soluble sugar contents of the treated plants were determined. Figure 2H shows that the trend of free proline and soluble sugars in the four treatment groups has an increasing trend. The free proline content in grapevine seedlings was markedly increased by 34.45% and 19.44% at 48 h and 96 h, respectively, in the EBR group compared with CK. Soluble sugar content of grape seedling leaves increased by 84.72%, 19.71%, and 5.29% at 24 h, 48 h, and 96 h, respectively, compared with CK. With the prolongation of cold stress, EBR-treated obviously increased soluble sugar content compared with CK. However, EBR + BRZ treatment had a better promotion effect. The soluble sugar content of grape seedlings increased by 129.19%, 27.83%, and 18.14% at 24 h, 48 h, and 96 h in the EBR + BRZ group compared with the CK group. The free proline and soluble sugar contents were significantly greater in the EBR group than CK after 96 h. The content of free proline and soluble sugar in grape seedlings increased with prolonged cold stress. When grapevine seedlings were subjected to exogenous EBR treatment and subsequently exposed to low-temperature stress, there was a notable rise in the levels of free proline and soluble sugars. This unique physiological regulation mechanism has shown its indispensable value in alleviating the damage caused by low temperature.

As shown in Figure 3A , the 23 physiological indices were correlated with five matrix types grouped by low-temperature stress time. Pn notably correlated with low-temperature stress at 0 h. Chl b, Chl total, carotenoids, Gs, NPQ, and APX were significantly correlated with stress at 12 h. H₂O₂, AsA, POD, and free proline were significantly correlated with stress at 96 h. However, no significant correlation was observed between 0 h and 48 h and the physical and chemical indices. Low-temperature stress treatments significantly affected the leaf photosynthetic pigments, photosynthetic gas exchange parameters, and chlorophyll fluorescence parameters of grapevine seedlings after 48 h. The results of this study are summarized as follows. Low-temperature stress treatment for 48 h to 96 h significantly affected the indices of ROS, antioxidant substances, antioxidant enzyme activity, and osmotic adjustment substances.

As shown in Figure 3B , the physiological and biochemical indices of grape cuttings after exogenous EBR spray treatment were subjected to principal component analysis (PCA). Three principal components with the highest eigenvalues, PC1 (97.9%), PC2 (0.9%), and PC3 (0.6%), were extracted. The samples treated with EBR and CK exhibited significant sample separation at different low-temperature treatment times.

As shown in Figure 3C , based on the standardized data of physiological indexes of EBR sprayed grapevine seedlings under different cold stress time treatments, the seedling leaf samples of five low-temperature periods were clustered by clustering heatmap analysis to study their stoichiometric characteristics. The results showed that the 20 treated samples could be categorized into three groups. The first group of treatment samples was CK and BRZ after 96 h. The second group of treatment samples was EBR for 48 h and 96 h and EBR + BRZ for 96 h. The third group of treatment samples was CK for 0 h, 12 h, 24 h, and 48 h; EBR for 0 h, 12 h, and 24 h; BRZ for 0 h, 12 h, 24 h, and 48 h; and EBR + BRZ for 0 h, 12 h, 24 h, and 48 h. The cluster analysis plot showed that the 23 physiological indicator samples could be categorized into two groups. The first group of indicator samples included soluble sugars, free proline, SOD, GSH, POD, APX, AsA, CAT, O₂·⁻, MDA, H₂O₂, and Ci. The second group of indicator samples comprised Fv/Fm, qP, NPQ, φPSII, Pn, Gs, Tr, carotenoid, Chl total, Chl b, and Chl a. Exogenous spraying of EBR effectively counteracted the low-temperature stress experienced by grapevine seedlings and was dominated by the modulation of osmoregulatory substances and antioxidant system indices in grape leaves.

The harm caused by low temperatures in plants is reflected in both phenotypic features and anatomical structures and physiological and biochemical indices. Phenotypic traits of plants experiencing low-temperature stress usually include symptoms such as plant wilting and leaf necrosis (Lu et al., 2019). Morphological changes in grapevine seedlings were investigated in four treatments, CK, EBR, BRZ, and EBR + BRZ, at 0 h, 12 h, 24 h, 48 h, and 96 h, respectively. There was a tendency for wilting and water loss in the leaves of seedlings in the CK group at 12 h, 24 h, 48 h, and 96 h, which were significantly damaged by low-temperature stress. Although no significant damage was observed in EBR, more significant damage was observed in BRZ. However, the EBR, BRZ, and EBR + BRZ groups were more resistant to cold stress than CK. In the physiological process of plants subjected to cold stress, the application EBR showed a significant protective effect. This discovery opened up new research ideas to improve the cold tolerance of plants. EBR regulated a series of complex pathways in plants to build up a physiological barrier against cold attack. It was worth pondering that the establishment of this protective mechanism is far more subtle than we have imagined, which not only is reflected in the improvement of the apparent morphology but also involves the maintenance of the stability of the cell membrane and the dynamic balance of osmotic regulation at a deeper level. The scientific findings revealed by the experimental study of Chen et al. (2019) coincide with this phenomenon, with its insights into the EBR processing mechanism showing significant mitigating effects in response to cold stress damage. The tightly arrange fenestrated tissues of plants exposed to cold stress could effectively maintain water retention in grapes (Lu et al., 2019). After the leaves were subjected to stress, the leaves fenestrated and spongy tissues were loosely arranged, and LT, PT, and ST were reduced. In the research field of oil tea seedling growth and development, Xu et al. (2022a) confirmed the scientific validity of this viewpoint through a series of experimental design. Exogenous EBR treatment resulted in a more complete leaf cell morphology, a tighter arrangement of fenestrated tissues, and no significant reduction in LT, ST, or SR. In contrast, the fenestrated tissue was loosely arranged in the CK group after 96 h. Exogenous spraying of EBR protected the leaf structure of grape seedlings and alleviated the stressful effects of adversity on grapes. In the study of Wang et al. (2022), the effects of EBR on the anatomical structure of oil tea leaves under low-temperature stress showed a similarity, a finding that not only corroborates the conclusions of the present study but also provides a new way of thinking for in-depth exploration of phytohormone regulatory mechanisms.

When plants suffer from low-temperature stress, photosynthesis, a central mechanism of plant life activity, inevitably gets disrupted (Guan et al., 2023; Lee et al., 2021). Accumulation of photosynthetic pigments in plants showed a significant attenuation under the stress of cold environments. It was worthwhile to pay attention to the fact that, by applying exogenous EBR, this unavoidable attenuation process seemed to be alleviated to a certain extent, so that the rate of loss of photosynthetic pigments showed a relatively moderate trend, which were consistent with Li (2020) and Anwar et al. (2018). The importance of photosynthesis as a fundamental way for plants to obtain energy cannot be overstated. It was worth pondering that when the ambient temperature is persistently low, this core system of plant survival will be inhibited, which might trigger a series of chain reactions affecting growth of plant (Yudina et al., 2020). In grape seedlings, cold stress resulted in a significant decrease in Pn, Tr, and Gs, and a significant increase in Ci. This diminished the leaves’ photosynthetic traits, resulting in reduced chloroplast activity, a finding that coincides with the experimental data of Yang et al. (2019). In contrast, exogenous EBR and EBR + BRZ pretreatments restored the levels of Tr, Pn, and Gs and reduced Ci levels. This showed that damage to the photosynthetic system of grape seedlings triggered by low temperatures was significantly alleviated by exogenous EBR and that the blockage of photoinhibition and the increase in photosynthetic capacity could lead to an increase in CO₂ uptake by the plants. These experimental data coincide with the findings reached by Chen et al. (2019). The electron transport chain of photosynthesis showed an irreversible blockage during low-temperature stress. This altered physiological mechanism upset the dynamic equilibrium between the absorption of light energy by the photosynthesis system and the energy consumption during metabolism, which was completely disrupted. It was worth pondering that this imbalance triggers a series of chain reactions: excessive excitation energy accumulated in the photosynthetic system inevitably induced photoinhibition of PSII (Jin et al., 2022). The values of Fv/Fm, qP, NPQ, and φPSII decreased with the increase in stress time. Cold stress inhibited the conversion efficiency of PSII primary light energy and the potential photosynthetic activity of PSII in grapevine seedling leaves. The chlorophyll fluorescence properties of grapevine seedling leaves responded strongly to cold stress. The decreases in Fv/Fm, NPQ, qP, and PSII in the leaves treated with EBR and EBR + BRZ were markedly lower than CK-treated leaves. EBR showed a significant protective effect under cold stress, which was negatively correlated with the degree of photosystem damage, when exploring the mechanism of the effect of exogenous EBR on the grapevine photosystem, aligning with the results of Stachurska et al. (2022) and Sadura and Janeczko (2022).

Plant cells maintain a delicate balance of reactive oxygen species, an equilibrium that is particularly vulnerable to cold stress. When the cold comes, the homeostasis inside the cell is ruthlessly broken, and O₂·⁻ and H₂O₂, two substances, begin to accumulate. It is worth pondering that these reactive oxygen molecules, which are cellular metabolites under normal physiological conditions, not only are the key bioindicators of oxidative stress in plants but also harness their strengths against various threats to life (Singh et al., 2024). The accumulation of MDA, an important product of lipid peroxidation in cell membranes during plant stress, has always been regarded as a key indicator of the degree of harm to membrane systems. By analyzing the content of MDA, the oxidative damage suffered by cell membranes can be evaluated, and this approach is of irreplaceable value in the study of plant adversity physiology (Zhao et al., 2023). In this study, low-temperature stress caused a notable rise in O₂·⁻ and H₂O₂ levels, alongside enhanced activities of SOD, POD, CAT, and APX, and an increase in AsA, GSH, and MDA levels. The homeostatic balance between ROS scavenging and accumulation was disrupted, and the ROS attack resulted in peroxidative damage to the cytoplasmic membrane. This aligns with the results reported by Amin et al. (2022). In contrast, the MDA content under EBR was obviously lower than CK, and the O₂·⁻ and H₂O₂ contents were lower than CK. Meanwhile, the activities of SOD, POD, CAT, and APX were notably higher than in the CK group, together with AsA and GSH. Exogenous EBR and EBR + BRZ treatments alleviated the extent of oxidative harm to the cell membrane of grapevine seedling leaves. Excess ROS were efficiently scavenged in the plants, whereas membrane lipid peroxidation was mitigated. These findings align with Chen et al. (2019) and Yang et al. (2019).

Stress regulatory responses are found in plants when they encounter an adverse environment, in which osmoregulation is a crucial pathway (Khanna et al., 2023). Low-temperature environments cause water condensation in plant cell tissues, dehydration of the protoplasm, denaturation of proteins, and irreversible mechanical damage (Kumari et al., 2022). The osmoregulatory functions of plants are stimulated by stress, and free proline and soluble sugars are important osmoregulators (Altaf et al., 2024). Low-temperature stress resulted in a substantial increase in the free proline and soluble sugar contents in grape. This cold environmental challenge caused varying extents of cell membrane injury, accompanied by membrane lipid peroxidation, which disrupted the structural and functional integrity of the cellular membrane system. Such observations align with the research outcomes reported by Guo et al. (2020) and Karimi (2020), underscoring the susceptibility of plant cellular structures to oxidative damage under cold stress conditions. In contrast, the free proline and soluble sugar contents of exogenous EBR and EBR+BRZ-treated seedling leaves were enhanced. The application of EBR mitigated the detrimental effects induced by low-temperature stress while exerting a notable regulatory influence on the concentrations of osmoregulatory substances. These results align closely with the observations documented by Yang et al. (2019) and Heidari et al. (2021), further substantiating the hypothesis regarding EBR in mitigating abiotic stress through physiological adjustments.