摘要
低温冷害危害玉米种子的萌发出苗和苗期生长,降低玉米产量,提高玉米苗期的耐寒性具有重要的意义。多胺是生物体代谢过程中产生的具有生物活性的低分子量脂肪含氮碱,在植物对环境胁迫的反应中起着重要作用。本文以不同耐寒型玉米自交系黄C(耐寒型)和Mo17(低温敏感型)为试验材料,研究了低温胁迫下玉米幼苗不同部位抗氧化酶活性、脯氨酸含量、多胺含量的变化及与其耐寒性的关系;采用响应面方法对腐胺(Put)浸种提高低温胁迫下玉米种子活力进行了优化研究;通过外施不同浓度多胺和ABA研究了不同成分多胺与膜完整性的关系;单独或组合施用多胺合成抑制剂D型精氨酸(D-Arg)、二氟甲基鸟氨酸(DFMO)、甲基乙二醛双脒基腙(MGBG)、氨基胍(Ag)研究了低温胁迫下玉米幼苗多胺含量的变化,探讨了Put的主要合成途径以及亚精胺(Spd)能否逆转生成Put及其与耐寒性的关系;同时探讨了两种不同耐寒型自交系杂交F_1代多胺的遗传特点和杂种优势。主要结果如下:
低温胁迫(5℃,3d)期间,耐寒型自交系黄C的中胚轴、胚芽鞘的相对生长率显著高于低温敏感型自交系Mo17;黄C幼苗3个部位(根、中胚轴、胚芽鞘)的过氧化氢酶(CAT)、过氧化物酶(POD)活性和脯氨酸(Pro)含量变化率均高于Mo17;与常温对照比,低温胁迫处理结束,两个自交系除Mo17根部外,其余各部位CAT活性和Pro含量均增加,黄C增加幅度大于Mo17;POD活性降低,黄C下降幅度小于Mo17。逐步回归分析表明黄C低温胁迫期间干物质的积累主要与Pro含量有关,而Mo17主要与POD活性有关,玉米中胚轴Pro含量变化率与玉米耐寒性关系最密切。
低温处理(5℃,3d)结束时,玉米幼苗3个部位除根部Put的含量以外,膜透性(用电导率表示),丙二醛(MDA)和Put的含量均显著增加,而Spd和Spin含量均显著下降。Put和Spd的相对变化量以及Spd占总多胺含量的比例黄C均高于Mo17,两自交系胚芽鞘均高于中胚轴。然而,冷害指标(电导率,MDA),黄C均低于Mo17,胚芽鞘均低于中胚轴。回归分析表明,玉米幼苗的冷害指标根部主要与Spd有关,中胚轴主要与Put和Spd有关,胚芽鞘主要与Spd和Spm有关。因此,根据耐寒型玉米幼曲多胺变化的特点并结合同归分析,能更有效的判断玉米自交系间耐寒性的差异。
采用响应面试验设计的二因素(Put浸种浓度和浸种时间)14个组合,对低温敏感型玉米自交系Mo17的种子进行处理,然后进行低温发芽试验。对得到的根和(地上)苗干重,相对生长速率利相对电导率进行响应面分析,找到了提高苗干重、根干重、苗相对生长速率和降低苗相对电导率的最佳处理组合。根据最佳组合找到适宜的Put浸种浓度和时间组合,浸种浓度为0.45-0.60 mM,浸种时间为11.5-17.9 h。
低温(5℃)处理前1d,用不同浓度的Put、Spd和Spm(分别为0.1mM、0.5mM、1.0mM和2.0mM)和ABA(5μM、10μM、50μM和100μM)处理玉米幼苗,研究对幼苗生长、膜稳定性(MDA含量为指标)以及叶绿素含量的影响。结果表明,低浓度0.5mM Put、0.1mM Spd和0.1mM Spm处理和5μM脱落酸(ABA)处理对改善幼苗生长、降低MDA含量和促进叶绿素合成的综合效果较好,且
Seed germination and seedling growth of maize (Zea mays L.) are susceptible to injury by low temperature stress. It is important to improve chilling tolerance of maize seedlings. Polyamines are small aliphatic polycationic nitrogenous compounds ubiquitous in higher plants. They are all involved in the response of plants to environmental stress. In present research, chilling-tolerant (HuangC) and chilling-sensitive (Mo 17) maize inbred lines were used to investigate the changes of the activities of catalase (CAT) and peroxidase (POD), proline (Pro) and polyamine concentration in different segments of seedlings and their relationship to chilling tolerance of maize under low temperature stress (5℃ 3 d);optimum condition on improving maize seed vigor by Put soaking was studied by employing the method of response surface analysis (RSA) under low temperature stress;relationship between polyamines, ABA and cellular membrane stability was investigated by appling exogenous polyamine, ABA with different concentrations;changes in polyamine concentration in different segments of maize seedlings, the main biosynthesis pathways of putrescine (Put) and the possibility of conversion of spermidine (Spd) to Put were investigated by applying polyamine biosynthesis inhibitors, D-arginine (D-Arg), difluoromethylormithine (DFMO), methylglyoxyl-bis- (guanyhydrazone) (MGBG) and aminoguanidine (Ag) under low temperature stress;and the genetic trait and heterosis of hybrid F1 of two maize inbred lines differing in chilling tolerance were researched. Main results were as follows:The relative growth rate (RGR) in the mesocotyl and coleoptile of HuangC significantly higher than that of Mo 17, the change rates of CAT and POD activities and Pro concentration in the three segments (root, mesocotyl and coleoptile ) of HuangC seedlings were higher than those in Mo 17 during low temperature stress (5℃, 3 d). Compared to seedlings at normal temperature, the activities of CAT and Pro concentrations in the other segments of chilled seedlings were increased except in the root of Mo 17, and the increasing extents of CAT activity and Pro concentrations in HuangC were higher than those in Mo 17. However, the POD activity in the three segments of chilled seedlings were reduced, and the decreasing extents of POD activity in HuangC was lower than those in Mo 17. The results of stepwise regression analysis showed that the accumulation of dry weights in chilling-tolerant line HuangC was mainly related to Pro concentration, however, in chilling-sensitive line Mo 17 was mainly related to POD activity during low temperature stress. The change rate of Pro concentration in mesocotyl was more closely correlation with chilling tolerance of maize inbred lines than the other physiological parameters in the other two segments.Membrane permeability (as measured by relative electrolyte (RE)), malondialdehyde (MDA) concentration and Put concentration in the three segments of the maize seedlings increased after low temperature stress (5℃, 3 d), except for Put concentration in root. However, Spd and Spermine (Spm) concentrations in the three seedling segments decreased after chilling stress. A relative change level in both Put and Spd, and the ratio of Spd to total polyamines in HuangC was higher than those of Mo 17, as compared different segments, coleoptile was higher than mesocotyl in both inbred lines. However, for chilling injury parameters, HuangC was lower than Mo 17, comparing different segments, coleoptile was lower than mesocotyl in both inbred lines. The results of regression analysis indicated that chilling injury
parameters in root were mainly correlated with Spd concentration, in mesocotyl were mainly with Put and Spd concentration and in coleoptile were mainly with Spd and Spm concentration. Therefore, the characteristics of polyamine changes in chilling-tolerant maize seedling combining with regression analysis will be more reliable for evaluating relative chilling tolerance among maize inbred lines.Two factors (concentrations of Put and soaking time) with 14 combinations of RSA experiment method was designed to treat chilling-sensitive Mo 17 seeds, then the low temperature germination experiment was carried out. After root and shoot dry weight, RGR and RE were analyzed by RSA, the optimum combinations of Put treatment for increasing shoot dry weight, root dry weight, shoot RGR and decreasing shoot RE was found. According to these optimum combinations, the optimum ranges for Put soaking concentration and soaking time were 0.45-0.60mM and 11.5-17.9 h.The effects treated seedlings by Put> Spd and Spm (O.lmM, 0.5mM, l.OmM, 2.0mM ) and abscisic acid (ABA) (5uM, lOuM, 50uM, lOOuM) before low temperature stress on seedling growth, membrane stability (indicator by MDA) and Chlorophyll (Chi) contents were investigated. The results indicated that low concentrations of 0.5mM Put, O.lmM Spd and O.lmM Spm treatments and 5uM ABA on the improving seedling growth, reducing MDA concentrations and increasing Chi contens had better effects. Among Put, Spd and Spm treatments, the most effective treatment was Spd, followed was Put, the worst was Spm. High concentrations of polyamine (2.0mM) and ABA (50uM, lOOuM) treatments were negative effects. It indicated that the relationship between polyamine and cellular membrane stability, the improvement of chilling tolerance of maize seedlings were all correlated with polyamines and ABA concentrations and components of polyamines.MGBG treatment reduced the Spd concentration, increased membrane permeability and MDA concentrations in three segments of both maize inbred lines seedlings before and after low temperature treatment (5°C, 3 d), and decreased the length and fresh weight of root and shoot, chl contents at the end of recovery growth. It suggested that MGBG treatment increased the maize seedling chilling injury. Put or Spd plus MGBG treatment alleviated the lipid peroxidation (indicator by MDA concentration), improved seedlings growth and increased Chl contents comparing to MGBG treatment, however, Spd plus MGBG was more effective than that of Put plus MGBG It suggested that Spd may be play an more important role in chilling tolerance.D-Arg and DFMO treatment inhibited the biosynthesis of Put in three segments of both inbred lines after suffering low temperature stress suggesting the Put biosynthesis may be affected by both ADC and ODC pathways. However, when maize seedlings treated by the same concentration of D-Arg or DFMO, the Put concentration in seedlings by DFMO treatment was significantly lower than that by D-Arg treatment. It indicated that biosynthesis of Put was mainly via ODC pathway, minorly via ADC pathway. Low concentration (10uM) of D-Arg and DFMO improved seedling growth, increased the length and fresh weight of root and shoot and Chl content of seedling leaves under low temperature, moreover, the effects of D-Arg treatment was better than that of DFMO treatment. It may be related to that DFMO inhibited Put synthesis more effective than D-Arg.Combinations of four polyamine inhibitors, D-Arg and DFMO (inhibiting the biosynthesis of Put), Ag (inhibiting the degradation of Put) and MGBG (inhibiting the biosynthesis of Spd and Spm from Put) with different concentration ((1) lOuM combinations included lOuM D-Arg, lOuM DFMO, lOuM MGBG, lOOuM Ag;(2) lOOuM combinations included lOOuM D-Arg, lOOuM DFMO, lOOuM MGBG, lOOOuM Ag;(3) 500uM combinations included 500uM D-Arg, 200uM DFMO, 500uM MGBG, 5mM Ag;(4) lOOOuM combinations included lOOOuM D-Arg, 200uM DFMO, lOOOuM MGBG, lOmM Ag) were applied to investigate whether Spd can convert to Put or not. Combination treatments of polyamine
inhibitors inhibited the biosynthesis of Put in three segments of both maize inbred lines. There was no significant difference in Put concentration between 500jtM and 1000/iM polyamine inhibitor combinations except for Mo 17 coleoptile. Therefore, Spd plus 1000/iM polyamine inhibitor combination was selected to treat maize seedlings sequentially, Put concentration in the treated root was 2.54-5.23 times, in mesoctyl was 1.34-1.48 times, in coleoptile was 1.47-1.49 times compared to the control of 1000/tM combination, respectively. It indicated that Spd may be converted to Put, moreover, this conversion in root was stronger than that in mesocotyl and colepoptile.Heterosis of hybrid Fl of two maize inbred lines differing in chilling tolerance was researched under low temperature stress. Length and fresh weight of root and shoot of the Fi seedlings were higher than those in the parents. Chi a, carotenoid and total Chi content in the Fi were all higher than those in the parents. After maize seedling growth for 4 days at normal temperature, Spd and total polyamine concentration in the three segments of the Fi seedlings had significant heterosis in heterosis of mid-parent, heterosis of over high-parent, and index of heterosis, however, Put and Spm no heterosis, total ployamine had heterosis in heterosis of mid-parent and index of heterosis. After suffering low temperature stress for 3 days, Spd and total polyamines concentrations in three segments of the Fi seedlings, except for total polyamines concentrations in coleoptile, heterosis in other segements was simlar to at normal temperature. After recovery growth at normal temperature for 3days, Spd and toatl polyamine concentration lost heterosis, only Put in mesoctyl had heterosis. MDA had a strong negative heterosis, sugesting that the hybrid Fj from chilling-tolerant crossed chilling-sensitive inbred lines reduced lipid peroxidtion may be related to the increase of Spd concentration in different segments of the Fi seedlings.
引文
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