Exogenous application of 5-aminolevulinic acid Improves Chilling Tolerance in Rapeseed (Brassica napus L.) Seedlings

doi:10.21203/rs.3.rs-2837181/v1

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Exogenous application of 5-aminolevulinic acid Improves Chilling Tolerance in Rapeseed (Brassica napus L.) Seedlings

https://doi.org/10.21203/rs.3.rs-2837181/v1

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Rapeseed is an important oil crop in China. Low winter temperatures (LT) often limit plant growth and cause seed yield losses in rapeseed. Although exogenous application of 5-aminolevulinic acid (ALA) has been shown to enhance plant tolerance to various abiotic stressors, its physiological mechanisms for improving cold tolerance in rapeseed are not yet fully understood. In this study we investigated the physiological responses to exogenous ALA using the chilling-tolerant cultivar Zayou15 (ZY15) and the chilling-sensitive cultivar Huiyou 49 (HY49) under low-temperature stress. The results showed that low-[A1] temperature stress caused a considerable decrease the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as in the contents of endogenous hormones GA, IAA, and CTK. These resulted in the accumulation of reactive oxygen species, with both the content of H₂O₂ and O₂^-showing considerable increases, as well as in membrane lipid peroxidation with considerable increases in both malondialdehyde content (MDA) and relative electrical conductivity (REC). Moreover, the contents of chlorophyll（Chl）b, Chl a/Chl b, transpiration rate (Tr), net photosynthesis (Pn), stomatal conductance (Gs), and intercellular CO₂ concentration (Ci) were considerably decreased by low-temperature stress, resulting in remarkable growth inhibition (with the aboveground fresh weight significantly decreased). Although, 20 mgL^-1 exogenous ALA significantly reduced O₂^-, H₂O₂, and MDA content, and REC in both cultivars; it did not increase POD, SOD, CAT, or ascorbic acid peroxidase (APX) under short-term low-temperature stress (12–48 h). However, ALA substantially increased the activities of GA, Pn, Tr, Gs and Ci, resulting in a considerable increase in aboveground fresh weight. Post-treatment spraying of rapeseed plants with 5 mL of 20 mg·L-1 ALA was more effective than pre-treatment spraying. Both cultivars sprayed with ALA post-treatment showed greater reduction in MDA content. The optimum ALA dosage and concentration were 5 mL and 20 mg·L-1, respectively, leading to considerable decrease in MDA, and REC content and increase in Chl content in leaves. In conclusion, our results demonstrate that exogenous application of ALA is an appropriate strategy for rapeseed to resist winter low-temperature stress.

Rapeseed

5-aminolevulinic acid (ALA)

Chilling tolerance

Low temperature stress

Physiological characteristics

Rapeseed (Brassica napus L.) is the third-largest oilseed crop in the world and the most important oilseed crop in China (Hu et al. 2021). In 2019, the rapeseed planting area in China was approximately 6.57 million ha, accounting for nearly 20% of the total rapeseed planting area worldwide (FAO 2019). In the Yangtze River Basin, the main rapeseed production region, delayed rice harvest often leads to late rapeseed sowing in late October or early November. This results in poor germination and seedling establishment owing to low temperatures (Wang et al. 2012; Luo et al. 2019; Xu et al. 2021). Rapeseed seedling biomass is significantly reduced during the overwintering stage, making them more susceptible to low-temperature stresses, such as chilling (0–15 °C) or freezing (<0 °C) (Sage and Kubien 2007; Wang et al. 2012; Zhang et al. 2016).

Chilling stress is a critical factor that limits crop growth, development, and productivity (Ding et al. 2017). Chilling injuries adversely affect plants, leading to the excessive accumulation of reactive oxygen species (ROS), reduced photosynthetic efficiency, membrane decomposition, protein degradation, hormonal imbalance, cell death, necrosis, chlorosis, and growth retardation (Willems et al. 2016;Wang et al. 2020;Bhat et al. 2022). Therefore, maintaining the balance of ROS, improving photosynthesis, reducing damage caused by low-temperature stress, and enhancing tolerance to chilling stress are crucial for rapeseed production (Soliman et al., 2018).

Our previous studies showed genotype-dependent differences in chilling stress tolerance (Zhu et al. 2021; Xu et al. 2022). Additionally, the application of plant growth regulators, such as abscisic acid (Zhu et al. 2021), brassinolide (Russinova et al. 2017), and salicylic acid (Zhang et al. 2021); osmoregulatory substances, such as soluble sugar (Fu et al. 2020), betaine (Park et al. 2006); and inorganic salt ions, such as Ca⁺ and K⁺ (Yang et al. 2016) can also enhance chilling tolerance of plants. 5-Aminolevulinic acid (ALA) is a universal precursor for the synthesis of tetrapyrroles, such as chlorophyll, heme, siroheme, vitamin B12, and phytochroobilin. It is found in various living organisms, including bacteria, algae, plants, and animals (Sasaki et al. 2002; Agrawal et al. 2020; Tan et al. 2022). ALA enhances plant growth, yield, and abiotic stress tolerance by regulating antioxidant mechanisms, photosynthesis, and nutrient uptake (Balestrasse et al. 2010). Exogenous ALA can enhance plant tolerance to diverse abiotic stresses, such as high temperature (Zhang et al. 2012), salinity (Zhang et al. 2013), drought (Li et al. 2011), waterlogging (An et al. 2016), weak light (Ali et al. 2020), UV-B stress (Ozkan et al. 2017), and heavy metals (Tian et al. 2014). Previous studies have shown that exogenous ALA can reduce the chilling injury in rice (Mohamed et al. 2017)，maize (Wang et al. 2018)，soybean (Balestrasse 2010), tomato(Liu et al. 2018), pepper (Wang et al. 2022), and cucumber (Ali et al. 2020).

However, few studies have investigated the effects of ALA on rapeseed with varying levels of low-temperature stress. In the present study, we examined the impact of exogenous ALA on seedling growth, chlorophyll content, photosynthetic capacity, antioxidant enzyme activity, ROS accumulation, and endogenous hormone levels in two rapeseed cultivars with varying low-temperature tolerance. This study aims to improve our understanding of rapeseed’s response to low-temperature stress and the mechanism by which ALA improves rapeseed resistance to chilling.

Plant material and Experimental design

The low-temperature-sensitive rapeseed cultivar cv. Huiyou 49 (HY49, National registration number: GPD rapeseed (2020) 340049), and low-temperature-tolerant cv. Zayou 15 (ZY15, National registration number: GPD rapeseed (2018)340063), provided by Anhui Agricultural University, was used in the experiments (Xu et al., 2022). Seedlings were cultivated as previously described (Zhu et al. 2021). The experimental treatments began at the five-leaf stage. Plants with similar appearance were selected for the experiments, which consisted of four parts.

Experiment 1: We investigated the effects of different concentrations of ALA on rapeseed tolerance to chilling stress and determined the optimal ALA level. The five-leaf-stage rapeseed seedlings were divided into the following six groups:

CK (Control; 20 ℃ day and night temperature)

A0 (0 mg/L ALA + 5 ℃ day and night temperature)

A10 (10 mg·L⁻¹ALA + 5 ℃ day and night temperature)

A20 (20 mg·L⁻¹ALA + 5 ℃ day and night temperature)

A40 (40 mg·L⁻¹ALA + 5 ℃ day and night temperature)

A80 (80 mg·L⁻¹ALA + 5 ℃ day and night temperature)

The CK group was untreated, sprayed with 5 mL distilled water and grown under 20 ℃ (day and night temperature), whereas treatment groups were treated with same amount of with distilled water (A0) or ALA solution (Sigma-Aldrich, St. Louis, MO, USA) at concentrations of 10(A10), 20(A20), 40(A40), or 80(A80) mg·L⁻¹. The experiment was repeated three times, with each treatment comprising three plants. The treated seedlings were exposed to a low temperature at 5 ℃ (day and night temperature). After a six-day treatment period, measurements of the contents of superoxide anion (O^2-), hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and relative electrical conductivity (REC) were taken from the fifth leaf of the seedlings in each group, along with determination of plant growth indices.

Experiment 2: To evaluate the effectiveness of ALA on reducing chilling injury before and after low-temperature stress, two groups of both rapeseed cultivars were used. One group was sprayed with 5 mL of 20 mg·L⁻¹ ALA per plant and placed at 5 °C for 24h (ALA before LT), whereas the other group was placed at 5 °C for 24h and then immediately sprayed with the same amount 20 mg·L⁻¹ ALA (ALA after LT). MDA, REC, and chlorophyll (Chl) levels were determined.

Experiment 3: Effects of different ALA doses on chilling stress were determined. Five-leaf-stage rapeseed seedlings were divided into three groups.

(1) A20, pre-sprayed with 5 mL of 20 mg·L^-1 ALA on the leaves of each rapeseed seedling.

(2) A20*2: Pre-spray 10 mL of 20 mg·L^-1ALA onto the leaves of each rapeseed seedling.

(3) A40: Pre-spray 5 mL of 40 mg·L^-1 ALA onto the leaves of each rapeseed seedling.

The rapeseed seedlings of the above three treatment groups were first placed under 20 °C for 12h, and then placed under 5 °C. MDA, REC, and Chl were determined 24 h later using the fifth leaf of rapeseed seedlings in each treatment.

Experiment 4: We determined the effects of ALA application on chlorophyll, antioxidant enzymes, endogenous hormones, and photosynthetic parameters in rapeseed seedlings under short-term low-temperature stress. The plants of HY49 were selected and divided into two treatment groups. In one group each plant was sprayed with 5 mL of 20 mg·L^-1 ALA solution, and in the other group each plant was sprayed with 5 mL of distilled water. Then, half of the two groups of seedlings were exposed to low temperature (5°C, day and night), whereas the rest of the groups were kept at normal temperatures (20 °C, day and night). Photosynthetic indices, chlorophyll content, antioxidant enzyme activity, and endogenous hormone levels were measured 12, 24, and 48 h after spraying.

Measurement of plant growth characteristics

In this experiment, the five-leaf stage seedlings per treatment were separated into shoots and roots after being washed with distilled water and blotted to remove topical water. The fresh weights of separated tissues were determined, and then the dry weights were obtained after drying for 15 min at 105 °C and then 80 °C in an oven until completely dry.

Measurement of chlorophyll, and photosynthetic index

Chlorophyll and photosynthetic indices were measured as described by Sami et al. (2020). A chlorophyll content analyzer (TYS-A, Zhejiang Top Cloud-Agri Technology Co., Ltd., China) was used to determine the chlorophyll content. Photosynthetic indices, such as photosynthetic gas exchange parameters (Ci, Pn, Gs, Tr) of rapeseed leaves were determined using a portable photosynthesis system (Li-6800; Li-Cor, USA).

Determination of MDA, and REC

MDA content was determined using the method described by Sami et al. (2020). REC was calculated as described in a previous study (Zhu et al. 2021). Three fresh leaves (approximately 0.1 g) were placed in glass tubes containing 10 mL deionized water. After soaking the leaves at room temperature for 12 h, the initial conductivity of the distilled solvent, referred to as R₁, was measured using a conductivity meter (DDS-307A; Shanghai Yoke Instruments Co. Ltd., Shanghai, China). The mixture was then heated in a boiling water bath for 30 min, placed on ice, and cooled to room temperature. After shaking, the conductivity of the extract was re-calculated and denoted as R₂. The calculation formula for REC was =R₁/R₂100%

Determination of O₂⁻ and H₂O₂ content

The O₂⁻ and H₂O₂ content in rapeseed leaves were detected using enzyme-linked immunosorbent assay (ELISA) kits (Jiangsu Meimian Industrial Co., Ltd., China). Approximately 0.1 g of fresh leaves were frozen in liquid nitrogen and extracted with 0.9 mL a sodium phosphate buﬀer (50mM, pH7.0). After centrifugation, the supernatant was used to measure O₂⁻and H₂O₂ contents by following the manufacturer’s instructions, respectively.

Assessment of antioxidant enzyme activity

Antioxidant enzymes (APX, POD, CAT, and SOD) were measured according to our previous study (Zhu et al. 2021).

Assessment of ABA, GA, IAA, and CTK

The endogenous hormones GA, CTK, IAA, and ABA in rapeseed leaves were measured as described previously (Xu et al. 2022).

Statistical analysis

The experimental design was a completely randomized block with three replicates. Each replicate consisted of 12 plants. Statistical analysis was performed using IBM SPSS Statistics 27. Analysis of variance (ANOVA) was performed using Tukey’s test at P <0.05.

The effect of different concentrations of ALA on the growth index of rapeseed seedlings under low-temperature stress

The results suggest that low-temperature stress had a negative impact on the growth of both rapeseed cultivars, as seen by the significant decreases in aboveground fresh weight (AFW), aboveground dry weight (ADW), underground fresh weight (UFW), and underground dry weight (UDW) in the A0 treatment compared to the control. In ZY15, AFW, ADW, UFW, and UDW decreased by 24.2%, 17.6%, 27.3%, and 9.1%, respectively, whereas in HY49, these values decreased by 55.3%,52.6%,31.4%, and 16.7%, respectively (Table 1). Therefore, larger decreases in growth parameters occurred under low-temperature stress in the low-temperature-sensitive rapeseed variety HY 49 than in the low-temperature-tolerant variety ZY15. Compared with the treatment of A0, exogenous application of ALA with 20 mg·L^− 1 significantly increased the aboveground fresh weight of ZY15. With an increase in ALA concentration, the AFW, ADW, UFW, and UDW first increased and then decreased compared to the A0 treatment. The optimal ALA concentration for both ZY15 and HY49 was 20 mg·L^− 1 (Table 1).

Table 1

Effects of different ALA concentrations on the growth index of rapeseed seedlings in ZY15 and HY49 under low temperature stress
Cultivars	Treatment	Aboveground		Underground
Cultivars	Treatment	Fresh weight(g)	Dry weight(g)	Fresh weight(g)	Dry weight(g)
ZY15	Control	2.60 ± 0.45a	0.17 ± 0.03a	0.77 ± 0.06a	0.11 ± 0.01a
	A0	1.97 ± 0.80b	0.14 ± 0.01ab	0.56 ± 0.10a	0.10 ± 0.02a
	A10	2.35 ± 0.82ab	0.13 ± 0.05ab	0.65 ± 0.26a	0.10 ± 0.01a
	A20	2.54 ± 0.31a	0.10 ± 0.02ab	0.69 ± 0.09a	0.11 ± 0.02a
	A40	2.11 ± 0.39ab	0.12 ± 0.01b	0.63 ± 0.15a	0.10 ± 0.04a
	A80	1.96 ± 0.71b	0.10 ± 0.02b	0.50 ± 0.26a	0.08 ± 0.03a
HY49	Control	3.71 ± 0.87a	0.19 ± 0.02b	0.86 ± 0.24a	0.12 ± 0.04a
	A0	1.66 ± 0.13b	0.09 ± 0.02b	0.59 ± 0.15a	0.10 ± 0.00a
	A10	1.95 ± 0.55b	0.13 ± 0.01b	0.65 ± 0.14a	0.1 ± 0.02a
	A20	2.15 ± 0.09ab	0.12 ± 0.03b	0.67 ± 0.16a	0.11 ± 0.01a
	A40	1.89 ± 0.37b	0.10 ± 0.02b	0.62 ± 0.08a	0.10 ± 0.02a
	A80	1.61 ± 0.52b	0.12 ± 0.04	0.58 ± 0.03a	0.10 ± 0.02a
Note: Control: Sprayed with distilled water on the leaves of rapeseed seedlings, and cultivate at room temperature (20℃/ 20℃); A0-A80: Use concentrations of 0, 10, 20, 40 or 80 mgL^− 1 for rapeseed seedlings. ALA solution was sprayed on leaves, and after 12 hours of incubation at room temperature, low temperature treatment (5℃/5℃) was started. The growth index of rapeseed seedlings was determined on the 6th day after low temperature treatment. Different letters in the same column represent significant differences between treatments at the P < 0.05 level.
Author Contributions: ZW, ZZ, and WL conceived the study. ZZ, WX, ZF, YY, and ZK designed the study. ZW, ZZ, WL, LJ,HJ ,QX and SM conducted the experiments. WL, ZZ, MJ, WM and LY analyzed the data and prepared the figures and tables. ZW, ZZ, and WL wrote the manuscript. All the authors have read and approved the submission of the manuscript.

Effect of different concentrations of ALA on the levels of H ₂ O ₂, O₂⁻, MDA and REC of rapeseed seedlings under low-temperature stress

Low-temperature stress significantly increased the levels of O₂⁻, H₂O₂, MDA content, and REC in ZY15 and HY49 compared to the control (Figs. 2 and 3). With the increase in exogenous ALA application concentration, the levels of O₂⁻, H₂O₂, MDA content, and REC first decreased and then increased compared to the A0 treatment. Furthermore, under low-temperature stress, compared to A0, treatment with 20 mg·L^− 1 ALA had the best effect on reducing O₂⁻, H₂O₂, MDA content, and REC in both rapeseed cultivars by 21.5%,26.4%, 21.9%, and 8.9%, respectively, in ZY15 and 25.0%, 24.6%, 14.2%, and 9.5%, respectively, in HY49 (Figs. 2 and 3).

Effects of ALA application time and dosage on the peroxidation damage of leaf membrane

In both rapeseed cultivars, post low-temperature stress treatment with ALA significantly reduced MDA content in the leaves compared to pre-stress treatment, however no significant difference in Chl content and REC were observed (Fig. 3). Under low-temperature stress, the MDA content in the leaves of both rapeseed cultivars sprayed with 5 mL of 20 mg·L^− 1 ALA (A20) resulted in relatively low MDA content in leaves of both cultivars, which was significantly lower than the treatment with 5 mL of 40 mg·L^− 1 ALA (A40). In HY49, the REC of A20 was significantly lower than the treatment sprayed with 10mL of 20 mg·L^− 1 ALA (A20*2), whereas there was no significant difference among the three treatments in ZY15. The Chl content of A20 was relatively high in both cultivars, and significantly higher than that of the A20*2 treatment (Fig. 4).

The effect of exogenously sprayed ALA on antioxidant enzyme activities in rapeseed seedling leaves under low-temperature stress

Compared with the control, short-term low-temperature stress for 12, 24 and 48h significantly reduced the activities of antioxidant enzymes (SOD, POD, and CAT) but significantly increased the activity of APX (Fig. 5). Exogenous application of ALA slightly increased SOD, POD, CAT, and APX activities under low-temperature stress but did not reach a significant level.

The effect of ALA on chlorophyll and photosynthesis

Short-term low-temperature stress at 12 and 24 h caused a slight decline in Chl a, Chl b, Chl a + b and Chl a/Chl b levels in HY49, however showed no significant difference compared with the control (Fig. 6). After 48 hours of low-temperature stress, the levels of Chl b and the ratio of Chl a/Chl b decreased significantly in the leaves of HY49, whereas the levels of Chl a and Chl a + b did not show a significant decrease compared to the control. Application of 20 mg·L^− 1 ALA enhanced the levels of Chl b and Chl a/Chl b under short-term low-temperature stress of 48h, and showed no significant difference from the control (Fig. 6).

Compared with the control, the transpiration rate (Tr), net photosynthesis (Pn), and stomatal conductance (Gs) significantly decreased under low-temperature stress for 12h, whereas intercellular CO₂ concentration (Ci) significantly decreased under low-temperature stress for 24h in leaves of HY49 leaves (Fig. 7). Exogenous application of ALA significantly increased and reduced Ci and Pn, respectively, but had no significant effect on Tr. After 12 hours of low-temperature stress, exogenous application of 20 mg·L^− 1 ALA significantly increased Pn, Tr, Gs and Ci (Fig. 7).

The effect of exogenous application of ALA on the content of endogenous hormones in rapeseed seedling leaves under low-temperature stress

Compared with the control, the contents of GA and CTK in the leaves decreased significantly under 12 h of low-temperature stress, whereas the contents of ABA and IAA increased significantly after 24 h of low-temperature treatment (Fig. 8). After 12 hours of low-temperature stress, exogenous spraying of 20 mg·L^− 1 ALA significantly increased the GA content in leaves, but significantly decreased the content of ABA. After 48 hours of low-temperature stress, application of 20 mg·L^− 1 ALA had no significant effect on the IAA and CTK content in leaves (Fig. 8).

Chilling stress seriously affects the growth of rapeseed (Brassica napus L.) cultivars, particularly at the seedling stage. Exogenous application of ALA of 20 mg·L^− 1 on rapeseed seedling leaves under low-temperature stress (5 ℃), improved plant growth and physiological characteristics, resulting in enhanced low-temperature tolerance. ALA enhanced photosynthesis by increasing chlorophyll content. By decreasing the MDA and REC content, ALA improved the osmotic regulation capacity and alleviated membrane lipid peroxidation and membrane injury in seedlings. ALA decreased the levels of H₂O₂ and O₂⁻ by increasing the activity of the antioxidant enzymes SOD, POD, CAT, and APX. In addition, the effect of ALA on the chilling tolerance of HY49(a low-temperature-sensitive cultivar) was observed to be higher than that of ZY1 (a low-temperature-tolerant cultivar).

Rapeseed yield is largely determined by plant growth during the vegetative growth stage. Low temperatures are among the most important abiotic factors that inhibit the growth and productivity of rapeseed (Brassica napus L.) (Yan et al. 2019). In this study, we found that the AFW, ADW, UFW, and UDW of the two rapeseed cultivar seedlings were significantly affected by low temperatures; these findings are consistent with our previous results (Zhu et al. 2021). Previous studies have suggested that exogenous application of ALA promotes plant growth and biomass accumulation in maize seedlings (Wang et al. 2018), pepper seedlings (Wang et al. 2022), cucumber (Ali et al. 2020), Elymus nutans (Fu et al. 2016), and tomato seedlings (Liu et al. 2018). In the present study, the exogenous application of ALA significantly decreased the inhibitory effects of low-temperature stress on rapeseed seedling growth in both cultivars. With an increase in ALA concentration, the AFW, ADW, UFW, and UDW first increased and then decreased. The optimal concentration of ALA to improve rapeseed growth under low-temperature stress of 5 ℃ was 20 mg·L^− 1 (Table 1). However, it is important to note that high concentrations of ALA can inhibit the growth of rapeseed under low-temperature stress, which requires further investigation.

Low-temperature stress leads to the formation and accumulation of ROS such as O₂⁻ and H₂O₂ (Cao et al. 2017). Excessive accumulation of ROS has detrimental effects on plant growth, as it can seriously disrupt normal metabolism through oxidative damage to lipids (Korkmaz et al. 2010; Cao et al. 2017). MDA is one of the most important products of oxidative damage to lipids and is generally considered indicative of the degree of membrane lipid peroxidation, which often occurs in plants under abiotic stress, such as low temperatures (Suzuki et al. 2012; Liu et al. 2018). Plant antioxidant enzyme systems, including APX, SOD, CAT, and POD, play key roles in balancing ROS regulation (Ali et al. 2018). SOD is the “first line of defense” of plants against oxidative damage, which helps to protect cellular components from O₂⁻ attack, decompose O₂⁻ into H₂O₂, and then decompose H₂O₂ into H₂O by enzymes such as, POD, CAT, and APX. This prevents excessive accumulation of O₂⁻ and H₂O₂ thereby preserving plant health (Chen and Asada 1989; Hai et al. 2022).

In the present study, low-temperature increased whereas ALA reduced the content of O₂ ⁻, H₂O₂, MDA, and REC (Fig. 1, 2 ). This finding is consistent with the results of Liu et al. (2018), who found that ALA can improve the antioxidant capacity of crops under low-temperature stress by regulating the activity of antioxidant enzymes, as well as inducing H₂O₂ to sense low temperature signals and reduce the cell membrane damage. However, the effect of different ALA treatment times on MDA content in rapeseed seedling leaves under low-temperature stress was not consistent with the results of Liu et al. (2018) in tomato, which could be owing to the varying susceptibility of rapeseed and tomato to low temperatures.

In our study, low temperatures did not increase the activities of SOD, POD, and CAT (Fig. 7), which is consistent with the results of Wang et al. (2022). In addition, the exogenous application of ALA slightly increased SOD, POD, CAT, and APX activities under low-temperature stress. This discrepancy may be related to the different treatment times and responses of different plants to low-temperature stress.

Chlorophyll is an important photosynthetic pigment in plants that absorbs light energy and provides energy for plant development through photosynthesis. Chlorophyll is sensitive to abiotic stresses and degrades easily (Wang et al. 2010), resulting in reduced plant photosynthetic capacity (Memon et al. 2009). In the present study, under low-temperature stress for 48h, Chl content (Chl b and Chl a/b) significantly decreased, and under low-temperature stress for 12h and 24h Chl content (Chl a, Chl b, Chl a + b, and Chl a/b) significantly increased. Further, ALA slightly increased chlorophyll content (Chl a, Chl b, and Chl a + b) after short-term low-temperature stress (12, 24, and 48h) (Fig. 6). Low-temperature stress significantly reduced Ci after 12, 24, and 48h, whereas it significantly increased the exogenous application of ALA. Additionally, short term low-temperature stress (12, 24, and 48h) significantly increased Pn, which significantly decreased after ALA application. These findings partially differ from those reported by Ali et al. (2018), who found that low-temperature stress induced a significant reduction in chlorophyll contents, whereas ALA increased chlorophyll content (Chl a, Chl b, and Chl a + b) and improved photosynthetic capacity, net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO₂ concentration (Ci), and transpiration rate (Tr). The inconsistent results of these studies may be related to the treatment duration.

In the present study, low-temperature stress significantly decreased GA and IAA, but significantly increased ABA content, whereas ALA significantly decreased ABA content under low-temperature stress for 12, 24, and 48h, which is consistent with the results of Ali et al. (2018). In addition, ALA significantly increased GA content under low-temperature stress for 12h, ALA could resist low-temperature stress by regulating the content of endogenous hormones in plants.

Under low-temperature stress, exogenous ALA enhances the ability of the antioxidant system to eliminate ROS, effectively reduces the accumulation of active oxygen and the degree of membrane lipid peroxidation in rapeseed seedlings, and promotes photosynthesis and growth in rapeseed seedlings. The effect was greatest when the ALA concentration was 20 mg·L^− 1. Exogenous application of ALA can effectively promote the removal of excessive O₂⁻ and H₂O₂ in rapeseed seedlings, and for the low-temperature-tolerant variety ZY15, exogenous ALA has a more evident low-temperature relief effect on the low-temperature-sensitive variety HY49 rapeseed seedlings. In addition, the effect of applying exogenous ALA after encountering low-temperature stress is better than applying exogenous ALA before stress, and the application of 5 mL of ALA with a concentration of 20 mg·L^− 1 has the best effect.

Competing interests: The authors declare no competing interests.

Author Contributions: ZW, ZZ, and WL conceived the study. ZZ, WX, ZF, YY, and ZK designed the study. ZW, ZZ, WL, LJ,HJ ,QX and SM conducted the experiments. WL, ZZ, MJ, WM and LY analyzed the data and prepared the figures and tables. ZW, ZZ, and WL wrote the manuscript. All the authors have read and approved the submission of the manuscript.

Acknowledgements

This work was supported by the Natural Science Foundation of Anhui Province(1908085MC68) and the Open Project of the Key Laboratory of Crop Quality Improvement of Anhui Province (2020ZW003).

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Exogenous application of 5-aminolevulinic acid Improves Chilling Tolerance in Rapeseed (Brassica napus L.) Seedlings

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Effects of ALA application time and dosage on the peroxidation damage of leaf membrane

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