【作者】 徐盛春；

【导师】 胡晋；

【作者基本信息】 浙江大学 ， 作物学， 2009， 博士

【摘要】 低温冷害影响烟草种子发芽、出苗和幼苗生长,最终降低烟叶的质量和产量,因此提高烟草苗期的耐寒性具有重要意义。当植物处于各种逆境条件下时,细胞内常会积累大量的多胺(Polyamines)。多胺是生物体内具有生物活性的一类低分子量脂肪族含氮碱,在对植物低温胁迫的反应中起着重要作用。本文在筛选出耐寒型烟草品种红花大金元和低温敏感型品种MSk326的基础上,以这两个品种为材料,开展了烟草种子和幼苗内多胺调控与耐寒性关系的研究。主要研究结果如下:对烟草主栽品种发芽和苗期的耐寒性进行了鉴定。在变温(20～30℃)和低温(11℃)胁迫下测定了种子发芽特性以及幼苗素质等指标,将各性状低温与常温下测定值的比值作为耐寒性评价指标。结果表明,对低温胁迫,幼苗地上部比根部敏感。相关性分析表明,相对发芽率、相对发芽指数、相对地上部高、相对幼苗干重之间显著相关,是较好的耐寒性评价指标;通过聚类分析,将5个主栽品种分为耐寒型、中等耐寒型和低温敏感型3类。研究结果将为烟草抗寒性机理研究和育种提供选材依据。在各个品种中,红花大金元耐寒性较好,MSk326则对低温较敏感。在低温胁迫(5℃)下,研究了烟草幼苗地上部与根部抗氧化酶活性与丙二醛(MDA)含量变化,探讨其与生长率的关系。结果表明,低温胁迫期间,2个品种幼苗根部低温期间相对生长率占恢复期间相对生长率的百分率显著高于地上部,说明低温对地上部的影响较大。与常温对照相比,在低温胁迫下,耐寒型品种红花大金元地上部的过氧化物酶(POD)活性增高,而超氧化物歧化酶(SOD)活性降低,过氧化氢酶(CAT)活性变化较小;低温胁迫显著增加了红花大金元根部SOD和CAT活性,而POD活性变化较小。对于低温敏感型品种MSk326,低温胁迫增加了地上部SOD活性,但降低了根部CAT活性。低温胁迫增高了两个品种幼苗中MDA含量,但在常温(25℃)恢复生长4 d后,MDA含量较低温时期有所下降。表明烟草幼苗对短期低温胁迫具有一定的恢复能力。通过逐步回归分析,发现低温胁迫下幼苗相对生长率与CAT活性关系密切。以红花大金元和MSk326为材料,研究了低温胁迫下烟草幼苗地上部和根部干重、生长速率、多胺含量、多胺合成酶(精氨酸脱羧酶和乌氨酸脱羧酶)和氧化酶(二胺氧化酶和多胺氧化酶)活性的变化,并采用逐步回归方法分析得到与两烟草品种不同部位生长指标关系最密切的多胺组分。结果发现,短期低温胁迫处理降低了两个品种幼苗地上部与根部干重和相对生长率,恢复生长4 d后低温伤害有所减轻。在低温下,红花大金元幼苗相对生长率显著高于MSk326。两个品种根部多胺含量均高于地上部,说明多胺在烟草植株的不同部位间存在差异,同时也表明地上部受低温伤害严重可能与其多胺含量较低有关。通过回归分析表明,低温敏感型烟草幼苗在低温胁迫下的相对生长率主要与腐胺(Put)有关,耐寒型烟草幼苗的生长指标与Put和亚精胺(Spd)有关。在低温胁迫下,多胺合成酶和氧化酶活性均上升,乌氨酸脱羧酶(ODC)活性强于精氨酸脱羧酶(ADC),似乎说明多胺合成主要以ODC途径为主,但需要进一步证实。以MSk326和红花大金元为材料,利用多胺抑制剂D-Arg和DFMO处理四叶期幼苗,发现低温胁迫下,两种多胺抑制剂不同程度地抑制了烟草幼苗不同部位Put的合成;而在D-Arg和DFMO中分别加入Put合成前体精氨酸(Agm)和Put后,处理幼苗,发现抑制效果得到缓解。说明在低温下ADC途径和ODC途径均影响了烟草幼苗Put的合成。同时,DFMO的抑制效果显著大于D-Arg,说明烟草幼苗多胺合成主要以ODC途径为主。对膜透性分析表明,抑制剂处理增加了低温对幼苗的伤害作用,通过加入Agm或Put能减缓此伤害,说明多胺的合成在一定程度上有利于促进烟草幼苗的抗寒能力。以不同浓度(0.01,0.1和1 mM)的两种外源多胺(Put和Spd)浸种处理MSk326和红花大金元种子,研究各处理对低温胁迫下种子发芽、幼苗素质、抗氧化酶活性和MDA含量的影响。结果表明,低温下,两个烟草品种的发芽率、发芽指数、幼苗地上部高、根长以及幼苗干鲜重均较对照显著增高,平均发芽时间显著下降。低浓度(0.01mM和0.1 mM)Put浸种处理显著提高幼苗中SOD、POD、CAT和抗坏血酸氧化酶(APX)活性;外源Put和Spd浸种处理均降低了MDA含量,表明适宜浓度的多胺浸种处理能增强幼苗的耐寒能力。0.01 mM Put对抗寒型品种的效果较好,而0.1 mM Put对低温敏感型品种的效果较好,表明提高不同耐寒型烟草品种抗低温胁迫的能力需要不同的多胺浓度。研究了低温胁迫下,4种外源Put种子丸化处理(Coat-1,Coat-2,Coat-3,Coat-4)对MSk326和红花大金元种子低温发芽和短期低温胁迫下幼苗体内的抗氧化酶活性、MDA含量及内源多胺的影响。与未添加Put的干种丸化对照比,Coat-2和Coat-4处理提高了低温11℃下两个品种的发芽率、发芽指数、全苗长和干重。在5℃短暂低温胁迫后,Coat-4的幼苗SOD、POD、CAT和APX活性显著高于对照,而MDA含量低于对照。同时,在低温胁迫和恢复生长后,烟草幼苗的Put、Spd和Spm含量也有提高。上述结果表明,Put丸化处理有利于增强烟草幼苗抵御低温逆境的能力。综合看,丸化配方Coat-4(0.1 mM Put浸种48 h后丸化)的效果最佳。但Put和丸化材料间是否存在互作而影响提高耐寒效果有待于进一步研究。更多还原

【Abstract】 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.更多还原