Mitigative effect of 5-aminolevulinic acid pretreatment on chilling injury of young leaves in peach

Abstract:【Objective】Peach (Prunus persica L.) is an important temperate deciduous fruit crop in China as well as the rest of the world. It is well-liked for its delicious and juicy fruit. However, peach trees are often vulnerable to be injured from the late frost due to its flowering in early spring, which will seriously affect the fruit yield and quality. Therefore, it is meaningful to study how to improve the trees’ ability to defend against cold temperature in early spring. 5-Aminolevulinic acid (ALA) is one of natural δ-amino acids, but not involved in the biosynthesis of proteins. It is an essential biosynthetic precursor of all tetrapyrrole compounds like chlorophyll and heme. Therefore, it plays an important role in photosynthesis and respiration of plants. ALA has also been suggested as a new plant growth regulator, which can be used to improve plant stress tolerance including cold resistance. However, it has never been reported before whether ALA can improve cold tolerance of peach in early spring. To understand the effect of ALA on improving peach cold tolerance, we artificially exerted low temperature stress at different days after spraying an exogenous ALA solution.【Methods】The young leaves of peach at bud break stage were sprayed with 50 mg·L^-1 ALA solution, then harvested at 3, 6, 9, and 12 d, and put in a freezer at -6 ℃ for 0, 2, 4, and 6 h. After then, the chlorophyll fast fluorescence was measured with a multiple plant efficiency analyzer (M-PEA), and the maximum photochemical efficiency and the electron transfer rate of the photosystem Ⅱ (PS Ⅱ) and the photosystem Ⅰ (PSⅠ) reaction centers were analyzed. Additionally, the leaves pretreated with ALA for 6 d were used to determine the relative electrical conductivity, the content of osmotic solutes (including soluble sugars, free proline and soluble proteins), antioxidant enzyme (including SOD, CAT and POD) activities and MDA content. And finally, the relative expressions of genes coding the proteins in PSII reaction center and the key genes in proline metabolism were detected by qRT-PCR.【Results】It was shown that 2-hour chilling at -6 ℃ did not significantly affect the chlorophyll fast fluorescence curves of young leaves, and 3 d pretreatment of ALA did not show enough protective potency against chilling stress. It was found that 4 or 6 hours chilling greatly increased the early rise and the late decrease in the kinetic curves, suggesting that the chlorophyll fluorescence properties were impaired by chilling stress, while ALA pretreatment for 6-12 d significantly inhibited the changes. The variance analysis of the maximum photochemical efficiency of PS Ⅱ (φPo) and PSⅠ (φRo) reaction center of peach leaves showed that longer-term stress caused significant decreases of φPo and φRo in the control whereas that in the pretreated situation for 6-12 d by ALA kept rather high level, suggesting that ALA pretreatment at least for 6 d significantly improved chilling tolerance for PSⅡ and PSⅠ reaction center. PSⅡ reaction center seemed to be more sensitive to chilling stress than PSⅠ. Similarly, the maximum oxidation rate of PSⅠ reaction center (VPSI) and the maximum reduction rate of PS Ⅰ reaction center by the electrons transferred from PSⅡ reaction center (VPSII- PSI) were significantly improved by ALA pretreatment. Once again, VPSII- PSI in the young leaves was more sensitive to chilling stress than VPSI, and the protective effect of ALA pretreatment was also greater on the inter-system electron transfer. Physiological and biochemical analyses showed that chilling stress resulted in dramatic increases of the relative electrical conductivity of peach leaves, while ALA pretreatment significantly prevented the rise amplitude. ALA pretreatment significantly improved the soluble sugar content in the young leaves compared with the control, implying a result of ALA improving leaf photosynthesis and thus accumulating more carbohydrate, which is crucial to enhance leaf chilling tolerance. Furthermore, ALA pretreatment improved the soluble proteins and free proline content in young leaves, which was more significant after chilling stress. 2 h chilling stress stimulated the activities of antioxidant enzymes including SOD and POD, which then decreased after longer time. The activity that ALA pretreatment stimulated increased more and kept higher for longer time. The activity of CAT in peach leaves did not increase during chilling stress. In fact, it depressed with chilling stress. However, ALA pretreatment caused the enzyme activity higher than the control after 4-6 h chilling. Therefore, the content of lipid peroxidation product MDA in the control increased significantly after 2 h chilling while that in the ALA pretreated leaves kept unchanged. The qRT-PCR analysis showed that the relative expression of PsbB under low- temperature stress was significantly higher than the control, and that of Psb28, PsbO, PsbR and PsbY in the ALA pretreatment was also higher than that of the control, especially under longer chilling stress. However, the PsbA gene expression in peach leaves was sensitive to chilling stress, whether with ALA pretreatment or not. On the other hand, the relative expressions of P5CS1 and P5CR, two key genes in proline biosynthetic route, were significantly higher in ALA pretreated leaves than that of the control, while the expression of PDH, an important gene for proline catabolism was down- regulated by ALA pretreatment, suggesting that ALA pretreatment promoted proline biosynthesis and blocked its catabolism during chilling stress.【Conclusion】Spraying peach leaves with ALA solution at least 6 d before chilling stress can improve the resistance to low temperature stress at -6 ℃ for 6 h. Exogenous ALA pretreatment can relieve the damage to PSII and PSI, maintain important protein gene expression of PSII reaction center, improve antioxidant enzyme activity, increase osmotic solute content, and enhance its ability to resist the harm of the late frost in early spring.