Physiological Regulation Role Of Polyamines In Chilling Tolerance Of Tomato Seedlings

Chilling stress is one of the abiotic stresses limiting crop growth. Tomato as a kind of warm-season vegetable, it is sensitive to low temperature. Chilling stress seriously affects the yield and quality of tomato, especially when it grown in the early spring and autumn and winter in the north of China. In this study, two tomato cultivars with different low temperature tolerance (chilling-tolerant cultivar’Mawa’and chilling-sensitive cultivar’Moneymaker’) were used to study changes of PAs metabolism in respose to cold acclimation and the relationship between PAs and osmotic regulation substances, product of lipid peroxidation. This study investigated the exogenous Put regulation function in tomato plant resisting to low temperature and analyzed the relationship between PAs and NO under chilling stress. Aim to explore the physiological mechainism of how PAs imporve the chilling tolerance of tomato and enrich the theoretical basis of polyamine improving tomato chilling tolerance, and then provide guidance for the production. The main results are as follows:1. In order to examine the role of PAs in tomato responses to chilling stress, we test PAs metabolism, the changes of osmotic regulation substances and membrane lipid peroxide level were measured in tomato seedlings during cold acclimation. The study found that putrescine (Put) accumulated obviously in leaves of both cultivars for 8 day under cold acclimation (the treatment temperature is 23/13 ℃,20/10 ℃,18/8 ℃, 15/6 ℃ (day/night), and each treatment for 2 days) and it was higher in cv. Mawa than that in cv. Moneymaker during cold acclimation. The spermidine (Spd) content also increased in both cultivars under cold acclimation, but the effect was not obvious. Interestingly, there was no regularity of spermine (Spm) level compared with control in cv. Mawa under cold acclimation, whereas the Spm level of cv. Moneymaker was consistently lower compared to that of control under cold acclimation.2. Arginine decarboxylase (ADC) activities increased in both cultivars in response to cold acclimation; however, almost no changes of ornithine decarboxylase (ODC) activities were observed in both cultivars. Furthermore, we found LeADC and LeODC expressions enhanced partly, but the LeADC1expression did not improve in two cultivars under cold acclimation. The activities of diamine oxidase (DAO), polyamine oxidase (PAO) as well as soluble sugar, soluble protein appeared a higher level in cv. Mawa than that in cv. Moneymaker under cold acclimation. PAs catabolism could influence proline accumulation in tomato leaves under cold acclimation. MDA content did not obviously increase in both cultivars except when the Put accumulation was reduced. These results suggested that Put as a kind of protective substance might have a closest relationship with the tomato chilling resistance, and the change of its content mainly depended on ADC in anabolism and DAO in catabolism.3. In order to further verify that Put could enhance tomato chilling resistance, we measured the effect of Put and D-arginine (D-Arg, a synthesis inhabitor of Put) on tomato seedling growth, PSII photochemical efficiency (Fv/Fm) and active oxygen metabolism, the content of endogenous free and conjugated PAs. The study shows that chilling treatment (15℃/6℃ (day/night)) significantly inhibited the increases of growing, dry and fresh weight of both cultivars after 8 day. By spraying 1 mM D-Arg, this effect was intensified, and by adding back 1 mM Put, this inhibition of chilling treatment was relieved significantly.4. During chilling stress, accumulation of hydrogen peroxide (H2O2) was obviously detected in the leaves of both cultivars, but it was fewer in cv. Mawa. Meanwhile, D-Arg caused more H2O2 accumulation in both cultivars, especially in cv. Moneymaker. By adding back Put to leaves, accumulation of H2O2 obviously reduced in two cultivars. Put could increase of Fv/Fm and decrease malondialdehyde (MDA) content in two cultivars under chilling stress.5. Despite the two cultivars displayed differential behavior towards enzymic antioxidants, enzymes and components of the ascorbate-glutathione (AsA-GSH) cycle in responses to chilling stress, D-Arg treatment diminished the enzyme activities and antioxidant contents induced by chilling stress and its reversion was performed by adding Put in both cultivars. During chilling stress, free and conjugated endogenous PA contents increased in two cultivars. D-Arg treatment inhibited the increases, and exogenously applied Put enhanced the increases in two cultivars. These results suggested that Put could relieve the damage caused chilling stress through improving antioxidant system efficiency, Fv/Fm, the content of free and conjugated PAs, and reducing the ROS production as well as the level of membrane lipid peroxidation in tomato seedlings.6. PAs also could improve plant chilling tolerance by inducing other regulating substances (such as NO). To study the effect of PAs on NO production, a series of relevant indicators were measured. The study found that during chilling stress (4℃), 1mM Spd and Spm application increased NO contents in cv. Moneymaker, but lmM Put application had no obvious impact on NO contents in leaves. The inductions of Spd and Spm on NO content were inhibited by adding CAT before applying PAs under chilling stress. In recover period, NO contents with PA pretreatment were back to the control levels.7. Under chilling treatment, 1mM Spd and Spm application increased NR and NOS activities, but Put had no influence on them. After recover period, compared with the control, NR and NOS activities had no obvious changes with PA pretreatment. LeNR and LeNOSl encode NR and NOS, respectively. During chilling stress, exogenously applied Spd and Spm increased LeNR relative expression, but LeNOSl relative expression was not improved. In recover period, their relative expression had no obvious changes, compared with the control. Under chilling stress and recover period, Put had no influence on LeNR and LeNOSl relative expressions.8. Under chilling stress, Spd and Spm pretreatment improved H2O2 content in leaves, and Put pretreatment did not have this effect. After recovery period, compared with the control, there were no changes in H2O2 content with PAs pretreatment. Under chilling treatment, 1mM Spd and Spm pretreatment improved DAO activity in leaves, and only Spm pretreatment improved PAO activity. However, they did not perform this effect after recovery period. In summary, we can concluded that Spd and Spm pre-treatment could improve the content of NO in tomato leaves under chilling stress, but Put has no effect. The induction effect on NO production might be achieved by improving the activities of NR and NOS. In addition, Spd and Spm pre-treatment can induce the generation of H2O2 via raising the activities of DAO and PAO respectively, and the H2O2 might act an upstream signal to promote NO generation.9. In order to examine the effect of NO on PA content in tomato leaves, the NO donor, SNP was applied exogenously on tomato seedlings, and then we measured the change of PAs content in tomato leaves. The study showed that during chilling stress (4℃), 1mM NO application improved Put and Spd contents in tomato leaves. Put content appeared increases first, and then Spd content began to rise. After recovery period, NO application still improved Put and Spd contents in leaves. After chilling treatment and recover period, exogenously applied NO did not improved Spm content. Thus, it is concluded that NO could improve the Put and the Spd content under chilling treatment and recovery period, the reason might be duo to NO directly induce the increases of Put and the Spd content; Or, NO only induced the increase of Put content. And Put as a diamine, it could transform to Spd that a kind of three amine.