Improving Photosynthetic Capacity, Alleviating Photosynthetic Inhibition and Oxidative Stress Under Low Temperature Stress With Exogenous Hydrogen Sulfide in Blueberry Seedlings

In this study, we investigated the mechanism of photosynthesis and physiological function of blueberry leaves under low temperature stress (4–6°C) by exogenous hydrogen sulfide (H₂S) by spraying leaves with 0.5 mmol·L^–1 NaHS (H₂S donor) and 200 μmol·L^–1 hypotaurine (Hypotaurine, H₂S scavenger). The results showed that chlorophyll and carotenoid content in blueberry leaves decreased under low temperature stress, and the photochemical activities of photosystem II (PSII) and photosystem I (PSI) were also inhibited. Low temperature stress can reduce photosynthetic carbon assimilation capacity by inhibiting stomatal conductance (G_s) of blueberry leaves, and non-stomatal factors also play a limiting role at the 5^th day of low temperature stress. Low temperature stress leads to the accumulation of Pro and H₂O₂ in blueberry leaves and increases membrane peroxidation. Spraying leaves with NaHS, a donor of exogenous H₂S, could alleviate the degradation of chlorophyll and carotenoids in blueberry leaves caused by low temperature and reduce the photoinhibition of PSII and PSI. The main reason for the enhancement of photochemical activity of PSII was that exogenous H₂S promoted the electron transfer from Q_A to Q_B on PSII acceptor side under low temperature stress. In addition, it promoted the accumulation of osmotic regulator proline under low temperature stress and significantly alleviated membrane peroxidation. H₂S scavengers (Hypotaurine) aggravated photoinhibition and the degree of oxidative damage under low temperature stress. Improving photosynthetic capacity as well as alleviating photosynthetic inhibition and oxidative stress with exogenous H₂S is possible in blueberry seedlings under low temperature stress.

Introduction

Hydrogen sulfide H₂S has dual effects on plant growth and development. A high concentration of H₂S can cause cytotoxicity, while a low concentration of H₂S does not cause toxicity to plants and may act as a signaling molecule (Duan et al., 2015; Li et al., 2016). Recently, many studies have found that H₂S can regulate plant growth and development, such as inducing plant seed germination (Liu and Lal, 2015), improving photosynthetic capacity (Coyne and Bingham, 1978; Chen et al., 2011), regulating stomatal movement (Lisjak et al., 2011; Scuffi et al., 2014), promoting the development of lateral roots (Jia et al., 2015; Fang T. et al., 2014), regulating secondary metabolism of sugar, polyamines, organic acids and amino acids (Shi et al., 2015; Chen et al., 2016), participating in protein modification (Mustafa et al., 2009), maintaining ion balance in plants (Wang et al., 2012; Lai et al., 2014), delaying ripening and senescence of postharvest fruits during storage (Fu et al., 2014; Hu et al., 2014), and improving antioxidant capacity (Luo et al., 2015). In addition, H₂S has been shown to participate in the regulation of resistance (Hua et al., 2010; Christou, 2013; Jin et al., 2018). The application of exogenous H₂S could promote plant growth and seed germination (Zhang et al., 2010a), increase the survival and regeneration ability of Nicotiana tabacum cells under heat stress, alleviate cell electrolyte leakage and malondialdehyde (MDA) accumulation after heat shock (Li et al., 2012), and alleviate the inhibition of heavy metal stress on plant root growth (Chen et al., 2013). H₂S also interacts with other hormones and signaling substances in plants (Hancock and Whiteman, 2016). H₂S can alleviate the inhibition of salt stress on the growth of Medicago sativa seedlings and is closely related to the increase of NO content (Wang et al., 2012). Under lead stress, H₂S and NO can improve the antioxidant system and mineral balance of sesame by interacting (Amooaghaie et al., 2017). H₂S can be used as upstream signaling molecule of H₂O₂ to promote mung bean (Vigna radiata) seed germination (Li and He, 2015). H₂S can be used as a signal molecule of salicylic acid (SA) to participate in Cd tolerance in Arabidopsis thaliana (Qiao et al., 2015). H₂S has a complex relationship with Ca²⁺ in regulating abiotic stressors such as high temperature (Li et al., 2012), Cr⁶⁺ (Fang H. et al., 2014), and drought (Jin et al., 2013). Cheng et al. (2013). found that H₂S could inhibit the production of reactive oxygen species and ethylene and alleviate the death of Pisum sativum root tip induced by hypoxia by simulating flooding and a hypoxic environment.

Low temperature is one of the most common adversities facing agricultural production in cold regions. Low temperature stress inhibits plant growth and physiological function, which is also related to the decrease of photosystem II (PSII) and photosystem I (PSI) activity (Shen et al., 1990), the limitation of assimilation synthesis (Strauss et al., 2010), the decrease of dark reaction-related enzymes activity, and the disturbance of active oxygen metabolism (Joanna et al., 2019). In the early spring in northern China, blueberries often suffer from low temperature damage, so improving low temperature tolerance is of great significance in the flowering and fruiting stages of blueberries. Sodium hydrosulfide (NaHS) can form H₂S in solution, and hypotaurine (Hypotaurine) can scavenge H₂S by directly binding with sulfides. Although a large number of studies have proved that exogenous NaHS with appropriate concentration can improve plant resistance to abiotic stresses, there are few studies on H₂S improving plant resistance to low temperature, especially on photosynthetic function of blueberry under low temperature stress.Therefore, NaHS and Hypotaurine are often used as the donor and scavenger of H₂S, respectively (Wang et al., 2012). In this paper, the effects of exogenous NaHS and Hypotaurine on the photosynthetic function and physiological characteristics of blueberry leaves under simulated low temperature stress were studied. The aim of the study was to explore the mechanism of exogenous H₂S regulating the physiological characteristics and photosynthetic function of blueberry leaves under low temperature stress and to provide theoretical basis for improving the low temperature tolerance of blueberry seedlings in the greenhouse and during transplanting.

Materials and Methods

Materials and Treatments

This study was conducted using annual seedlings of Meiden, a lowbush blueberry cultivar with strong cold resistance, which is popular in northern China, at the College of Horticulture, Jilin Agricultural University, Jilin, China in 2018. The seedlings were seeded in pots with a top diameter of 20 cm, a bottom diameter of 16 cm, and a height of 20 cm. The pots were filled with well mixed turf soil and vermiculite (volume ratio 2:1). Plants were grown in an artificial climate chamber with a temperature of 25°C, a light intensity of 400 μmol·m^–2·s^–1, and a light cycle of 12 h light, 12 h dark.

Thirty seedlings with similar growth were selected for the experiment. The treatment group was sprayed with 0.5 mmol·L^–1 NaHS and 200 μmol·L^–1 Hypotaurine, respectively, and the control group was treated with distilled water. The leaves were sprayed uniformly on both sides until the solution on the leaves formed fine mist-like droplets. Each treatment contained 10 plants as repeats. After spraying NaHS and Hypotaurine, the droplets on the leaf surface were allowed to dry naturally. After three days, all groups were removed to the temperature controlled growth cabinet and the cabinet was maintained at 4–6°C. Light intensity and humidity were identical for all treatments. Physiological indexes were determined before the treatment (marked as day 0) and at the 2^nd and 5^th days after the treatment.

Parameters and Methods of Determination

Determination of Fast Chlorophyll Fluorescence Induction Curve (OJIP) and 820 nm Light Reflection Curve

The unfolded penultimate leaves of blueberry in different treatments were selected and dark adapted for 30 min by dark adaptation clips. The OJIP curves and 820 nm light reflection curves of leaves after dark adaptation were measured using a Hansatech M-PEA (Multi-Function Plant Efficiency Analyser). Five repetitions were carried out for each treatment (biological experiments). According to the formulas V_O–P=(F_t–F_o)/(F_P–F_o) and V_O–J=(F_t–F_o)/(F_J–F_o), OJIP curves were standardized by O–P and O–J to obtain V_O–P and V_O–J curves. The relative variable fluorescence V_J of J point (2 ms) on V_O–P curve and the relative variable fluorescence V_K of K point (0.3 ms) on the V_O–J curve were also defined. In the formula, F_t is the relative fluorescence intensity at each time point on the OJIP curve, while F_o, F_J and F_P represent the relative fluorescence intensity at 0.01, 2, and 1,000 ms time points, respectively. The standard V_O–P and V_O–J curves of blueberry leaves in different treatments were compared with those of CK curves and expressed as △V_O–P and △V_O–J. A JIP-test analysis was conducted on the OJIP curve to obtain the maximum photochemical efficiency of PSII (F_v/F_m), the performance index of PSII based on absorption (PI_ABS), and the JIP-test analysis of OJIP curves following the method described by Strasser et al. (1995). The activity of the PSI reaction center is reflected by the relative decrease (△I/I_o) of the 820 nm light reflection curve (MR820 nm) signal and the slope of the MR820 nm curve as it descends in the initial stage (1–2.5 ms). I_o and △I represent the maximum of the reflected signal and the difference between the maximum and minimum reflected signals in the 820 nm light reflection curve, respectively (Zhang et al., 2018b).

Determination of Photosynthetic Gas Exchange Parameters and Carboxylation Efficiency (CE)

The unfolded penultimate leaves of blueberry in different treatments were selected to measure the photosynthetic gas exchange parameters by Li-6800 photosynthetic system (Licor Corporation, UK). The net photosynthetic rate (P_n), stomatal conductance (G_s), transpiration rate (T_r), and intercellular CO₂ concentration (C_i) of blueberry leaves in different treatments were measured under the conditions of 400 μmol·mol^–1 CO₂ fixed by CO₂ cylinder and 1,000 μmol·m^–2·s^–1 PFD set by built-in light source. The measurements were repeated five times (biological experiments). The light intensity PFD was fixed to 1,500 μmol·m^–2·s^–1 (saturated light intensity) using the built-in light source of the Li-6400 photosynthetic system. The CO₂ concentration (Ci) was controlled by CO₂ cylinders to 400, 300, 200, 150, 100, and 50 μmol·mol^–1, respectively, to obtain the corresponding P_n. The initial slope of P_n–Ci response curve was considered the carboxylation efficiency (CE).

Determination of Physiological Indexes

The content of chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoids (Car) was determined by visible spectrophotometry with 80% acetone extraction (Lichtenthaler, 1987). The proline (Pro) content was measured by acidic ninhydrin colorimetry with 3% sulfosalicylic acid boiling water extraction (Bates et al., 1973). The measurement of H₂O₂ content followed the methods described by Alexieva et al. (2001). To monitor lipid peroxidation and membrane integrity, malondialdehyde (MDA) concentration was determined with fresh leaves as described previously (Wang et al., 2010). All physiological indexes were repeated three times (biological experiments).

Statistical Analysis

Excel (2003) and SPSS (22.0) software were used for statistical analysis. All data were the means ± standard error (SE). One-way ANOVA and least significant difference (LSD) were used for the comparison of the differences between different datasets. A P value less than 0.05 was considered statistically significant.

Results and Analysis

Chlorophyll and Carotenoid Content

As shown in Figure 1, the Chl a, Chl b, Chl (a+b), and Car content in blueberry leaves decreased significantly under low temperature stress, and the extent of the reduction increased with the increased duration of low temperature stress. The Chl a, Chl b, and Chl (a+b) content in blueberry leaves treated with NaHS under low temperature stress increased to varying degrees compared with those treated with LT, but the difference was not significant (P > 0.05). The Chl a, Chl b, and Chl content after treatment with Hypotaurine was significantly lower than that of the LT treatment (P < 0.05). Car content of blueberry leaves treated with NaHS was 17.14% (P < 0.05) and 23.25% (P < 0.05) higher than the leaves treated with LT at the 2^nd and 5^th day of low temperature, respectively. In contrast, the Car content in blueberry treated with Hypotaurine was 8.29% (P > 0.05) and 38.59% (P < 0.05) lower than that in the LT treatment, respectively.

FIGURE 1

Figure 1 Effects of exogenous NaHS and Hypotaurine on chlorophyll a (A), chlorophyll b (B), total chlorophyll a (C), and carotenoid (D) contents in blueberry leaves under low temperature stress. The data in the figure are from three replicated experiments (n = 3), and represent means ± standard error (SE). Different small letters show significant differences (P < 0.05). CK: room temperature control at 25°C; LT: low temperature treatment; LT + NaHS: low temperature treatment at 4–6°C after spraying 0.5 mmol·L^–1 NaHS; LT + Hypotaurine: low temperature treatment at 4-6°C after spraying leaves with 200 μmol·L^–1 hypotaurine.

OJIP Curve and Photochemical Efficiency of PSII

The results in Figure 2 showed that low temperature stress significantly changed the OJIP curve of blueberry leaves. The relative fluorescence intensity F_o of point O changed little, whereas the F_p of point P decreased significantly, and the variation at the 5^th day of low temperature treatment was significantly larger than that at the 2^nd day. NaHS treatment significantly alleviated the decrease of F_p in blueberry leaves under low temperature stress, whereas the application of Hypotaurine increased the reduction of F_p.

FIGURE 2

Figure 2 Effects of exogenous NaHS and Hypotaurine on OJIP curves of blueberry leaves under low temperature stress at the 2^nd (A) and 5^th (B) day. The data in the figure are from five replicated experiments (n = 5). Different small letters show significant differences (P < 0.05). CK: room temperature control at 25°C; LT: low temperature treatment; LT + NaHS: low temperature treatment at 4–6°C after spraying 0.5 mmol·L^–1 NaHS; LT + Hypotaurine: low temperature treatment at 4-6°C after spraying leaves with 200 μmol·L^–1 hypotaurine.

With the increased duration of the low temperature treatment, F_v/F_m and PI_ABS of blueberry leaves showed a decreasing trend. PI_ABS showed a greater decrease than F_v/F_m (Figure 3). Under low temperature stress, there was no significant difference of F_v/F_m between the LT+NaHS treatment and the LT treatment, but PI_ABS of blueberry leaves in the LT+NaHS treatment was higher than that in LT treatment by 65.35% (P < 0.05) and 36.51% (P > 0.05), respectively. Spraying with Hypotaurine resulted in an increase in the reduction of F_v/F_m and PI_ABS.

FIGURE 3

Figure 3 Effects of exogenous NaHS and Hypotaurine on F_v/F_m (A) and PI_ABS (B) of blueberry leaves under low temperature stress. The data in the figure are from five replicated experiments (n = 5), and represent means ± standard error (SE). Different small letters show significant differences (P < 0.05).CK: room temperature control at 25°C; LT: low temperature treatment; LT + NaHS: low temperature treatment at 4–6°C after spraying 0.5 mmol·L^–1 NaHS; LT + Hypotaurine: low temperature treatment at 4–6°C after spraying leaves with 200 μmol·L^–1 hypotaurine.

Standardized O–P Curve and Standardized O–J Curve

OJIP curves of blueberry leaves in different treatments were standardized by O-P (V_O-P) (Figures 4A, B). The difference (△V_O–P) between V_O–P and CK (Figures 4C, D) showed that the relative variable fluorescence V_J at 2 ms of the V_O–P curve increased significantly under low temperature stress, and the increase was greater at the 5^th day than at the 2^nd day. Under low temperature stress, the V_J of blueberry leaves in the NaHS treatment was lower than that in LT treatment by 23.04% (P > 0.05) and 17.55% (P > 0.05) at the 2^nd and 5^th day, respectively, while the Hypotaurine treatment further increased V_J (Figure 5A).

FIGURE 4

Figure 4 Effects of exogenous NaHS and Hypotaurine on V_O-P and V_O-J curves of blueberry leaves under low temperature stress. The data in the figure are from five replicated experiments (n = 5). CK: room temperature control at 25°C; LT: low temperature treatment; LT + NaHS: low temperature treatment at 4–6°C after spraying 0.5 mmol·L^–1 NaHS; LT + Hypotaurine: low temperature treatment at 4–6°C after spraying leaves with 200 μmol·L^–1 hypotaurine. Effects of exogenous NaHS and Hypotaurine on OJIP curves of blueberry leaves under low temperature stress. OJIP curves of blueberry leaves in different treatments were standardized by O-P (VO-P) (A, B). The difference (ΔVO-P) between VO-P and CK (C, D). OJIP curves were standardized by O-J (VO-J) (E, F). The difference(ΔVO-J) between the VO-J curve and the CK (G, H). The data in the figure are from five replicated experiments (n = 5). CK: room temperature control at 25°C; LT: low temperature treatment; LT + NaHS: low temperature treatment at 4–6°C after spraying 0.5 mmol•L-1 NaHS; LT + Hypotaurine: low temperature treatment at 4-6°C after spraying leaves with 200 μmol•L-1 hypotaurine.

FIGURE 5

Figure 5 Effects of exogenous NaHS and Hypotaurine on V_J (A) and V_K (B) of blueberry leaves under low temperature stress. The data in the figure are from five replicated experiments (n = 5), and represent means ± standard error (SE). Different small letters show significant differences (P < 0.05). CK: room temperature control at 25°C; LT: low temperature treatment; LT + NaHS: low temperature treatment at 4–6°C after spraying 0.5 mmol·L^–1 NaHS; LT + Hypotaurine: low temperature treatment at 4–6°C after spraying leaves with 200 μmol·L^–1 hypotaurine.

OJIP curves were standardized by O–J (V_O–J) (Figures 4E, F). The difference (△V_O–J) between the V_O–J curve and the CK (Figures 4G, H) revealed that low temperature stress had little effect on V_K at 0.3 ms, and there was no significant difference between V_K and CK at the 2^nd and 5^th day of low temperature treatment. The effect of NaHS and Hypotaurine treatments on V_K was also not significant (Figure 5B).

The Modulated Reflected Signal 820 nm (MR820 nm)

Under low temperature stress, the amplitude of the MR820 nm curve of blueberry leaves decreased (Figures 6A, B), and the slope of the MR820 nm curve at the initial stage (1–2.5 ms) decreased compared with the CK (Figures 6C, D). The decrease of the MR820 nm curve at the 5^th day of low temperature treatment was greater than that at the 2^nd day. Exogenous NaHS significantly alleviated the amplitude of the MR820 nm curve and minimized the decrease of the initial slope. In contrast, treatment with Hypotaurine showed the opposite effect. Quantitative analysis of △I/I_o changes (Figure 7) showed that △I/I_o of blueberry leaves decreased by 18.78% (P < 0.05) and 46.16% (P < 0.05) on the 2^nd and 5^th day of low temperature treatment, respectively. The decrease of △I/I_o in the LT + NaHS treatment was significantly lower than that in the LT treatment, whereas the Hypotaurine treatment maximized the decrease of △I/I_o under low temperature stress.

FIGURE 6

Figure 6 Effects of exogenous NaHS and Hypotaurine on the modulated reflected signal of 820 nm (MR820 nm) in blueberry leaves at the 2^nd (A) and 5^th (B) day of low temperature treatment and on the slope of the MR820 nm curve at the initial stage (1–2.5 ms) of decline at the 2^nd (C) and 5^th (D) day of treatment. The data in the figure are from five replicated experiments (n = 5). CK: room temperature control at 25°C; LT: low temperature treatment; LT + NaHS: low temperature treatment at 4–6°C after spraying 0.5 mmol·L^–1 NaHS; LT + Hypotaurine: low temperature treatment at 4–6°C after spraying leaves with 200 μmol·L^–1 hypotaurine.

FIGURE 7

Figure 7 Effects of exogenous NaHS and Hypotaurine on △I/I_o of blueberry leaves under low temperature stress. The data in the figure are from five replicated experiments (n = 5), and represent means ± standard error (SE). Different small letters show significant differences (P < 0.05). CK: room temperature control at 25°C; LT: low temperature treatment; LT + NaHS: low temperature treatment at 4–6°C after spraying 0.5 mmol·L^–1 NaHS; LT + Hypotaurine: low temperature treatment at 4–6°C after spraying leaves with 200 μmol·L^–1 hypotaurine.

Gas Exchange Parameters of Photosynthesis

The results in Figure 8 showed that the P_n, G_s, and T_r of blueberry leaves decreased significantly under low temperature stress; however, the decrease of P_n, G_s, and T_r was alleviated to varying degrees after spraying with exogenous NaHS. After spraying with Hypotaurine, P_n, G_s, and T_r showed a more evident decrease compared with the control. C_i in blueberry leaves did not change significantly at the 2^nd day of low temperature, but increased significantly at the 5^th day. Exogenous NaHS had no significant effect on C_i in blueberry leaves under low temperature stress, but Hypotaurine treatment increased C_i significantly.

FIGURE 8

Figure 8 Effects of exogenous NaHS and Hypotaurine on net photosynthetic rate (A), stomatal conductance (B), transpiration rate (C), and intercellular CO₂ concentration (D) of blueberry leaves under low temperature stress. The data in the figure are from five replicated experiments (n=5), and represent means ± standard error (SE). Different small letters show significant differences (P < 0.05).CK: room temperature control at 25°C; LT: low temperature treatment; LT + NaHS: low temperature treatment at 4–6°C after spraying 0.5 mmol·L^–1 NaHS; LT + Hypotaurine: low temperature treatment at 4–6°C after spraying leaves with 200 μmol·L^–1 hypotaurine.

Change in the Activity of the Dark Reaction

The determination of the initial slope of the CO₂ response curve (Figures 9A, B) and CE (Figure 9C) showed that CE in blueberry leaves decreased significantly under low temperature stress. However, CE in the LT + NaHS treatment was significantly greater than that in the LT treatment on the 2^nd and 5^th day of cold treatment (P < 0.05), whereas the decrease of CE in LT+ Hypotaurine treatment was significantly greater than that in LT treatment.

FIGURE 9

Figure 9 Effects of exogenous NaHS and HT on the initial slope (A, B) and CE (C) of the CO₂ response curve of blueberry leaves under low temperature stress. The data in the figure are from five replicated experiments (n=5), and represent means ± standard error (SE). Different small letters show significant differences (P < 0.05).CK: room temperature control at 25°C; LT: low temperature treatment; LT + NaHS: low temperature treatment at 4–6°C after spraying 0.5 mmol·L^–1 NaHS; LT + Hypotaurine: low temperature treatment at 4–6°C after spraying leaves with 200 μmol·L^–1 hypotaurine.

Pro, H₂O₂, and MDA Content

With the prolongation of the low temperature treatment, the Pro, H₂O₂, and MDA content in blueberry leaves increased obviously (Figure 10). At the 2^nd and 5^th day of low temperature treatment, the Pro content of blueberry leaves treated with LT + NaHS increased by 32.69% (P < 0.05) and 19.05% (P > 0.05), respectively, compared with the plants treated with LT. The MDA content in the LT + NaHS treatment was 19.15% (P > 0.05) and 15.36% (P > 0.05) lower than that in LT treatment on the 2^nd and 5^th day of low temperature treatment, respectively. Therefore, spraying blueberry leaves with Hypotaurine significantly decreased Pro content and increased the accumulation of H₂O₂ and MDA content in blueberry leaves under low temperature stress.

FIGURE 10

Figure 10 Effects of exogenous NaHS and Hypotaurine on proline (Pro) (A), H₂O₂ (B), and MDA (C) content in blueberry leaves under low temperature stress. The data in the figure are from three replicated experiments (n=3), and represent means ± standard error (SE). Different small letters show significant differences (P < 0.05). CK: room temperature control at 25°C; LT: low temperature treatment; LT + NaHS: low temperature treatment at 4–6°C after spraying 0.5 mmol·L^–1 NaHS; LT + Hypotaurine: low temperature treatment at 4–6°C after spraying leaves with 200 μmol·L^–1 hypotaurine.

Discussion

Chloroplasts are the main site of plant photosynthesis and one of the organelles that is most sensitive to stress. The decrease of chlorophyll content in the chloroplast inhibits the absorption and utilization of light energy by plants (Zhang et al., 2016). In our study, the Chla, Chlb, and Chla+b content in blueberry leaves were significantly decreased under low temperature stress (Figures 1A–C), which indicated that low temperature stress could lead to chlorophyll degradation or inhibit chlorophyll synthesis. The addition of exogenous NaHS could promote chlorophyll synthesis or alleviate its degradation rate (Chen et al., 2011), and exogenous NaHS could also promote chlorophyll synthesis and chloroplast development in maize under iron deficient conditions (Chen et al., 2015). Our results are consistent with these reports. The treatment with exogenous NaHS prior to low temperature stress significantly alleviated the decrease of chlorophyll content. In contrast, the application of exogenous Hypotaurine increased the reduction of chlorophyll content, indicating that exogenous H₂S could prevent the degradation of chlorophyll in blueberry leaves under low temperature stress. Carotenoids are involved in the absorption and transmission of light energy by plants, as well as have strong antioxidant capacity (Zhai et al., 2016), and beneficial to the photosystem II assembly and function (Zakar et al., 2016). In the carotenoid-reduced Arabidopsis szl1 mutant, the sensitivity of PSI and PSII to low temperature increased significantly (Cazzaniga et al., 2012). Low temperature stress induced the decrease of carotenoid content in blueberry leaves (Figure 1D) and exogenous H₂S alleviated the degradation of Car in blueberry leaves under low temperature stress.

Low temperature stress often leads to the decrease of PSII activity in plants. The photoinhibition of PSII decreases linearly with the decrease of temperature in the range of 4 to 25°C (Sonoike, 2011). The relative fluorescence intensity at point P of the OJIP curve decreased significantly under low temperature stress, and F_v/F_m and PI_ABS showed a decreasing trend, especially PI_ABS (Figure 3), indicating that low temperature led to the decrease of photochemical activity of PSII, and even photoinhibition. In addition, V_J increased significantly, whereas V_K did not change significantly. The increase of V_J reflects the inhibition of electron transfer from Q_A to Q_B on the PSII acceptor side (Zhang et al., 2017; Zhang et al., 2018a), while the increase of V_K is considered to be a specific marker of damage to oxygen-evolving complex on the PSII donor side (Zhang et al., 2018b). However, the change of V_K is not only affected by injury on the PSII donor side, but also by damage on PSII acceptor side. When the injury on the acceptor side is greater than that on the donor side, V_K does not increase significantly (Zhang et al., 2018c; Zhang et al., 2019b). Therefore, although the electron transfer from Q_A to Q_B on the PSII acceptor side of blueberry leaves was inhibited by low temperature, low temperature had little effect on the oxygen-evolving complex of the PSII donor side as V_K did not change. The inhibition of electron transport from Q_A to Q_B in the PSII acceptor side under stress conditions was mainly related to the degradation of D1 protein, while exogenous H2S can accelerate the turnover of D1 protein in wheat leaves under drought stress to improve the drought resistance of PSII function (Li et al., 2015). Therefore, exogenous NaHS significantly alleviated the increase of V_J under low temperature stress, while exogenous Hypotaurine increased V_J, suggesting that exogenous H₂S might protect the D1 protein in blueberry leaves under low temperature stress. Ultimately, this alleviates the photoinhibition of PSII.

In addition to the photoinhibition of PSII, PSI is also an important photoinhibition site under low temperature stress, especially in cold-sensitive plants. Low temperature stress makes PSI more prone to photoinhibition than PSII, and its degree of photoinhibition is often greater than PSII, and more challenging to recover (Terashima et al., 1994; Sonoike and Terashima, 1994; Zhang et al., 2010). We found that the PSI activity of blueberry leaves decreased under low temperature stress. The decrease of PSI activity was significantly alleviated by the exogenous application NaHS, while PSI activity was further decreased by Hypotaurine, an H₂S scavenger, under low temperature stress. These data indicated that exogenous H₂S could increase the PSI activity under low temperature stress. A previous study has reported that the photoinhibition of PSI is mainly related to the increase of reactive oxygen species in PSI (Sonoike, 1996). Thus, the accumulation of H₂O₂ under low temperature stress caused by Hypotaurine (Figure 10B) is an important reason for the increase of PSI photoinhibition.

Some studies have reported that exogenous H₂S improved the drought resistance of Arabidopsis thaliana by inducing stomatal closure (Jin et al., 2017), which was mainly due to the reduction of the stomatal diameter caused by H₂S (Jin et al., 2011) or the increase in the expression of the mitogen activated protein kinase gene to prevent over opening of stomata under low temperature stress (Du et al., 2017). However, other studies have shown that exogenous H₂S improved photosynthetic capacity of rice leaves by increasing stomatal aperture and density, and NaHS induced stomatal opening in Arabidopsis thaliana by inhibiting NO production (Lisjak et al., 2010). Therefore, the function of H₂S in plant stomatal movement is still controversial and requires further study. The G_s of blueberry leaves decreased rapidly under low temperature stress, leading to the decrease of T_r and P_n.

After spraying leaves with exogenous NaHS, the decrease of G_s and T_r was significantly lower than that of the control. Moreover, the decrease of P_n was also alleviated to varying degrees with the application of NaHS, whereas spraying with Hypotaurine aggravated stomatal closure and further decreased the photosynthetic rate under low temperature stress (Figure 8). This indicates that exogenous H₂S could improve photosynthetic capacity of blueberry leaves under low temperature stress by promoting stomatal opening. Although the G_s in the exogenous NaHS treatment was significantly higher than that in non-sprayed NaHS treatment, the variation between the P_n was not significant. These results indicated that the application of exogenous H₂S also increased photosynthetic capacity under low temperature stress via non-stomatal factors. Non-stomatal factors, such as the decrease of photosynthetic enzyme activity under stress, are also important factors that limit plant photosynthesis. Under severe stress, non-stomatal factors often play a major role in limiting plant photosynthesis (Zhang et al., 2019b). At the 5^th day of low temperature stress, the C_i increased significantly (Figure 8D), indicating that the reason for the decrease of photosynthetic capacity caused by long-term (5 d) low temperature stress was due to the limitation of non-stomatal factors (Arena and Vitale, 2018; Zhang et al., 2019a) Exogenous H₂S can promote the transport of CO₂ (Espie et al., 1989), the expression of photosynthesis-related enzymes, and the redox modification of thiol groups to improve photosynthesis Application of exogenous H₂S can also promote the protein and gene expression of Ribulose-1,5-Bisphosphate Carboxylase (Rubisco) and phenol pyruvate carboxylase in maize leaves under iron deficiency conditions (Chen et al., 2011). In our study, spraying NaHS increased the CE under low temperature stress, while spraying Hypotaurine decreased CE (Figure 9). Therefore, the reason why exogenous H₂S can increase photosynthetic capacity of blueberry leaves under low temperature stress is not only related to the increase of induced stomatal conductance, but also possibly related to the fact that exogenous H₂S is beneficial to CO₂ fixation in dark reaction under low temperature stress, which may be related to the protection of dark reaction-related enzymes.

H₂S also interacts with other hormones and signaling substances in plants, such as NO (Chen et al., 2011; Wang et al., 2012), SA (Amooaghaie et al., 2017), and Ca²⁺ (Li et al., 2012; Qiao et al., 2015). In addition, as a very important cell signaling molecule, H₂O₂ content is regulated by H₂S in many physiological processes during plant growth and development (Fang H. et al., 2014; Zhang et al., 2008). H₂S can be used as an upstream signaling molecule of H₂O₂ to promote the seed germination as described for mung bean (Li et al., 2012). Li et al. found that H₂S could improve salt tolerance in Arabidopsis thaliana roots, and this process required the active participation of H₂O₂ (Li et al., 2015). With the increased duration of low temperature stress, H₂O₂ content in blueberry leaves increased significantly, although exogenous NaHS treatment promoted the increase of H₂O₂ content in blueberry leaves to some extent, the difference was not significant, which may be related to the increase of antioxidant enzyme activity or accumulation of antioxidants by NaHS under low temperature stress. Exogenous Hypotaurine significantly increased H₂O₂ content under low temperature stress. A low concentration of H₂O₂ can be used as a signaling substance in plant response to stress, and a high concentration of H₂O₂ can cause oxidative damage to plant cells (Li et al., 2014). Therefore, exogenous Hypotaurine aggravates oxidative damage under low temperature stress.

H₂S can alleviate the membrane peroxidation under low temperature stress by regulating the activity of antioxidant enzymes in hawthorn fruits (Cheng et al., 2016), mitigate the oxidative damage caused by Al by increasing the antioxidant capacity of wheat (Aghdam et al., 2018), and reduce the MDA content by enhancing the activity of antioxidant enzymes in alfalfa seedlings under Cd stress (Zhang et al., 2010). In addition, H₂S can also induce the accumulation of ascorbic acid and glutathione in plants to improve its antioxidant capacity (Cui et al., 2014). Under low temperature stress, the increase of MDA content of blueberry leaves was significantly alleviated in NaHS treatment, while the membrane peroxidation of leaves was intensified in Hypotaurine treatment, which was consistent with the change of H₂O₂ content (Figure 10B).

Under stress, plant cells actively accumulate small molecules to regulate their osmotic potential and maintain their normal water content (Shan et al., 2011). Exogenous H₂S could control the water potential and relative water content of spinach leaves by regulating the synthesis of soluble sugar, polyamine, and glycine betaine to enhance the adaptability of spinach seedlings to drought (Kaur and Asthir, 2015). The accumulation of Pro plays an important role in improving stress resistance of plants (Chen et al., 2016). Pro is also an inducer of osmotic stress-related genes and is a scavenger of reactive oxygen species (Hong et al., 2000; Theocharis et al., 2012), which plays an important role in improving the stability of plant cell membranes under stress (Lu and Becker, 2015). Luo et al. (2015) has found that exogenous H2S could increase the Pro content of banana under low temperature stress and significantly enhances their cold tolerance, which was mainly related to H₂S increasing the activity of 1-pyrroline-5-carboxylate synthetase and decreasing the activity of proline dehydrogenase (Mansour, 2013). Li et al. (2015) also demonstrated that exogenous H₂S improved the heat tolerance of maize, which was related to the Pro accumulation induced by exogenous H₂S. The results of the present experiment are consistent with these previous reports. Under low temperature stress, the accumulation of Pro leaves increased, and spraying with exogenous NaHS promoted the Pro accumulation, while spraying with Hypotaurine had the opposite effect (Figure 10A). Therefore, the accumulation of Pro is an adaptive mechanism to low temperature stress, and the accumulation of Pro induced by exogenous H₂S plays an active role in improving its low temperature tolerance. The mechanism of exogenous H₂S donor (NaHS) alleviating photosynthesis inhibition under low temperature stress is summarized in Figure 11.

FIGURE 11

Figure 11 Mechanisms of exogenous H₂S donor (NaHS) in alleviating photosynthesis inhibition under low temperature stress.

Conclusions

NaHS, an exogenous H₂S donor, significantly alleviated the degradation of chlorophyll and carotenoids in blueberry leaves under low temperature stress. NaHS also increased the activities of PSII and PSI, of which the electron transfer from Q_A to Q_B on the acceptor side of PSII may be the site of primary activity of H2S. Exogenous H₂S also promoted stomatal opening and photosynthetic carbon assimilation ability under low temperature stress. Promoting the accumulation of Pro plays an important role in improving the low temperature tolerance of blueberry by exogenous H₂S. In contrast, spraying blueberry leaves with Hypotaurine, an H₂S scavenger, aggravated the photoinhibition and oxidative damage of blueberry leaves. In conclusion, the application of exogenous H₂S improved the tolerance of blueberry to low temperature stress, which was mainly related to the improvement of photosynthetic capacity and the accumulation of Pro in blueberry leaves.

Data Availability Statement

All datasets generated for this study are included in the article/supplementary material.

Author Contributions

XT, BA and XL conceived and designed experiments; All the authors performed the experiments and analyzed the data; XT and BA wrote the manuscript and prepared the figures and/or tables. XT, BA and XL reviewed drafts of the paper. XT and BA contributed equally to this work.

Funding

This work was supported by National key Research and Development Program of China (2017YFC0504205); Science and Technology Development Program of Jilin Province in China (2016041201XH).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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