Introduction

Loquat (Eriobotrya japonica Lindl.), an evergreen fruit tree native to subtropical regions of China, is sensitive to low-temperature stress; this is an important factor affecting the geographical distribution and production of loquats (Wu et al. 2016). Tropical loquat varieties cultivated in the southern subtropical and tropical marginal areas (e.g., Fujian and Guangdong provinces in China) display poor low-temperature tolerance in the southern areas of China. In particular, the major loquat cultivar Zaozhong No. 6 suffers from severe frost damage, even leading to a total loss of yield (Wu et al. 2010). Northern China has cold weather, thus making it unsuitable for loquat cultivation. In recent years, small-scale production of loquat has been developed in some northern cities, such as Beijing and Yingkou, using cultivation facilities according to the local conditions. Therefore, it is necessary to investigate how to improve the frost resistance in loquat.

Nitric oxide (NO) and Ca²⁺ are ubiquitous second messengers in plants, which play important roles in regulating various physiological and metabolic processes in response to stress (Lamattina et al. 2003; Reddy et al. 2011; Zhang et al. 2019). It was found that sodium nitroprusside (SNP) treatment significantly enhanced the activities of antioxidant enzymes, reduced the level of reactive oxygen species (ROS), and decreased the level of membrane lipid peroxidation in ginger leaves, which alleviated the heat damage of ginger leaves and increased the heat resistance of ginger plants (Li et al. 2014). SNP treatment obviously increased antioxidant enzyme activities in pumpkin seedlings under cold stress, decreased the accumulation of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), showed protective effects against oxidative damage of the seedlings, and enhanced the adaptability of the plants to cold stress (Wu et al. 2016). In previous studies, we found that appropriate SNP treatment of young fruit induced protective enzyme activities, decreased the level of membrane lipid peroxidation, and improved the fruit’s cold resistance (Wu et al. 2010).

Numerous studies have shown that exogenous Ca²⁺ improves plant resistance to various stresses, including low temperature, high temperature and drought, which are associated with ROS metabolism (Ramanjulu and Sudhakar 2001; Sulochana and Rao 2002). Calcium treatment significantly increased the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in tomato under high-temperature stress and prevented the damage of photosynthetic organs caused by heat-induced ROS, thereby improving the heat resistance of the plants (Qi et al. 2015). Calcium treatment also enhanced the activities of SOD, CAT and POD in muskmelon and alleviated the peroxidation damage of membrane lipids due to the accumulation of ROS under cold stress, thus enhancing its cold resistance (Li et al. 2011). We found that calcium treatment activated SOD and CAT activities in loquat seedlings exposed to low-temperature stress and decreased the level of membrane lipid peroxidation, thus increasing the frost resistance of loquat seedlings (Wu et al. 2016). Although it has been confirmed that NO and Ca²⁺ play regulatory roles in the frost resistance in many plant species, it is still not clear whether the NO and Ca²⁺ pathways are crosslinked in response to low-temperature stress in loquat. In this study, we investigated the association between Ca²⁺ and NO signalling pathways in loquat seedlings in response to low-temperature stress. The results could provide a theoretical basis for the prevention of frost damage to loquat.

Materials and Methods

Plant materials and treatments

Plant materials: Two-year-old container seedlings of loquat (Eriobotrya japonica Lindl. cv. Zaozhong No. 6) with normal and uniform growth were provided by the Putian Institute of Pomology and used as test materials.

Treatments: Sodium nitroprusside (SNP, an exogenous nitric oxide [NO] donor), CaCl₂, 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, a NO scavenger), and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7, a calmodulin [CaM] antagonist) were used to treat loquat seedlings, respectively. The seedlings were randomly divided into eight groups that were subjected to different treatments: Control (CK), H₂O; Treatment 1 (T1), CaCl₂; T2, 2-4-carboxyphenyl-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) + CaCl₂; T3, SNP; T4, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7) + SNP; T5, CaCl₂ + SNP; T6, cPTIO + CaCl₂ + SNP; and T7, W7 + CaCl₂ + SNP. The treatments were performed according to the methods of Yang et al. (2015), Wu et al. (2016) and Zhang et al. (2018). The final concentration of SNP, CaCl₂, cPTIO and W7 in different treatments were 0.5, 5, 0.5 and 0.5 mmol·L^-1 (each treatment solution was supplemented with 1% Tween-20), respectively. These treatment solutions were sprayed on the leaves of plants at room temperature (25°C) at 17:00 until it dripped off the leaves. Spraying was carried out every two days and repeated three times. Twenty-four h after spraying, the container seedlings were exposed to low-temperature stress at -3°C as described by Wu et al. (2010). Fifteen container seedlings from each treatment group were placed in an artificial climate chamber (relative humidity: 70%; light intensity: 20 W/m²), and the temperature was lowered to -3°C for 6 h; the temperature was then increased to 25°C and maintained at that level for 12 h before the samples were taken. The sampling was performed according to Wu et al. (2016). The 3^rd to the 5^th leaves from the top were collected, frozen in liquid nitrogen and stored in a -70°C freezer. Three biological replicates were collected, and each replicate was a mixed sample of five container seedlings. SNP (a NO donor), W7 (a calmodulin [CaM] antagonist), and cPTIO (a NO scavenger) were purchased from Sigma (Merck KGaA, Darmstadt, Germany). Other chemicals used in this study were analytical grade reagents from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Determination of H₂O₂, MDA and CaM

The H₂O₂ content was determined based on the method of Patterson et al. (1984). For this purpose, 2 g of leaf tissue was ground in liquid nitrogen and homogenised with chilled 100% acetone and then centrifuged at 10,000 × g for 20 min at 4°C. The supernatant was used for analysis of H₂O₂, which was expressed as μmol g^-1 FW (fresh weight). The content of MDA was analysed following the method of Hodges et al. (1999). Two grams of leaf tissue was ground in liquid nitrogen and homogenised with 80% ethanol, followed by centrifugation at 10, 000 × g for 20 min. An appropriate volume of supernatant was added to the reaction mixture. The supernatant was appropriately added to a reaction mixture containing 20% (w/v) trichloroacetic acid and 0.01% butylated hydroxytoluene. The reactions were mixed and heated at 95°C for 25 min, followed by centrifuging at 3000 × g for 10 min after cooling. The absorbances were read at 440 nm, 532 nm and 600 nm. The MDA content was calculated and expressed as mmol g^-1 FW. The CaM content was measured by enzyme-linked immunosorbent assay (ELISA) using the Plant CaM ELISA Kit (Kmaels, Shanghai, China) according to the user manual. Each assay was repeated three times. The CaM content was expressed as μg g^-1 FW.

Enzyme activity determination

Enzyme extraction and SOD activity analysis were performed according to Stewart and Bewley (1980) method. Two grams of leaf tissue were ground in liquid nitrogen and homogenised in cold 50 mM phosphate buffer (pH 7.0). The homogenate was then centrifuged at 15,000 × g for 30 min. The supernatant was retained as the enzyme extract. Fifty micrograms of enzyme extract was added to 1 mL of solution for the measurement of enzyme activity. The measurement of SOD activity was assayed in a reaction mixture containing 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 μm nitroblue tetrazolium (NBT), 2 μm riboflavin, 100 nM EDTA and the enzyme extract. The amount of enzyme required to inhibit the reduction of NBT by 50% was defined as one activity unit (U) of SOD. The activities of CAT and POD were measured as described by Cakmak and Marschner (1992), and the activity of ascorbate peroxidase (APX) was based on the method of Nakano and Asada (1981). CAT activity was assayed in a reaction mixture containing 25 mM phosphate buffer (pH 7.0), 10 mM H₂O₂ and the enzyme extract. The POD activity was assayed in a reaction mixture containing 25 mM phosphate buffer (pH 7.0), 0.05% guaiacol, 10 mM H₂O₂ and the enzyme extract. The APX activity was assayed in a reaction mixture containing 50 mM phosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.1 mM H₂O₂, 100 μM EDTA and the enzyme extract. One activity unit (U) of CAT, POD and APX was defined as the amount of enzyme that produced a change of 0.01 in optical density (OD) at 240 nm, 470 nm and 290 nm, respectively, in 1 min. Each assay was repeated three times.

Data analysis

Each treatment group included 15 container seedlings and three replicates were set randomly for the determinations of H₂O₂, MDA, CaM and enzyme activity. Significance tests of differences between groups and correlation analyses were performed using Excel 2003 and SPSS 19.0 statistical software; the means of three replicates were used in the analyses.

Results

Effects of treatments on H₂O₂ content

Fig. 1a shows that in the seedlings that received the single treatments CaCl₂ and SNP, and in those that received co-treatment CaCl₂ + SNP, the leaf H₂O₂ contents were significantly lower than that of control plants (p < 0.05); the H₂O₂ contents of the leaves were in the order CK (H₂O) > CaCl₂ > SNP > CaCl₂ + SNP, indicated that exogenous SNP or CaCl₂ treatment effectively reduced the intracellular accumulation of H₂O₂. Co-treatment with SNP + CaCl₂ had a notable synergistic effect, while the H₂O₂ scavenging capacity of CaCl₂ was lower than that of SNP. The leaf H₂O₂ content of seedlings treated with cPTIO + CaCl₂ + SNP was slightly higher than that of the seedlings in the CaCl₂ group, indicated that cPTIO, a NO scavenger, not only abolished the ability of SNP to scavenge cellular H₂O₂ but also weakens the ability of CaCl₂ to scavenge cellular H₂O₂. The leaf H₂O₂ content of plants that received the W7 + SNP treatment or W7 + CaCl₂ + SNP treatment was significantly higher than that of CK (H₂O), indicating that W7, a CaM antagonist, not only inhibits the effects induced by exogenous CaCl₂ and SNP but also weakens the ability of the cells to resist low-temperature oxidative stress; as a result, under low-temperature stress conditions, cellular H₂O₂ may be not removed in a timely manner and thus remains at a high level.

Effect of treatments on MDA content

Fig. 1b shows that when seedlings were exposed to low-temperature stress at -3°C, the leaf MDA content of the seedlings that received the single treatments CaCl₂) or SNP or the co-treatment CaCl₂ + SNP was significantly lower than that of the seedlings in the CK group (p < 0.05); the leaf MDA content showed the order SNP > CaCl₂ > CaCl₂ + SNP, indicating that the treatments had inhibitory effects on membrane lipid peroxidation in the leaf cells and that the inhibitory abilities of the three treatments differed. The inhibitory effect of CaCl₂ + SNP was higher than those of CaCl₂ and SNP. The MDA contents of leaves treated with cPTIO + CaCl₂ and cPTIO + CaCl₂ + SNP, both co-treatments, were significantly higher than those of plants treated with CaCl₂ and CaCl₂ + SNP to varying degrees (p < 0.01). The MDA content of the leaves of seedlings treated with W7 + SNP and W7 + CaCl₂ + SNP was significantly higher than the plants treated with SNP and CaCl₂ + SNP, and the differences were significant (p < 0.01). These results indicated that cPTIO and W7 promoted cell membrane lipid peroxidation and weakened the inhibitory effects of CaCl₂ and SNP on cell membrane lipid peroxidation; the inhibitory effect of the NO scavenger cPTIO was weaker than that of the CaM antagonist W7. The leaf MDA content of seedlings treated with cPTIO + CaCl₂ was higher than that of seedlings treated with CaCl₂, whereas the leaf MDA contents of seedlings treated with W7 + SNP and W7 + CaCl₂ + SNP were significantly higher than those of seedlings treated with CaCl₂, SNP, CaCl₂ + SNP and CK (p < 0.01). The results indicated that the inhibitory effect of CaCl₂ on membrane lipid peroxidation was partially mitigated by cPTIO, whereas W7 not only abolishes the inhibitory effects of SNP and CaCl₂ on cell membrane lipid peroxidation but also exacerbates oxidative damage to the membrane.

Effects of treatments on CaM content

Fig. 2 showed that under low-temperature stress conditions, the leaf CaM content of seedlings treated with CaCl₂), SNP) or CaCl₂ + SNP was significantly higher than that of the CK seedlings (p < 0.05), indicating that CaCl₂ and SNP treatment improves the content of CaM. CaCl₂ treatment had the most profound upregulating effect, whereas co-treatment with CaCl₂ + SNP produced a synergistic effect. The difference in the leaf CaM content of seedlings treated with the co-treatments cPTIO + CaCl₂ and cPTIO + CaCl₂ + SNP was slight, and the leaf CaM contents of seedlings treated with cPTIO + CaCl₂ and cPTIO + CaCl₂ + SNP were lower than those of seedlings treated with CaCl₂, SNP and CaCl₂ + SNP, indicating that cPTIO treatment inhibits the expression of CaM. The leaf CaM

Fig. 1: Effects of different treatments on the H₂O₂ and MDA contents of loquat leaves under low-temperature stress. (a) H₂O₂; (b) MDA. CK: H₂O; T1: CaCl₂; T2: cPTIO + CaCl₂; T3: SNP; T4: W7 + SNP; T5: CaCl₂ + SNP; T6: cPTIO + CaCl₂ + SNP; T7: W7 + CaCl₂ + SNP; FW: fresh weight. Different letters indicate significant differences at the 0.05 level via a Tukey-Kramer test for multiple comparisons

Fig. 2: Effects of different treatments on the CaM content of loquat leaves under low-temperature stress. CK: H₂O; T1: CaCl₂; T2: cPTIO + CaCl₂; T3: SNP; T4: W7 + SNP; T5: CaCl₂ + SNP; T6: cPTIO + CaCl₂ + SNP; T7: W7 + CaCl₂ + SNP. Different letters indicate significant differences at the 0.05 level via a Tukey-Kramer test for multiple comparisons

content of seedlings treated with W7 + SNP was lower than that of seedlings treated with CK, indicating that W7 not only eliminates the increased CaM expression induced by SNP but also further inhibits CaM expression. The leaf CaM content of seedlings treated with W7 + CaCl₂ + SNP was significantly higher than that of seedlings treated with W7 + SNP or CK (p < 0.01), indicating that W7 inhibits the expression of CaM. However, the CaCl₂ used in the T7 (W7 + CaCl₂ + SNP) co-treatment promoted the cellular production of CaM, leading to higher leaf CaM content in seedlings treated with W7 + CaCl₂ + SNP compared to seedlings treated with W7 + SNP or CK. CaCl₂ and SNP promote the synthesis of CaM in leaf cells, while cPTIO and W7 inhibit its synthesis.

Treatments effects on the activities of antioxidant enzymes

Under low-temperature conditions, the leaf CAT activities of seedlings treated with CaCl₂, SNP or CaCl₂ + SNP were higher than that of the CK seedlings; CAT activity showed the order CaCl₂ + SNP > SNP > CaCl₂ > CK, indicating that CaCl₂ and SNP have the physiological effect of improving cellular CAT activity and that SNP enhances CAT activity more effectively than CaCl₂ (Fig. 3a). Co-treatment with CaCl₂ + SNP produced a synergistic effect in regulating and activating CAT activity. The leaf CAT activities of seedlings treated with cPTIO + CaCl₂, W7 + SNP, cPTIO + CaCl₂ + SNP and W7 + CaCl₂ + SNP were significantly lower than seedlings treated with CaCl₂, SNP and CaCl₂ + SNP, and the leaf CAT activity of seedlings treated with W7 + CaCl₂ + SNP was lower than seedlings treated with cPTIO + CaCl₂ + SNP, indicated that cPTIO and W7 inhibited leaf CAT activity and that the latter had a more profound inhibitory effect.

Fig. 3b showed that, under cold stress conditions, the leaf POD activity of seedlings treated with cPTIO + CaCl₂, W7 + SNP, cPTIO + CaCl₂ + SNP and W7 + CaCl₂ + SNP was lower than that of the seedlings treated with CaCl₂, SNP and CaCl₂ + SNP. Moreover, leaf POD activity of seedlings treated with W7 + SNP and W7 + CaCl₂ + SNP was lower than that of seedlings treated with cPTIO + CaCl₂ and cPTIO + CaCl₂ + SNP. The results indicated that both cPTIO and W7 inhibited leaf POD activity, but the inhibitory abilities of the two compounds were different; compared with cPTIO, while W7 showed a stronger inhibitory effect on leaf POD activity. The leaf POD activities showed the order CaCl₂ + SNP > SNP > CaCl₂ > CK, but the differences were non-significant (p > 0.05), indicated that the effect of SNP and CaCl₂ on leaf POD activity was non-significant, and co-treatment with SNP + CaCl₂ indicated no synergistic effect on leaf POD activity.

Fig. 3c showed that the leaf SOD activity exhibited the order CaCl₂ + SNP > CaCl₂ > SNP > CK; the leaf SOD activity of seedlings treated with CaCl₂, SNP or CaCl₂ + SNP were significantly higher than that of CK, indicated that SNP and CaCl₂ had an activating effect on leaf SOD activity and that the activating effect of CaCl₂ on SOD activity was higher than that of SNP, while co-treatment with SNP + CaCl₂ produces a noticeable synergistic effect. The leaf SOD activity of seedlings treated with W7 + CaCl₂ + SNP or W7 + SNP were significantly lower than those of seedlings treated with CaCl₂, SNP, CaCl₂ + SNP or CK, while the leaf SOD activities of seedlings treated with cPTIO + CaCl₂ or cPTIO + CaCl₂ + SNP were significantly lower than those treated with SNP or CaCl₂ but significantly higher than that of CK, indicating that cPTIO and W7 inhibited leaf SOD activity and the inhibitory effect of cPTIO was lower than that of W7. The leaf SOD activities of seedlings treated with cPTIO + CaCl₂ and cPTIO + CaCl₂ + SNP were similar, indicating that, during co-treatment of cPTIO + CaCl₂ + SNP, the activating effect of SNP on cellular SOD activity was mitigated by cPTIO and that the enhancing effect of CaCl₂ on cellular SOD activity was also weakened. The leaf SOD activities of seedlings treated with W7 + SNP and W7 + CaCl₂ + SNP were significantly lower than those of seedlings treated with CaCl₂, SNP and CaCl₂ + SNP, indicated that the stimulatory effects of SNP and CaCl₂ on SOD activity were strongly suppressed by W7.

Data revealed that under low-temperature stress conditions, the leaf APX activities of seedlings treated with CaCl₂, SNP or CaCl₂ + SNP were significantly higher than that of CK (p < 0.01); the leaf APX activities showed the order CaCl₂ + SNP > SNP > CaCl₂ > CK, indicated that the SNP and CaCl₂ treatments significantly promoted leaf APX activity in plants undergoing low-temperature stress (Fig. 3d). The activating effect of SNP on APX activity is higher than that of CaCl₂, and the two treatments produce a synergistic effect. The leaf APX activity of seedlings treated with cPTIO + CaCl₂ was significantly higher than that of seedlings treated with W7 + CaCl₂ + SNP (p < 0.01), and the synergistic effect of SNP + CaCl₂ treatment on APX activity was inhibited by NO scavenger cPTIO and the CaM antagonist W7. The greater inhibitory effect of W7 may be because the upstream and downstream signal regulatory sites affected by W7 differ from those affected by cPTIO; this idea is supported by the observation that the leaf APX activity of seedlings treated with cPTIO + CaCl₂ was higher than that of seedlings treated with W7+SNP.

Correlation analysis of physiological indexes

Correlations among different physiological indexes are high and significant at the 0.01 level (p < 0.01). The CaM content and CAT, POD, SOD and APX activities were significantly positively correlated, and the correlation coefficients were all higher than 0.55 (Talbe 1). In contrast, the CaM contents and the activities of the four protective enzymes were negatively correlated with the H₂O₂ and MDA contents. The results indicate that the CaM content could be regulated collaboratively by the Ca²⁺ and NO signal pathways, thereby regulating the antioxidant abilities of loquat seedlings under low-temperature stress (Table 1).

Discussion

Table 1: Correlation analysis of different physiological indexes

Variables	CaM	H2O2	MDA	CAT	POD	SOD
H2O2	-0.556**
MDA	-0.525**	0.963**
CAT	0.652**	-0.886**	-0.766**
POD	0.558**	-0.987**	-0.913**	0.941**
SOD	0.754**	-0.946**	-0.893**	0.909**	0.937**
APX	0.603**	-0.962**	-0.893**	0.940**	0.977**	0.932**

The Pearson’s indexes were calculated by analyze-correlate-bivariate of SPSS 19.0

** The correlation was significant at the 0.01 level（p < 0.01)

Fig. 3: Effects of different treatments on the protective enzyme activities of loquat leaves under low-temperature stress. (a) CAT; (b) POD; (c) SOD; (d) APX. CK: H₂O; T1: CaCl₂; T2: cPTIO + CaCl₂; T3: SNP; T4: W7 + SNP; T5: CaCl₂ + SNP; T6: cPTIO + CaCl₂ + SNP; T7: W7 + CaCl₂ + SNP. Different letters indicate significant differences at the 0.05 level via a Tukey-Kramer test for multiple comparisons

When exposed to low-temperature stress, plant cells generated large amounts of H₂O₂ (Okuda et al. 1991; Guo et al. 2006). The excessive H₂O₂ accumulation triggered an imbalance in ROS metabolism in the cells, which could aggravate peroxidative damage to membrane lipids and cause the degradation of biomacromolecules, thereby leading to the frost damage of plants (Guo et al. 2006; Mishra et al. 2013). The activity of SOD, POD, CAT and APX in loquat leaves and young fruits were reduced after suffering low-temperature stress, while the MDA content was increased (Zheng et al. 2009; Wu et al. 2010). Various methods, including physical methods (e.g., cold acclimation), chemical methods (e.g., application of exogenous Ca²⁺, NO, SA, etc.), and biological methods (e.g., genetic improvement), have been employed to increase the activities of cellular antioxidant enzymes, to reduce the accumulation of active oxygen in the cells and to alleviate the peroxidative damage to the membrane lipids in plants under low-temperature stress, thus improving the cold resistance of plants. Application of appropriate amounts of exogenous NO to ryegrass (Lolium perenne) seedlings increased the activities of protective enzymes such as SOD, CAT and POD, thus enhancing the cold tolerance of ryegrass under low-temperature stress (Ma et al. 2005). In previous studies, we found that appropriate exogenous CaCl₂ and NO treatment increased the activity of the protective enzymes (SOD, CAT and POD) and reduced the peroxidative damage of membrane lipids in different loquat organs under low-temperature stress, thereby improving the cold resistance in loquat (Wu et al. 2010; 2016). In this study, loquat seedlings under low-temperature stress were treated with exogenous CaCl_2, SNP, and CaCl₂ + SNP, and it was found that all three treatments increased the activities of the protective enzymes (CAT, POD, SOD and APX) and facilitated the removal of endogenous active oxygen to varying degrees, thereby decreasing the intracellular H₂O₂ and MDA contents and maintaining them at low levels. The results indicated that treatment with both exogenous CaCl₂ and SNP effectively enhances the antioxidant capacity and thus the cold tolerance of loquat seedlings.

Treatment of seedlings with different exogenous agents induced similar biological effects, especially the synergistic effect of CaCl₂ and SNP co-treatment. The correlation between the CaCl₂-induced and SNP-induced enhancement of frost resistance in loquat seedlings is currently unclear. It was reported that the activity of NO synthase in plants required Ca²⁺ (or CaM) as a cofactor, and NO could also induce an increase in intracellular Ca²⁺ concentration (Zottini et al. 2007; Jeandroz et al. 2016). Studies have shown that both NO and Ca²⁺ signals were both involved in a series of physiological and biochemical processes induced by heavy metal stress in Ulva compressa and Pisum sativum (Rodríguez-Serrano et al. 2009; González et al. 2012). Therefore, in this study, the NO scavenger cPTIO and the CaM antagonist W7 were used in combination with CaCl₂ and SNP to treat loquat seedlings before the exposure to low-temperature stress. The results showed that the activities of CAT, POD, SOD and APX and the CaM content of the leaves of seedlings co-treated with cPTIO + CaCl₂ were lower than those of seedlings treated with CaCl₂ alone, leading to higher leaf MDA and H₂O₂ contents. These results indicated that when cPTIO removed intracellular NO, it might also inhibit the activity of NO synthase by blocking the synthesis of CaM and thus synergistically reducing the intracellular NO level, which directly or indirectly led to decreased activity of protective enzymes in the cells. Therefore, during low-temperature stress, the H₂O₂ was not scavenged in a timely manner, thereby resulting in aggravated membrane lipid peroxidation (i.e., increase in the MDA content). These results illustrated that the NO scavenger cPTIO inhibited the CaCl₂-induced positive regulation of cold resistance of loquat seedlings, which implied NO might be involved in these processes.

By comparing the results of co-treatment with W7 + SNP with those obtained after treatment with SNP alone, we found that the CAT, POD, SOD and APX activities and the CaM content of the leaves of seedlings treated with W7 + SNP were lower than those of seedlings treated with SNP, and the H₂O₂ and MDA contents were higher. In addition, the inhibitory effect of the CaM antagonist W7 on the antioxidant capacity of loquat seedlings under low-temperature stress was higher than that of the NO scavenger cPTIO. These observations suggested that W7 inhibited the SNP-induced positive regulation of the freezing resistance of loquat seedlings by causing a malfunction of the Ca²⁺-CaM messenger system. Ca²⁺ might be one of the indispensable components of the NO signalling pathway in loquat plants under low-temperature stress. Therefore, we predicted that NO and Ca²⁺ signals were both involved in a series of responses of loquat seedlings to low-temperature stress. This was confirmed by co-treatment with SNP + CaCl₂, which showed a positive regulatory synergistic effect on the cold resistance of loquat seedlings.

The results obtained with two different combinations of co-treatments, i.e., cPTIO + CaCl₂ + SNP and W7 + CaCl₂ + SNP, revealed that although both cPTIO and W7 had inhibitory effects on the increased antioxidant capacity of loquat seedlings induced by co-treatment with CaCl₂ + SNP, the degree of inhibition differed in that the inhibitory effect of the CaM antagonist W7 was greater than that of the NO scavenger cPTIO. It implied the presence of signalling interactions between NO and Ca²⁺ in response to low-temperature stress. Different signalling pathways ultimately acted on target enzymes, such as CAT, POD, SOD and APX, through the Ca²⁺-CaM messenger system, which in turn regulated the antioxidant activity in loquat cells and affected the frost resistance of loquat seedlings.

Conclusion

CaCl₂ and SNP treatment increased the CaM content and the activities of SOD, CAT, POD and APX in the leaves of seedlings under low-temperature stress and reduced the cellular accumulation of H₂O₂ and MDA. Co-treatment with CaCl₂ + SNP showed a synergistic effect. In two treatment combinations cPTIO + CaCl₂ and W7 + SNP, both cPTIO and W7 inhibited the physiological effects that were otherwise induced by SNP and CaCl₂ treatment while blocking the synergistic effect of CaCl₂ + SNP co-treatment and exacerbating oxidative damage to seedlings exposed to low temperature. The results suggested that interactions in Ca²⁺ and NO signal transduction occur in loquat seedlings in response to freezing stress.

Acknowledgement

This research was funded by the Natural Science Foundation of Fujian Province (2017J01644, 2017J01645, 2019J01809), the Education and Research Project of Young and Middle-Aged Teachers of Fujian Province (JAT160434, JA15454), the College of Outstanding Young Researchers Cultivation Program of the Fujian Education Department (2017), the Key Research Project of Putian Science and Technology Bureau (2018ZP03, 2018ZP07, 2018ZP08), the Research and Innovation Special Foundation of Putian University (2016CX001), the Scientific Research Project of Putian University (2018064) and the Open Foundation of Key Laboratory of Loquat Germplasm Innovation and Utilization (Putian University), Fujian Province University(2016001, 2017005). This manuscript was edited by American Journal Experts (AJE) for proper English language use.

References

Cakmak I, H Marschner (1992). Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves. Plant Physiol 98:1222‒1227

González A, MDlA Cabrera, MJ Henrıquez, RA Contreras, B Morales, A Moenne (2012). Cross talk among calcium, hydrogen peroxide, and nitric oxide and activation of gene expression involving calmodulins and calcium-dependent protein kinases in Ulva compressa exposed to copper excess. Plant Physiol 158:1451‒1462

Guo Z, WZ Ou, SY Lu, Q Zhong (2006). Differential responses of antioxidative system to chilling and drought in four rice cultivars differing in sensitivity. Plant Physiol Biochem 44:828‒836

Hodges DM, JM DeLong, CF Forney, RK Prange (1999). Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207:604‒611

Jeandroz S, D Wipf, DJ Stuehr, L Lamattina, M Melkonian, Z Tian, Y Zhu, EJ Carpenter, GK Wong, D Wendehenne (2016). Occurrence, structure, and evolution of nitric oxide synthase-like proteins in the plant kingdom. Sci Signal 9:re2

Lamattina L, C Garcíamata, M Graziano, G Pagnussat (2003). Nitric oxide: The versatility of an extensive signal molecule. Annu Rev Plant Biol 54:109‒136

Li T, Y Zhang, K Zhao (2011). Effect of Ca²⁺ on the growth and the cold resistance under low night temperature stress in muskmelon. J Shenyang Agric Univ 42:152‒156

Li X, B Gong, X Kun (2014). Effects of exogenous nitric oxide on reactive oxygen metabolism in ginger leaves under heat stress. Acta Hortic Sin 41:277‒284

Ma X, X Wei, R Long, WJ Cui, Y Wan (2005). Studies on mechanism of enhancing the chilling resistance of annual ryegrass by exogenous nitric oxide. Acta Ecol Sin 25:1269‒1274

Mishra MK, P Chaturvedi, R Singh, G Singh, LK Sharma, V Pandey, N Kumari, P Misra (2013). Overexpression of WsSGTL1 gene of Withania somnifera enhances salt tolerance, heat tolerance and cold acclimation ability in transgenic Arabidopsis plants. PLoS One 8:e63064

Nakano Y, K Asada (1981). Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867‒880

Okuda T, Y Matsuda, A Yamanaka, S Sagisaka (1991). Abrupt increase in the level of hydrogen peroxide in leaves of winter wheat is caused by cold treatment. Plant Physiol 97:1265‒1267

Patterson BD, EA Macrae, IB Ferguson (1984). Estimation of hydrogen peroxide in plant extracts using titanium(IV). Anal Biochem 139:487‒492

Qi M, D Wang, H Qi, Y Liu, T Li (2015). Effect of calcium regent on photosynthesis and calcium content of tomato leaves under heat stress. J Shenyang Agric Univ 46:277‒283

Ramanjulu S, C Sudhakar (2001). Alleviation of NaCl salinity stress by calcium is partly related to the increased proline accumulation in mulberry (Morus alba L.) callus. J Plant Biol 28:203‒206

Reddy ASN, GS Ali, H Celesnik, IS Day (2011). Coping with stresses: Roles of calcium- and calcium/calmodulin-regulated gene expression. Plant Cell 23:2010‒2032

Rodríguez-Serrano M, MC Romero-Puertas, DM Pazmino, PS Testillano, MC Risueno, LA Río, Del, LM Sandalio (2009). Cellular response of pea plants to cadmium toxicity: cross talk between reactive oxygen species, nitric oxide, and calcium. Plant Physiol 150:229‒243

Stewart RR, JD Bewley (1980). Lipid peroxidation associated with accelerated aging of soybean axes. Plant Physiol 65:245‒248

Sulochana C, TJVS Rao (2002). Effect of calcium on water stress amelioration through calmodulin and scavenging enzymes in groundnut. Ind J Plant Physiol 7:0 152‒158

Wu J, W Chen, L Cai, C Xie, S Huang, L Liangjin, Y Meilan (2010). Effects of exogenous nitric oxide on anti-oxidation capacities in young loquat fruits under low temperature stress. Sci Silvae Sin 46:73‒78

Wu J, Y Chen, B Wu, L Huang, W Zhang, F Zheng (2016). Effects of calcium on Ca²⁺-ATPase activity and lipid peroxidation level of loquat seedling under low temperature stress. J Northwest A & F Univ 44:121‒128

Wu XH, CM Lv, JM Feng (2016). Protective effect of exogenous nitric oxide against oxidative damage in pumpkin seedlings under chilling stress. Acta Pratacult Sin 25:161‒169

Yang Y, X Yang, C Zhang, G Fu, T Chen, L Tao (2015). Effects of nitric oxide on drought stress-induced physiological characteristics in leaves of nipponbare (Oryza sativa L.). Chin J Rice Sci 29:65‒72

Zhang Z, L Suo, P Wu, H Liu, J Cui (2018). Ca²⁺ involved in antioxidant enzymes activities of cucumber seedling leaves enhanced by exogenous nitric oxide under low temperature stress. J Nucl Agric Sci 32:600‒608

Zhang H, H Yin, G Jin (2019). Function of nitric oxide in chitosan oligosaccharide-induced resistance to tobacco mosaic virus. Intl J Agric Biol 21:85‒92

Zheng GH, HY Zhang, XR Zhong (2009). Changes in cell ultra-structure, membrane permeability and protective enzyme activity in Eriobotrya japonica Lindl. leaves under cold stress. Chin J Eco-Agric 17:739‒745

Zottini M, A Costa, MR De, M Ruzzene, F Carimi, SF Lo (2007). Salicylic acid activates nitric oxide synthesis in Arabidopsis. J Exp Bot 58:1397‒1405