Modulation Of Polyamines In Seeds And Seedlings Of Tobacco And Its Relationship With Chilling Tolerance

Chilling stress affects seed germination and seedling growth of tobacco (Nicotiana tabacum L.), finally causes the loss of tobacco quality and yield. It is important to improve chilling tolerance of tobacco seedlings. Polyamines (PAs) accumulated in plants after suffering stress conditions. PAs are biologically ubiquitous aliphatic amines with low molecular weight that involved in the response of plants to environmental stress. In present research, the chilling-tolerant variety (Honghuadajinyuan) and chilling-sensitive variety (MSk326) of tobacco were used to investigate the modulation of polyamines in seeds and seedlings of tobacco and its relationship with chilling tolerance. The main results were as follows:The chilling tolerance of five tobacco cultivars, that were extensively planted, at germination and seedling growth stages was investigated by determining germination characteristics and seedling quality at normal temperature (20-30℃) and low temperature (11℃) stress. The ratios of trait values at low temperature to those at normal temperature were used to assess chilling tolerance. The results showed that the shoot of tobacco was more sensitive than the root to low temperature. The correlation analyses showed that the relative values of germination percentage, germination index, shoot height and seedling dry weight were significantly correlated with each other; they were suitable parameters for evaluating chilling tolerance. After the cluster analysis, tobacco cultivars were clustered into three groups: chilling tolerance, intermediate chilling tolerance and chilling sensitivity. The results will provide basis for material selection during research of cold resistance mechanism and breeding in tobacco. Honghuadajijnyuan (HHDJY) and MSk326 were selected as chilling-tolerant and chilling-sensitive varieties, respectively.Growth rate, antioxidant enzymes and malondialdehyde (MDA) of seedlings of two tobacco, cv. MSk326 (chilling sensitive variety) and HHDJY (chilling tolerant variety) under chilling stress (5℃) were studied. The results showed that the ratio of relative growth rate in chilling period to that in recovery period was significantly higher in root than that in shoot in the both cultivars, suggesting that shoot growth was more easily affected by chilling stress. Chilling stress increased peroxidase (POD) activity and reduced superoxide dismutase (SOD) activity in shoot of HHDJY, and catalase (CAT) activity was little affected. In the root of HHDJY, chilling stress increased SOD and CAT activities, and had little effect on POD activity. For MSk326, chilling treatment increased SOD activity in shoot and declined CAT activity in root. MDA concentration of both varieties was increased suffering the chilling stress, while it was decreased after seedlings were recovery growth for 4 days at normal temperature (25℃). It showed that tobacco seedlings might have the capacity of recovering from chilling injury for a short term. The relationship between the growth rate and antioxidant enzyme activity was analyzed by stepwise regression, and it was found that there was a close relationship between relative growth rate of tobacco seedlings and CAT activity under chilling condition.MSk326 and HHDJY were used to study the effects of chilling stress (5℃) on dry weight, relative growth rate and PAs concentration, as well as the activities of ormithine decarboxylase (ODC), arginine decarboxylase (ADC), polyamine oxidase (PAO) and diamine oxidase (DAO) in shoots and roots of seedlings. The results showed that the seedling dry weight and relative growth rates of shoots and roots were decreased under chilling stress. And the chilling damage could be alleviated when seedlings were recovery growth at normal temperature (25℃) for 4 d. The seedling relative growth rate of HHDJY was significantly higher than that of MSk326 during chilling stress. PAs concentreation in roots was higher than that in shoots, suggesting PAs concentration was different in different parts of tobacco seedlings. It also indicated that the reason that shoots were more eazily affected by low temperature than roots was related with PAs concentration. The results of stepwise regression analysis showed that the relative growth rate in chilling-tolerant variety, HHDJY was mainly related with Put concentration, and it was mainly related with the concentration of Put and Spd in chilling-sensitive variety, MSk326 during chilling stress. The activities of polyamine synthase (ODC and ADC) and oxidase (PAO and DAO) increased during the chilling stress. ODC activity was higher than ADC activity, it indicated that Put might be formed mainly from the ODC pathway in tobacco seedlings.Tobacco seedlings of four-leaf age were treated by inhibitors, D-arginine (D-Arg) and difluoromethylornithine (DFMO), before chilling stress. D-Arg and DFMO treatments inhibited the biosynthesis of Put in shoots and roots of both varieties after suffering chilling stress. After treated with D-Arg + agmatine (Agm) and DFMO + Put, the inhibition of Put biosynthesis were alleviated. It suggested that Put biosynthesis was affected by both ADC and ODC pathways. The Put concentration in seedlings by DFMO treatment was significantly lower than that by D-Arg treatment, suggesting that biosynthesis of Put was mainly by ODC pathway, minorly by ADC pathway. Moreover, D-Arg and DFMO increased the membrane permeability of tobacco seedlings under chilling stress. And the chilling damage illustrated by the membrane permeability could be recovered when Agm or Put were added to the inhibitor solution. It indicated that the synthesis of PAs could improve the tolerance of tobacco seedling to chilling stress. Seeds of MSk326 and HHDJY were used to investigate the effects of seed soaking treatments with different concentrations (0.01, 0.1 and 1 mM) of Put and Spd on seed germination, seedling growth, antioxidant enzymes activities, as well as MDA concentration under chilling stress. The results showed that germination percentage, germination index, mean germination time, shoot and root length and seedling weight of soaking treatments with PAs were significantly higher than those of the untreated seeds under chilling stress in both varieties. The soaking treatments with low concentration (0.01 mM and 0.1 mM) of Put significantly increased the activities of antioxidant enzymes, including SOD, POD, CAT and ascorbate peroxidase (APX) in tobacco seedlings. During seedling growth, soaking treatments with PAs significantly reduced MDA accumulation. The results suggested that tobacco seeds soaking with PAs at a suitable concentration could improve germination and seedling growth under chilling stress condition. 0.01 mM Put treatment was better than other treatments for improving the chilling tolerance of chilling tolerant variety, while 0.1 mM Put was more effective for chilling sensitive variety. It suggested that different concentrations of PAs were needed for improving the tolerance of different chilling sensitive variety.Seeds of MSk326 and HHDJY were used to investigate the effects of four seed pelleting treatments with Put (Coat-1, Coat-2, Coat-3 and Coat-4) on inducing chilling tolerance of tobacco. Seed germination, antioxidant enzymes activities and MDA concentration, as well as PAs concentration were determined. During chilling stress at 11℃, Coat-2 and Coat-4 significantly increased germination percentage, germination index, seedling length and dry weight of both varieties compared to the control, that dry seed was pelleted without Put. When seedlings of four-leaf stage suffered a short chilling stress (5℃), Coat-4 improved the activities of antioxidant enzymes including SOD, POD, CAT and APX, increased endogenous Put, Spd and Spm concentration and decreased the MDA concentration. The results showed that Put pelleting treatments were available to enhance the chilling tolerance of tobacco seedlings. The optimal treatment of Put was Coat-4 (seed soaked in 0.1 mM Put for 48 h, and then pelleted). Moreover, the effects of Put may have interaction with pelleting materials. This link warrants further study.